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The Circulating Proteome - Technological Developments, Current Challenges, and Future Trends

Journal of Proteome Research, 2024

Philipp E Geyer, Daniel Hornburg, Maria Pernemalm, Stefanie M Hauck, Krishnan K Palaniappan, Vincent Albrecht, Laura F Dagley, Robert L Moritz, Xiaobo Yu, Fredrik Edfors, Yves Vandenbrouck, Johannes B Mueller-Reif, Zhi Sun, Virginie Brun, Sara Ahadi, Gilbert S Omenn, Eric W Deutsch, Jochen M Schwenk

Recent improvements in proteomics technologies have fundamentally altered our capacities to characterize human biology. There is an ever-growing interest in using these novel methods for studying the circulating proteome, as blood offers an accessible window into human health. However, every methodological innovation and analytical progress calls for reassessing our existing approaches and routines to ensure that the new data will add value to the greater biomedical research community and avoid previous errors. As representatives of HUPO’s Human Plasma Proteome Project (HPPP), we present our 2024 survey of the current progress in our community, including the latest build of the Human Plasma Proteome PeptideAtlas that now comprises 4608 proteins detected in 113 data sets. We then discuss the updates of established proteomics methods, emerging technologies, and investigations of proteoforms …

Plasma Proteome Profiling to detect and avoid sample‐related biases in biomarker studies

EMBO molecular medicine 11 (11), e10427, 2019

Philipp E Geyer, Eugenia Voytik, Peter V Treit, Sophia Doll, Alisa Kleinhempel, Lili Niu, Johannes B Müller, Marie‐Luise Buchholtz, Jakob M Bader, Daniel Teupser, Lesca M Holdt, Matthias Mann

Plasma and serum are rich sources of information regarding an individual's health state, and protein tests inform medical decision making. Despite major investments, few new biomarkers have reached the clinic. Mass spectrometry (MS)‐based proteomics now allows highly specific and quantitative readout of the plasma proteome. Here, we employ Plasma Proteome Profiling to define quality marker panels to assess plasma samples and the likelihood that suggested biomarkers are instead artifacts related to sample handling and processing. We acquire deep reference proteomes of erythrocytes, platelets, plasma, and whole blood of 20 individuals (> 6,000 proteins), and compare serum and plasma proteomes. Based on spike‐in experiments, we determine sample quality‐associated proteins, many of which have been reported as biomarker candidates as revealed by a comprehensive literature survey. We …

High‐resolution serum proteome trajectories in COVID‐19 reveal patient‐specific seroconversion

EMBO molecular medicine 13 (8), e14167, 2021

Philipp E Geyer, Florian M Arend, Sophia Doll, Marie‐Luise Louiset, Sebastian Virreira Winter, Johannes B Müller‐Reif, Furkan M Torun, Michael Weigand, Peter Eichhorn, Mathias Bruegel, Maximilian T Strauss, Lesca M Holdt, Matthias Mann, Daniel Teupser

A deeper understanding of COVID‐19 on human molecular pathophysiology is urgently needed as a foundation for the discovery of new biomarkers and therapeutic targets. Here we applied mass spectrometry (MS)‐based proteomics to measure serum proteomes of COVID‐19 patients and symptomatic, but PCR‐negative controls, in a time‐resolved manner. In 262 controls and 458 longitudinal samples of 31 patients, hospitalized for COVID‐19, a remarkable 26% of proteins changed significantly. Bioinformatics analyses revealed co‐regulated groups and shared biological functions. Proteins of the innate immune system such as CRP, SAA1, CD14, LBP, and LGALS3BP decreased early in the time course. Regulators of coagulation (APOH, FN1, HRG, KNG1, PLG) and lipid homeostasis (APOA1, APOC1, APOC2, APOC3, PON1) increased over the course of the disease. A global correlation map provides a system …

Revisiting biomarker discovery by plasma proteomics

Molecular systems biology 13 (9), 942, 2017

Philipp E Geyer, Lesca M Holdt, Daniel Teupser, Matthias Mann

Clinical analysis of blood is the most widespread diagnostic procedure in medicine, and blood biomarkers are used to categorize patients and to support treatment decisions. However, existing biomarkers are far from comprehensive and often lack specificity and new ones are being developed at a very slow rate. As described in this review, mass spectrometry (MS)‐based proteomics has become a powerful technology in biological research and it is now poised to allow the characterization of the plasma proteome in great depth. Previous “triangular strategies” aimed at discovering single biomarker candidates in small cohorts, followed by classical immunoassays in much larger validation cohorts. We propose a “rectangular” plasma proteome profiling strategy, in which the proteome patterns of large cohorts are correlated with their phenotypes in health and disease. Translating such concepts into clinical practice …

The proteome landscape of the kingdoms of life

Nature 582 (7813), 592-596, 2020

Johannes B Müller, Philipp E Geyer, Ana R Colaco, Peter V Treit, Maximilian T Strauss, Mario Oroshi, Sophia Doll, Sebastian Virreira Winter, Jakob M Bader, Niklas Köhler, Fabian Theis, Alberto Santos, Matthias Mann

Proteins carry out the vast majority of functions in all biological domains, but for technological reasons their large-scale investigation has lagged behind the study of genomes. Since the first essentially complete eukaryotic proteome was reported, advances in mass-spectrometry-based proteomics have enabled increasingly comprehensive identification and quantification of the human proteome, , –. However, there have been few comparisons across species,, in stark contrast with genomics initiatives. Here we use an advanced proteomics workflow—in which the peptide separation step is performed by a microstructured and extremely reproducible chromatographic system—for the in-depth study of 100 taxonomically diverse organisms. With two million peptide and 340,000 stringent protein identifications obtained in a standardized manner, we double the number of proteins with solid experimental evidence known to …

BoxCar acquisition method enables single-shot proteomics at a depth of 10,000 proteins in 100 minutes

Nature methods 15 (6), 440-448, 2018

Florian Meier, Philipp E Geyer, Sebastian Virreira Winter, Juergen Cox, Matthias Mann

Great advances have been made in sensitivity and acquisition speed on the Orbitrap mass analyzer, enabling increasingly deep proteome coverage. However, these advances have been mainly limited to the MS2 level, whereas ion beam sampling for the MS1 scans remains extremely inefficient. Here we report a data-acquisition method, termed BoxCar, in which filling multiple narrow mass-to-charge segments increases the mean ion injection time more than tenfold as compared to that of a standard full scan. In 1-h analyses, the method provided MS1-level evidence for more than 90% of the proteome of a human cancer cell line that had previously been identified in 24 fractions, and it quantified more than 6,200 proteins in ten of ten replicates. In mouse brain tissue, we detected more than 10,000 proteins in only 100 min, and sensitivity extended into the low-attomolar range.

Plasma proteome profiling to assess human health and disease

Cell systems 2 (3), 185-195, 2016

Philipp E Geyer, Nils A Kulak, Garwin Pichler, Lesca M Holdt, Daniel Teupser, Matthias Mann

Proteins in the circulatory system mirror an individual's physiology. In daily clinical practice, protein levels are generally determined using single-protein immunoassays. High-throughput, quantitative analysis using mass-spectrometry-based proteomics of blood, plasma, and serum would be advantageous but is challenging because of the high dynamic range of protein abundances. Here, we introduce a rapid and robust "plasma proteome profiling" pipeline. This single-run shotgun proteomic workflow does not require protein depletion and enables quantitative analysis of hundreds of plasma proteomes from 1 μl single finger pricks with 20 min gradients. The apolipoprotein family, inflammatory markers such as C-reactive protein, gender-related proteins, and >40 FDA-approved biomarkers are reproducibly quantified (CV <20% with label-free quantification). Furthermore, we functionally interpret a 1,000-protein …

Proteomics reveals the effects of sustained weight loss on the human plasma proteome

Molecular systems biology 12 (12), 901, 2016

Philipp E Geyer, Nicolai J Wewer Albrechtsen, Stefka Tyanova, Niklas Grassl, Eva W Iepsen, Julie Lundgren, Sten Madsbad, Jens J Holst, Signe S Torekov, Matthias Mann

Sustained weight loss is a preferred intervention in a wide range of metabolic conditions, but the effects on an individual's health state remain ill‐defined. Here, we investigate the plasma proteomes of a cohort of 43 obese individuals that had undergone 8 weeks of 12% body weight loss followed by a year of weight maintenance. Using mass spectrometry‐based plasma proteome profiling, we measured 1,294 plasma proteomes. Longitudinal monitoring of the cohort revealed individual‐specific protein levels with wide‐ranging effects of losing weight on the plasma proteome reflected in 93 significantly affected proteins. The adipocyte‐secreted SERPINF1 and apolipoprotein APOF1 were most significantly regulated with fold changes of −16% and +37%, respectively (P < 10−13), and the entire apolipoprotein family showed characteristic differential regulation. Clinical laboratory parameters are reflected in the plasma …

Advances and utility of the human plasma proteome

Journal of proteome research 20 (12), 5241-5263, 2021

Eric W Deutsch, Gilbert S Omenn, Zhi Sun, Michal Maes, Maria Pernemalm, Krishnan K Palaniappan, Natasha Letunica, Yves Vandenbrouck, Virginie Brun, Sheng-ce Tao, Xiaobo Yu, Philipp E Geyer, Vera Ignjatovic, Robert L Moritz, Jochen M Schwenk

The study of proteins circulating in blood offers tremendous opportunities to diagnose, stratify, or possibly prevent diseases. With recent technological advances and the urgent need to understand the effects of COVID-19, the proteomic analysis of blood-derived serum and plasma has become even more important for studying human biology and pathophysiology. Here we provide views and perspectives about technological developments and possible clinical applications that use mass-spectrometry(MS)- or affinity-based methods. We discuss examples where plasma proteomics contributed valuable insights into SARS-CoV-2 infections, aging, and hemostasis and the opportunities offered by combining proteomics with genetic data. As a contribution to the Human Proteome Organization (HUPO) Human Plasma Proteome Project (HPPP), we present the Human Plasma PeptideAtlas build 2021-07 that comprises …

Mass spectrometry-based plasma proteomics: considerations from sample collection to achieving translational data

Journal of proteome research 18 (12), 4085-4097, 2019

Vera Ignjatovic, Philipp E Geyer, Krishnan K Palaniappan, Jessica E Chaaban, Gilbert S Omenn, Mark S Baker, Eric W Deutsch, Jochen M Schwenk

The proteomic analysis of human blood and blood-derived products (e.g., plasma) offers an attractive avenue to translate research progress from the laboratory into the clinic. However, due to its unique protein composition, performing proteomics assays with plasma is challenging. Plasma proteomics has regained interest due to recent technological advances, but challenges imposed by both complications inherent to studying human biology (e.g., interindividual variability) and analysis of biospecimens (e.g., sample variability), as well as technological limitations remain. As part of the Human Proteome Project (HPP), the Human Plasma Proteome Project (HPPP) brings together key aspects of the plasma proteomics pipeline. Here, we provide considerations and recommendations concerning study design, plasma collection, quality metrics, plasma processing workflows, mass spectrometry (MS) data acquisition …

Plasma proteome profiling discovers novel proteins associated with non‐alcoholic fatty liver disease

Molecular systems biology 15 (3), e8793, 2019

Lili Niu, Philipp E Geyer, Nicolai J Wewer Albrechtsen, Lise L Gluud, Alberto Santos, Sophia Doll, Peter V Treit, Jens J Holst, Filip K Knop, Tina Vilsbøll, Anders Junker, Stephan Sachs, Kerstin Stemmer, Timo D Müller, Matthias H Tschöp, Susanna M Hofmann, Matthias Mann

Non‐alcoholic fatty liver disease (NAFLD) affects 25% of the population and can progress to cirrhosis with limited treatment options. As the liver secretes most of the blood plasma proteins, liver disease may affect the plasma proteome. Plasma proteome profiling of 48 patients with and without cirrhosis or NAFLD revealed six statistically significantly changing proteins (ALDOB, APOM, LGALS3BP, PIGR, VTN, and AFM), two of which are already linked to liver disease. Polymeric immunoglobulin receptor (PIGR) was significantly elevated in both cohorts by 170% in NAFLD and 298% in cirrhosis and was further validated in mouse models. Furthermore, a global correlation map of clinical and proteomic data strongly associated DPP4, ANPEP, TGFBI, PIGR, and APOE with NAFLD and cirrhosis. The prominent diabetic drug target DPP4 is an aminopeptidase like ANPEP, ENPEP, and LAP3, all of which are up‐regulated …

Urinary proteome profiling for stratifying patients with familial Parkinson’s disease

EMBO molecular medicine 13 (3), e13257, 2021

Sebastian Virreira Winter, Ozge Karayel, Maximilian T Strauss, Shalini Padmanabhan, Matthew Surface, Kalpana Merchant, Roy N Alcalay, Matthias Mann

The prevalence of Parkinson's disease (PD) is increasing but the development of novel treatment strategies and therapeutics altering the course of the disease would benefit from specific, sensitive, and non‐invasive biomarkers to detect PD early. Here, we describe a scalable and sensitive mass spectrometry (MS)‐based proteomic workflow for urinary proteome profiling. Our workflow enabled the reproducible quantification of more than 2,000 proteins in more than 200 urine samples using minimal volumes from two independent patient cohorts. The urinary proteome was significantly different between PD patients and healthy controls, as well as between LRRK2 G2019S carriers and non‐carriers in both cohorts. Interestingly, our data revealed lysosomal dysregulation in individuals with the LRRK2 G2019S mutation. When combined with machine learning, the urinary proteome data alone were sufficient to classify …

A paired liver biopsy and plasma proteomics study reveals circulating biomarkers for alcohol-related liver disease

BioRxiv, 2020.10. 16.337592, 2020

Lili Niu, Maja Thiele, Philipp E Geyer, Ditlev Nytoft Rasmussen, Henry Emanuel Webel, Alberto Santos, Rajat Gupta, Florian Meier, Maximilian Strauss, Maria Kjaergaard, Katrine Lindvig, Suganya Jacobsen, Simon Rasmussen, Torben Hansen, Aleksander Krag, Matthias Mann

Existing tests for detecting liver fibrosis, inflammation and steatosis, three stages of liver disease that are still reversible are severely hampered by limited accuracy or invasive nature. Here, we present a paired liver-plasma proteomics approach to infer molecular pathophysiology and to identify biomarkers in a cross-sectional alcohol-related liver disease cohort of nearly 600 individuals. Metabolic functions were downregulated whereas fibrosis-associated signaling and novel immune responses were upregulated, but only half of tissue proteome changes were transmitted to the circulation. Machine learning models based on our biomarker panels outperformed existing tests, laying the foundation for a generic proteomic liver health assessment.

Plasma proteome profiling reveals dynamics of inflammatory and lipid homeostasis markers after Roux-En-Y gastric bypass surgery

Cell systems 7 (6), 601-612. e3, 2018

Nicolai J Wewer Albrechtsen, Philipp E Geyer, Sophia Doll, Peter V Treit, Kirstine N Bojsen-Møller, Christoffer Martinussen, Nils B Jørgensen, Signe S Torekov, Florian Meier, Lili Niu, Alberto Santos, Eva C Keilhauer, Jens J Holst, Sten Madsbad, Matthias Mann

Obesity-related diseases affect half of the global population, and bariatric surgery is one of the few interventions with long-lasting weight loss and cardio-metabolic effects. Here, we investigated the effect of Roux-en-Y gastric bypass surgery on the plasma proteome, hypothesizing that specific proteins or protein patterns may serve as key mediators and markers of the metabolic response. We performed mass spectrometry (MS)-based proteomics on two longitudinal studies encompassing 47 morbidly obese patients, generating quantitative information on more than 1,700 proteins. A global correlation matrix incorporating about 200,000 relationships revealed functional connections between proteins and assigned them to physiological processes. The main classes of significantly altered proteins were markers of systemic inflammation and those involved in lipid metabolism. Our data highlight robust correlative and …

EASI-tag enables accurate multiplexed and interference-free MS2-based proteome quantification

Nature Methods 15 (7), 527-530, 2018

Sebastian Virreira Winter, Florian Meier, Christoph Wichmann, Juergen Cox, Matthias Mann, Felix Meissner

We developed EASI-tag (easily abstractable sulfoxide-based isobaric-tag), a new type of amine-derivatizing and sulfoxide-containing isobaric labeling reagents for highly accurate quantitative proteomics analysis using mass spectrometry. We observed that EASI-tag labels dissociate at low collision energy and generate peptide-coupled, interference-free reporter ions with high yield. Efficient isolation of 12C precursors and quantification at the MS2 level allowed accurate determination of quantitative differences between up to six multiplexed samples.

Proteome profiling of cerebrospinal fluid reveals biomarker candidates for Parkinson’s disease

Cell Reports Medicine 3 (6), 2022

Ozge Karayel, Sebastian Virreira Winter, Shalini Padmanabhan, Yuliya I Kuras, Duc Tung Vu, Idil Tuncali, Kalpana Merchant, Anne-Marie Wills, Clemens R Scherzer, Matthias Mann

Parkinson's disease (PD) is a growing burden worldwide, and there is no reliable biomarker used in clinical routines to date. Cerebrospinal fluid (CSF) is routinely collected in patients with neurological symptoms and should closely reflect alterations in PD patients' brains. Here, we describe a scalable and sensitive mass spectrometry (MS)-based proteomics workflow for CSF proteome profiling. From two independent cohorts with over 200 individuals, our workflow reproducibly quantifies over 1,700 proteins from minimal CSF amounts. Machine learning determines OMD, CD44, VGF, PRL, and MAN2B1 to be altered in PD patients or to significantly correlate with clinical scores. We also uncover signatures of enhanced neuroinflammation in LRRK2 G2019S carriers, as indicated by increased levels of CTSS, PLD4, and HLA proteins. A comparison with our previously acquired urinary proteomes reveals a large overlap …

Rapid proteomic analysis for solid tumors reveals LSD 1 as a drug target in an end‐stage cancer patient

Molecular oncology 12 (8), 1296-1307, 2018

Sophia Doll, Maximilian C Kriegmair, Alberto Santos, Michael Wierer, Fabian Coscia, Helen Michele Neil, Stefan Porubsky, Philipp E Geyer, Andreas Mund, Philipp Nuhn, Matthias Mann

Recent advances in mass spectrometry (MS)‐based technologies are now set to transform translational cancer proteomics from an idea to a practice. Here, we present a robust proteomic workflow for the analysis of clinically relevant human cancer tissues that allows quantitation of thousands of tumor proteins in several hours of measuring time and a total turnaround of a few days. We applied it to a chemorefractory metastatic case of the extremely rare urachal carcinoma. Quantitative comparison of lung metastases and surrounding tissue revealed several significantly upregulated proteins, among them lysine‐specific histone demethylase 1 (LSD1/KDM1A). LSD1 is an epigenetic regulator and the target of active development efforts in oncology. Thus, clinical cancer proteomics can rapidly and efficiently identify actionable therapeutic options. While currently described for a single case study, we envision that it can …

Proteomic Characterization of Undifferentiated Small Round Cell Sarcomas with EWSR1-and CIC:: DUX4-Translocations Reveals Diverging Tumor Biology and Distinct Diagnostic Markers

Modern Pathology, 100511, 2024

Sophia Doll, Lisa Schweizer, Christine Bollwein, Katja Steiger, Nicole Pfarr, Maria Walker, Klaus Wörtler, Carolin Knebel, Ruediger von Eisenhart-Rothe, Wolfgang Hartmann, Wilko Weichert, Matthias Mann, Peer-Hendrik Kuhn, Katja Specht

Undifferentiated small round cell sarcomas of bone and soft tissue (USRS) are a group of tumors with heterogenic genomic alterations sharing similar morphology. In the present study, we performed a comparative large-scale proteomic analysis of USRS (n=42) with diverse genomic translocations including classic Ewing sarcomas with EWSR1::FLI1 fusions (n=24) or EWSR1::ERG – fusions (n=4), sarcomas with an EWSR1 – rearrangement (n=2), CIC::DUX4 fusion (n=8), as well as tumors classified as USRS with no genetic data available (n=4). Proteins extracted from formalin-fixed, paraffin-embedded (FFPE) pretherapeutic biopsies were analyzed qualitatively and quantitatively using shot gun mass spectrometry (MS). More than 8000 protein groups could be quantified using data-independent acquisition. Unsupervised hierarchical cluster analysis based on proteomic data allowed stratification of the 42 cases …

MaxQuant. Live enables global targeting of more than 25,000 peptides

Molecular & Cellular Proteomics 18 (5), 982-994, 2019

Christoph Wichmann, Florian Meier, Sebastian Virreira Winter, Andreas-David Brunner, Jürgen Cox, Matthias Mann

Mass spectrometry (MS)-based proteomics is often performed in a shotgun format, in which as many peptide precursors as possible are selected from full or MS1 scans so that their fragment spectra can be recorded in MS2 scans. Although achieving great proteome depths, shotgun proteomics cannot guarantee that each precursor will be fragmented in each run. In contrast, targeted proteomics aims to reproducibly and sensitively record a restricted number of precursor/fragment combinations in each run, based on prescheduled mass-to-charge and retention time windows. Here we set out to unify these two concepts by a global targeting approach in which an arbitrary number of precursors of interest are detected in real-time, followed by standard fragmentation or advanced peptide-specific analyses. We made use of a fast application programming interface to a quadrupole Orbitrap instrument and real-time …

Region and cell-type resolved quantitative proteomic map of the human heart

Nature communications 8 (1), 1-13, 2017

Sophia Doll, Martina Dressen, Philipp E Geyer, Daniel N Itzhak, Christian Braun, Stefanie A Doppler, Florian Meier, Marcus-Andre Deutsch, Harald Lahm, Ruediger Lange, Markus Krane, Matthias Mann

The heart is a central human organ and its diseases are the leading cause of death worldwide, but an in-depth knowledge of the identity and quantity of its constituent proteins is still lacking. Here, we determine the healthy human heart proteome by measuring 16 anatomical regions and three major cardiac cell types by high-resolution mass spectrometry-based proteomics. From low microgram sample amounts, we quantify over 10,700 proteins in this high dynamic range tissue. We combine copy numbers per cell with protein organellar assignments to build a model of the heart proteome at the subcellular level. Analysis of cardiac fibroblasts identifies cellular receptors as potential cell surface markers. Application of our heart map to atrial fibrillation reveals individually distinct mitochondrial dysfunctions. The heart map is available at maxqb. biochem. mpg. de as a resource for future analyses of normal heart function …

LAMP-Seq: population-scale COVID-19 diagnostics using combinatorial barcoding

biorxiv, 2020.04. 06.025635, 2020

Jonathan L Schmid-Burgk, Ricarda M Schmithausen, David Li, Ronja Hollstein, Amir Ben-Shmuel, Ofir Israeli, Shay Weiss, Nir Paran, Gero Wilbring, Jana Liebing, David Feldman, Mikołaj Słabicki, Bärbel Lippke, Esther Sib, Jacob Borrajo, Jonathan Strecker, Julia Reinhardt, Per Hoffmann, Brian Cleary, Michael Hölzel, Markus M Nöthen, Martin Exner, Kerstin U Ludwig, Aviv Regev, Feng Zhang

The ongoing SARS-CoV-2 pandemic has already caused devastating losses. Exponential spread can be slowed by social distancing and population-wide isolation measures, but those place a tremendous burden on society, and, once lifted, exponential spread can re-emerge. Regular population-scale testing, combined with contact tracing and case isolation, should help break the cycle of transmission, but current detection strategies are not capable of such large-scale processing. Here we present a protocol for LAMP-Seq, a barcoded Reverse-Transcription Loop-mediated Isothermal Amplification (RT-LAMP) method that is highly scalable. Individual samples are stabilized, inactivated, and amplified in three isothermal heat steps, generating barcoded amplicons that can be pooled and analyzed en masse by sequencing. Using unique barcode combinations per sample from a compressed barcode space enables extensive pooling, potentially further reducing cost and simplifying logistics. We validated LAMP-Seq on 28 clinical samples, empirically optimized the protocol and barcode design, and performed initial safety evaluation. Relying on world-wide infrastructure for next-generation sequencing, and in the context of population-wide sample collection, LAMP-Seq could be scaled to analyze millions of samples per day.

Transparent exploration of machine learning for biomarker discovery from proteomics and omics data

Journal of Proteome Research 22 (2), 359-367, 2022

Furkan M Torun, Sebastian Virreira Winter, Sophia Doll, Felix M Riese, Artem Vorobyev, Johannes B Mueller-Reif, Philipp E Geyer, Maximilian T Strauss

Biomarkers are of central importance for assessing the health state and to guide medical interventions and their efficacy; still, they are lacking for most diseases. Mass spectrometry (MS)-based proteomics is a powerful technology for biomarker discovery but requires sophisticated bioinformatics to identify robust patterns. Machine learning (ML) has become a promising tool for this purpose. However, it is sometimes applied in an opaque manner and generally requires specialized knowledge. To enable easy access to ML for biomarker discovery without any programming or bioinformatics skills, we developed “OmicLearn” (http://OmicLearn.org), an open-source browser-based ML tool using the latest advances in the Python ML ecosystem. Data matrices from omics experiments are easily uploaded to an online or a locally installed web server. OmicLearn enables rapid exploration of the suitability of various ML …

The case for proteomics and phospho‐proteomics in personalized cancer medicine

PROTEOMICS–Clinical Applications 13 (2), 1800113, 2019

Sophia Doll, Florian Gnad, Matthias Mann

The concept of personalized medicine is predominantly been pursued through genomic and transcriptomic technologies, leading to the identification of multiple mutations in a large variety of cancers. However, it has proven challenging to distinguish driver and passenger mutations and to deal with tumor heterogeneity and resistant clonal populations. More generally, these heterogeneous mutation patterns do not in themselves predict the tumor phenotype. Analysis of the expressed proteins in a tumor and their modification states reveals if and how these mutations are translated to the functional level. It is already known that proteomic changes including posttranslational modifications are crucial drivers of oncogenesis, but proteomics technology has only recently become comparable in depth and accuracy to RNAseq. These advances also allow the rapid and highly sensitive analysis of formalin‐fixed and paraffin …

Noninvasive proteomic biomarkers for alcohol-related liver disease

Nature Medicine 28 (6), 1277-1287, 2022

Lili Niu, Maja Thiele, Philipp E Geyer, Ditlev Nytoft Rasmussen, Henry Emanuel Webel, Alberto Santos, Rajat Gupta, Florian Meier, Maximilian Strauss, Maria Kjaergaard, Katrine Lindvig, Suganya Jacobsen, Simon Rasmussen, Torben Hansen, Aleksander Krag, Matthias Mann

Alcohol-related liver disease (ALD) is a major cause of liver-related death worldwide, yet understanding of the three key pathological features of the disease—fibrosis, inflammation and steatosis—remains incomplete. Here, we present a paired liver–plasma proteomics approach to infer molecular pathophysiology and to explore the diagnostic and prognostic capability of plasma proteomics in 596 individuals (137 controls and 459 individuals with ALD), 360 of whom had biopsy-based histological assessment. We analyzed all plasma samples and 79 liver biopsies using a mass spectrometry (MS)-based proteomics workflow with short gradient times and an enhanced, data-independent acquisition scheme in only 3 weeks of measurement time. In plasma and liver biopsy tissues, metabolic functions were downregulated whereas fibrosis-associated signaling and immune responses were upregulated. Machine …

LAMP-Seq enables sensitive, multiplexed COVID-19 diagnostics using molecular barcoding

Nature biotechnology 39 (12), 1556-1562, 2021

Kerstin U Ludwig, Ricarda M Schmithausen, David Li, Max L Jacobs, Ronja Hollstein, Katja Blumenstock, Jana Liebing, Mikołaj Słabicki, Amir Ben-Shmuel, Ofir Israeli, Shay Weiss, Thomas S Ebert, Nir Paran, Wibke Rüdiger, Gero Wilbring, David Feldman, Bärbel Lippke, Nina Ishorst, Lara M Hochfeld, Eva C Beins, Ines H Kaltheuner, Maximilian Schmitz, Aliona Wöhler, Manuel Döhla, Esther Sib, Marius Jentzsch, Eva-Maria C Moench, Jacob D Borrajo, Jonathan Strecker, Julia Reinhardt, Brian Cleary, Matthias Geyer, Michael Hölzel, Rhiannon Macrae, Markus M Nöthen, Per Hoffmann, Martin Exner, Aviv Regev, Feng Zhang, Jonathan L Schmid-Burgk

Frequent testing of large population groups combined with contact tracing and isolation measures will be crucial for containing Coronavirus Disease 2019 outbreaks. Here we present LAMP-Seq, a modified, highly scalable reverse transcription loop-mediated isothermal amplification (RT–LAMP) method. Unpurified biosamples are barcoded and amplified in a single heat step, and pooled products are analyzed en masse by sequencing. Using commercial reagents, LAMP-Seq has a limit of detection of ~2.2 molecules per µl at 95% confidence and near-perfect specificity for severe acute respiratory syndrome coronavirus 2 given its sequence readout. Clinical validation of an open-source protocol with 676 swab samples, 98 of which were deemed positive by standard RT–qPCR, demonstrated 100% sensitivity in individuals with cycle threshold values of up to 33 and a specificity of 99.7%, at a very low material cost …

A knowledge graph to interpret clinical proteomics data

Nature biotechnology 40 (5), 692-702, 2022

Alberto Santos, Ana R Colaço, Annelaura B Nielsen, Lili Niu, Maximilian Strauss, Philipp E Geyer, Fabian Coscia, Nicolai J Wewer Albrechtsen, Filip Mundt, Lars Juhl Jensen, Matthias Mann

Implementing precision medicine hinges on the integration of omics data, such as proteomics, into the clinical decision-making process, but the quantity and diversity of biomedical data, and the spread of clinically relevant knowledge across multiple biomedical databases and publications, pose a challenge to data integration. Here we present the Clinical Knowledge Graph (CKG), an open-source platform currently comprising close to 20 million nodes and 220 million relationships that represent relevant experimental data, public databases and literature. The graph structure provides a flexible data model that is easily extendable to new nodes and relationships as new databases become available. The CKG incorporates statistical and machine learning algorithms that accelerate the analysis and interpretation of typical proteomics workflows. Using a set of proof-of-concept biomarker studies, we show how the CKG …

Structure-guided multivalent nanobodies block SARS-CoV-2 infection and suppress mutational escape

Science 371 (6530), eabe6230, 2021

Paul-Albert Koenig, Hrishikesh Das, Hejun Liu, Beate M Kümmerer, Florian N Gohr, Lea-Marie Jenster, Lisa DJ Schiffelers, Yonas M Tesfamariam, Miki Uchima, Jennifer D Wuerth, Karl Gatterdam, Natalia Ruetalo, Maria H Christensen, Caroline I Fandrey, Sabine Normann, Jan MP Tödtmann, Steffen Pritzl, Leo Hanke, Jannik Boos, Meng Yuan, Xueyong Zhu, Jonathan L Schmid-Burgk, Hiroki Kato, Michael Schindler, Ian A Wilson, Matthias Geyer, Kerstin U Ludwig, B Martin Hällberg, Nicholas C Wu, Florian I Schmidt

The global scale and rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pose unprecedented challenges to society, health care systems, and science. In addition to effective and safe vaccines, passive immunization by antibody-related molecules offers an opportunity to harness the vertebrate immune system to fight viral infections in high-risk patients. Variable domains of heavy-chain–only antibodies (VHHs), also known as nanobodies, are suitable lead molecules in such efforts, as they are small, extremely stable, easy to engineer, and economic to produce in simple expression systems.We engineered improved multivalent nanobodies neutralizing SARS-CoV-2 on the basis of two principles: (i) detailed structural information of their epitopes and binding modes to the viral spike protein and (ii) mechanistic insights into viral fusion with cellular …

Designer nuclease-mediated generation of knockout THP1 cells

TALENs: Methods and Protocols, 261-272, 2016

Tobias Schmidt, Jonathan L Schmid-Burgk, Thomas S Ebert, Moritz M Gaidt, Veit Hornung

Recent developments in the field of designer nucleases allow the efficient and specific manipulation of genomic architectures in eukaryotic cell lines. To this end, it has become possible to introduce DNA double strand breaks (DSBs) at user-defined genomic loci. If located in critical coding regions of genes, thus induced DSBs can lead to insertions or deletions (indels) that result in frameshift mutations and thereby the knockout of the target gene. In this chapter, we describe a step-by-step workflow for establishing knockout cell clones of the difficult-to-transfect suspension cell line THP1. The here described protocol encompasses electroporation, cell cloning, and a deep sequencing-based genotyping step that allows the in-parallel analysis of 96 cell clones per gene of interest. Furthermore, we describe the use of the analysis tool OutKnocker that allows rapid identification of cell clones with all-allelic frameshift …

Cell intrinsic immunity spreads to bystander cells via the intercellular transfer of cGAMP

Nature 503 (7477), 530-534, 2013

Andrea Ablasser, Jonathan L Schmid-Burgk, Inga Hemmerling, Gabor L Horvath, Tobias Schmidt, Eicke Latz, Veit Hornung

The innate immune defence of multicellular organisms against microbial pathogens requires cellular collaboration. Information exchange allowing immune cells to collaborate is generally attributed to soluble protein factors secreted by pathogen-sensing cells. Cytokines, such as type I interferons (IFNs), serve to alert non-infected cells to the possibility of pathogen challenge. Moreover, in conjunction with chemokines they can instruct specialized immune cells to contain and eradicate microbial infection. Several receptors and signalling pathways exist that couple pathogen sensing to the induction of cytokines, whereas cytosolic recognition of nucleic acids seems to be exquisitely important for the activation of type I IFNs, master regulators of antiviral immunity. Cytosolic DNA is sensed by the receptor cyclic GMP-AMP (cGAMP) synthase (cGAS), which catalyses the synthesis of the second messenger cGAMP(2′-5 …

RNA-guided DNA insertion with CRISPR-associated transposases

Science 365 (6448), 48-53, 2019

Jonathan Strecker, Alim Ladha, Zachary Gardner, Jonathan L Schmid-Burgk, Kira S Makarova, Eugene V Koonin, Feng Zhang

CRISPR-Cas nucleases are powerful tools for manipulating nucleic acids; however, targeted insertion of DNA remains a challenge, as it requires host cell repair machinery. Here we characterize a CRISPR-associated transposase from cyanobacteria Scytonema hofmanni (ShCAST) that consists of Tn7-like transposase subunits and the type V-K CRISPR effector (Cas12k). ShCAST catalyzes RNA-guided DNA transposition by unidirectionally inserting segments of DNA 60 to 66 base pairs downstream of the protospacer. ShCAST integrates DNA into targeted sites in the Escherichia coli genome with frequencies of up to 80% without positive selection. This work expands our understanding of the functional diversity of CRISPR-Cas systems and establishes a paradigm for precision DNA insertion.

Engineering of CRISPR-Cas12b for human genome editing

Nature communications 10 (1), 212, 2019

Jonathan Strecker, Sara Jones, Balwina Koopal, Jonathan Schmid-Burgk, Bernd Zetsche, Linyi Gao, Kira S Makarova, Eugene V Koonin, Feng Zhang

The type-V CRISPR effector Cas12b (formerly known as C2c1) has been challenging to develop for genome editing in human cells, at least in part due to the high temperature requirement of the characterized family members. Here we explore the diversity of the Cas12b family and identify a promising candidate for human gene editing from Bacillus hisashii, BhCas12b. However, at 37 °C, wild-type BhCas12b preferentially nicks the non-target DNA strand instead of forming a double strand break, leading to lower editing efficiency. Using a combination of approaches, we identify gain-of-function mutations for BhCas12b that overcome this limitation. Mutant BhCas12b facilitates robust genome editing in human cell lines and ex vivo in primary human T cells, and exhibits greater specificity compared to S. pyogenes Cas9. This work establishes a third RNA-guided nuclease platform, in addition to Cas9 and Cpf1/Cas12a …

A genome-wide CRISPR (clustered regularly interspaced short palindromic repeats) screen identifies NEK7 as an essential component of NLRP3 inflammasome activation

Journal of Biological Chemistry 291 (1), 103-109, 2016

Jonathan L Schmid-Burgk, Dhruv Chauhan, Tobias Schmidt, Thomas S Ebert, Julia Reinhardt, Elmar Endl, Veit Hornung

Inflammasomes are high molecular weight protein complexes that assemble in the cytosol upon pathogen encounter. This results in caspase-1-dependent pro-inflammatory cytokine maturation, as well as a special type of cell death, known as pyroptosis. The Nlrp3 inflammasome plays a pivotal role in pathogen defense, but at the same time, its activity has also been implicated in many common sterile inflammatory conditions. To this effect, several studies have identified Nlrp3 inflammasome engagement in a number of common human diseases such as atherosclerosis, type 2 diabetes, Alzheimer disease, or gout. Although it has been shown that known Nlrp3 stimuli converge on potassium ion efflux upstream of Nlrp3 activation, the exact molecular mechanism of Nlrp3 activation remains elusive. Here, we describe a genome-wide CRISPR/Cas9 screen in immortalized mouse macrophages aiming at the unbiased …

Inflammasomes: current understanding and open questions

Cellular and Molecular Life Sciences 68, 765-783, 2011

Franz Bauernfeind, Andrea Ablasser, Eva Bartok, Sarah Kim, Jonathan Schmid-Burgk, Taner Cavlar, Veit Hornung

The innate immune system relies on its capability to detect invading microbes, tissue damage, or stress via evolutionarily conserved receptors. The nucleotide-binding domain leucine-rich repeat (NLR)-containing family of pattern recognition receptors includes several proteins that drive inflammation in response to a wide variety of molecular patterns. In particular, the NLRs that participate in the formation of a molecular scaffold termed the “inflammasome” have been intensively studied in past years. Inflammasome activation by multiple types of tissue damage or by pathogen-associated signatures results in the autocatalytic cleavage of caspase-1 and ultimately leads to the processing and thus secretion of pro-inflammatory cytokines, most importantly interleukin (IL)-1β and IL-18. Here, we review the current knowledge of mechanisms leading to the activation of inflammasomes. In particular, we focus on the …

Optical pooled screens in human cells

Cell 179 (3), 787-799. e17, 2019

David Feldman, Avtar Singh, Jonathan L Schmid-Burgk, Rebecca J Carlson, Anja Mezger, Anthony J Garrity, Feng Zhang, Paul C Blainey

Genetic screens are critical for the systematic identification of genes underlying cellular phenotypes. Pooling gene perturbations greatly improves scalability but is not compatible with imaging of complex and dynamic cellular phenotypes. Here, we introduce a pooled approach for optical genetic screens in mammalian cells. We use targeted in situ sequencing to demultiplex a library of genetic perturbations following image-based phenotyping. We screened a set of 952 genes across millions of cells for involvement in nuclear factor κB (NF-κB) signaling by imaging the translocation of RelA (p65) to the nucleus. Screening at a single time point across 3 cell lines recovered 15 known pathway components, while repeating the screen with live-cell imaging revealed a role for Mediator complex subunits in regulating the duration of p65 nuclear retention. These results establish a highly multiplexed approach to image-based …

Plasma proteome profiles treatment efficacy of incretin dual agonism in diet‐induced obese female and male mice

Diabetes, Obesity and Metabolism 23 (1), 195-207, 2021

Stephan Sachs, Lili Niu, Philipp Geyer, Sigrid Jall, Maximilian Kleinert, Annette Feuchtinger, Kerstin Stemmer, Markus Brielmeier, Brian Finan, Richard D DiMarchi, Matthias H Tschöp, Nicolai Wewer Albrechtsen, Matthias Mann, Timo D Müller, Susanna M Hofmann

Unimolecular peptides targeting the receptors for glucagon‐like peptide‐1 (GLP‐1) and glucose‐dependent insulinotropic polypeptide (GIP) (GLP‐1/GIP co‐agonist) have been shown to outperform each single peptide in the treatment of obesity and cardiometabolic disease in preclinical and clinical trials. By combining physiological treatment endpoints with plasma proteomic profiling (PPP), we aimed to identify biomarkers to advance non‐invasive metabolic monitoring of compound treatment success and exploration of ulterior treatment effects on an individual basis.We performed metabolic phenotyping along with PPP in body weight‐matched male and female diet‐induced obese (DIO) mice treated for 21 days with phosphate‐buffered saline, single GIP and GLP‐1 mono‐agonists, or a GLP‐1/GIP co‐agonist.GLP‐1R/GIPR co‐agonism improved obesity, glucose …

Caspase‐4 mediates non‐canonical activation of the NLRP3 inflammasome in human myeloid cells

European journal of immunology 45 (10), 2911-2917, 2015

Jonathan L Schmid‐Burgk, Moritz M Gaidt, Tobias Schmidt, Thomas S Ebert, Eva Bartok, Veit Hornung

Inflammasome activation culminates in activation of caspase‐1, which leads to the maturation and subsequent release of cytokines of the interleukin 1 (IL‐1) family and results in a particular form of cell death known as pyroptosis. In addition, in the murine system, a so‐called non‐canonical inflammasome involving caspase‐11 has been described that directly responds to cytosolic LPS. Here, we show that the human monocytic cell line THP1 activates the inflammasome in response to cytosolic LPS in a TLR4‐independent fashion. This response is mediated by caspase‐4 and accompanied by caspase‐1 activation, pyroptosis, and IL‐1β maturation. In addition to caspase‐4, efficient IL‐1β conversion upon intracellular LPS delivery relies on potassium efflux, NLRP3, ASC, and caspase‐1, indicating that although caspase‐4 activation alone is sufficient to induce pyroptosis, this process depends on the NLRP3 …

A conserved histidine in the RNA sensor RIG-I controls immune tolerance to N1-2′ O-methylated self RNA

Immunity 43 (1), 41-51, 2015

Christine Schuberth-Wagner, Janos Ludwig, Ann Kristin Bruder, Anna-Maria Herzner, Thomas Zillinger, Marion Goldeck, Tobias Schmidt, Jonathan L Schmid-Burgk, Romy Kerber, Steven Wolter, Jan-Philip Stümpel, Andreas Roth, Eva Bartok, Christian Drosten, Christoph Coch, Veit Hornung, Winfried Barchet, Beate M Kümmerer, Gunther Hartmann, Martin Schlee

The cytosolic helicase retinoic acid-inducible gene-I (RIG-I) initiates immune responses to most RNA viruses by detecting viral 5′-triphosphorylated RNA (pppRNA). Although endogenous mRNA is also 5′-triphosphorylated, backbone modifications and the 5′-ppp-linked methylguanosine (m7G) cap prevent immunorecognition. Here we show that the methylation status of endogenous capped mRNA at the 5′-terminal nucleotide (N1) was crucial to prevent RIG-I activation. Moreover, we identified a single conserved amino acid (H830) in the RIG-I RNA binding pocket as the mediator of steric exclusion of N1-2′O-methylated RNA. H830A alteration (RIG-I(H830A)) restored binding of N1-2′O-methylated pppRNA. Consequently, endogenous mRNA activated the RIG-I(H830A) mutant but not wild-type RIG-I. Similarly, knockdown of the endogenous N1-2′O-methyltransferase led to considerable RIG-I stimulation …

Early IFN-α signatures and persistent dysfunction are distinguishing features of NK cells in severe COVID-19

Immunity 54 (11), 2650-2669. e14, 2021

Benjamin Krämer, Rainer Knoll, Lorenzo Bonaguro, Michael ToVinh, Jan Raabe, Rosario Astaburuaga-García, Jonas Schulte-Schrepping, Kim Melanie Kaiser, Gereon J Rieke, Jenny Bischoff, Malte B Monin, Christoph Hoffmeister, Stefan Schlabe, Elena De Domenico, Nico Reusch, Kristian Händler, Gary Reynolds, Nils Blüthgen, Gudrun Hack, Claudia Finnemann, Hans D Nischalke, Christian P Strassburg, Emily Stephenson, Yapeng Su, Louis Gardner, Dan Yuan, Daniel Chen, Jason Goldman, Philipp Rosenstiel, Susanne V Schmidt, Eicke Latz, Kevin Hrusovsky, Andrew J Ball, Joe M Johnson, Paul-Albert Koenig, Florian I Schmidt, Muzlifah Haniffa, James R Heath, Beate M Kümmerer, Verena Keitel, Björn Jensen, Paula Stubbemann, Florian Kurth, Leif E Sander, Birgit Sawitzki, Janine Altmüller, Angel Angelov, Anna C Aschenbrenner, Robert Bals, Alexander Bartholomäus, Anke Becker, Matthias Becker, Daniela Bezdan, Michael Bitzer, Conny Blumert, Ezio Bonifacio, Peer Bork, Bunk Boyke, Helmut Blum, Nicolas Casadei, Thomas Clavel, Maria Colome-Tatche, Markus Cornberg, Inti Alberto De La Rosa Velázquez, Andreas Diefenbach, Alexander Dilthey, Nicole Fischer, Konrad Förstner, Sören Franzenburg, Julia-Stefanie Frick, Gisela Gabernet, Julien Gagneur, Tina Ganzenmueller, Marie Gauder, Janina Geißert, Alexander Goesmann, Siri Göpel, Adam Grundhoff, Hajo Grundmann, Torsten Hain, Frank Hanses, Ute Hehr, André Heimbach, Marius Hoeper, Friedemann Horn, Daniel Hübschmann, Michael Hummel, Thomas Iftner, Angelika Iftner, Thomas Illig, Stefan Janssen, Jörn Kalinowski, René Kallies, Birte Kehr, Andreas Keller, Oliver T Keppler, Sarah Kim-Hellmuth, Christoph Klein, Michael Knop, Oliver Kohlbacher, Karl Köhrer, Jan Korbel, Peter G Kremsner, Denise Kühnert, Ingo Kurth, Markus Landthaler, Yang Li, Kerstin U Ludwig, Oliwia Makarewicz, Federico Marini, Manja Marz, Alice C McHardy, Christian Mertes, Maximilian Münchhoff, Sven Nahnsen, Markus Nöthen, Francine Ntoumi, Peter Nürnberg, Stephan Ossowski, Jörg Overmann, Silke Peter, Klaus Pfeffer, Isabell Pink, Anna R Poetsch, Ulrike Protzer, Alfred Pühler, Nikolaus Rajewsky, Markus Ralser, Kristin Reiche, Olaf Rieß, Stephan Ripke, Ulisses Nunes da Rocha, Philip Rosenstiel, Antoine-Emmanuel Saliba, Leif Erik Sander, Simone Scheithauer, Philipp Schiffer, Jonathan Schmid-Burgk, Wulf Schneider, Eva-Christina Schulte, Joachim L Schultze, Alexander Sczyrba, Mariam L Sharaf, Yogesh Singh, Michael Sonnabend, Oliver Stegle, Jens Stoye, Fabian Theis, Thomas Ulas, Janne Vehreschild

Longitudinal analyses of the innate immune system, including the earliest time points, are essential to understand the immunopathogenesis and clinical course of coronavirus disease (COVID-19). Here, we performed a detailed characterization of natural killer (NK) cells in 205 patients (403 samples; days 2 to 41 after symptom onset) from four independent cohorts using single-cell transcriptomics and proteomics together with functional studies. We found elevated interferon (IFN)-α plasma levels in early severe COVD-19 alongside increased NK cell expression of IFN-stimulated genes (ISGs) and genes involved in IFN-α signaling, while upregulation of tumor necrosis factor (TNF)-induced genes was observed in moderate diseases. NK cells exert anti-SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) activity but are functionally impaired in severe COVID-19. Further, NK cell dysfunction may be relevant …

A streamlined mass spectrometry–based proteomics workflow for large‐scale FFPE tissue analysis

The Journal of pathology 251 (1), 100-112, 2020

Fabian Coscia, Sophia Doll, Jacob Mathias Bech, Lisa Schweizer, Andreas Mund, Ernst Lengyel, Jan Lindebjerg, Gunvor Iben Madsen, José MA Moreira, Matthias Mann

Formalin fixation and paraffin‐embedding (FFPE) is the most common method to preserve human tissue for clinical diagnosis, and FFPE archives represent an invaluable resource for biomedical research. Proteins in FFPE material are stable over decades but their efficient extraction and streamlined analysis by mass spectrometry (MS)–based proteomics has so far proven challenging. Herein we describe a MS‐based proteomic workflow for quantitative profiling of large FFPE tissue cohorts directly from histopathology glass slides. We demonstrate broad applicability of the workflow to clinical pathology specimens and variable sample amounts, including low‐input cancer tissue isolated by laser microdissection. Using state‐of‐the‐art data dependent acquisition (DDA) and data independent acquisition (DIA) MS workflows, we consistently quantify a large part of the proteome in 100 min single‐run analyses. In an …

Proteomik in kardiovaskulärer Forschung

BIOspektrum 24, 168-171, 2018

Sophia Doll, Michael Wierer

Cardiovascular diseases are the leading cause of death worldwide. The molecular mechanisms involved in the underlying pathophysiologies of atherosclerosis and heart related disorders are still poorly known. A closer understanding would greatly benefit clinical outcome predictions and treatment options in future. Two recent studies by Matthias Mann and his team, presented in this review, have addressed cardiovascular diseases using high-resolution mass spectrometry-based proteomics.

Single fiber proteomics of respiratory chain defects in mitochondrial disorders

bioRxiv, 421750, 2018

Marta Murgia, Jing Tan, Philipp E Geyer, Sophia Doll, Matthias Mann, Thomas Klopstock

Mitochondrial DNA mutations progressively compromise the respiratory chain of skeletal muscle, resulting in a mosaic of metabolically healthy and defective fibers. The single fiber investigation of this important diagnostic feature has been beyond the capability of large-scale technologies so far. We used laser capture microdissection (LCM) to excise thin sections of individual muscle fibers from frozen biopsies of patients suffering from chronic progressive external ophthalmoplegia. We then applied a highly sensitive mass spectrometry (MS)-based proteomics workflow to analyze healthy and defective muscle fibers within the same biopsy. We quantified more than 4000 proteins in each patient, covering 75% of all respiratory chain subunits, and compared their expression in metabolically healthy and defective muscle fibers. Our findings show that mitochondrial disease causes extensive proteomic rearrangements, affecting the OPA1-dependent cristae remodeling pathway and mitochondrial translation. We provide fiber type-specific information showing that increased expression of fatty acid oxidation enzymes occurs in defective slow but not fast muscle fibers. Our findings shed light on compensatory mechanisms in muscle fibers that struggle with energy shortage and metabolic stress.

Quantitative proteomics reveals fundamental regulatory differences in oncogenic HRAS and isocitrate dehydrogenase (IDH1) driven astrocytoma

Molecular & Cellular Proteomics 16 (1), 39-56, 2017

Sophia Doll, Anatoly Urisman, Juan A Oses-Prieto, David Arnott, Alma L Burlingame

Glioblastoma multiformes (GBMs) are high-grade astrocytomas and the most common brain malignancies. Primary GBMs are often associated with disturbed RAS signaling, and expression of oncogenic HRAS results in a malignant phenotype in glioma cell lines. Secondary GBMs arise from lower-grade astrocytomas, have slower progression than primary tumors, and contain IDH1 mutations in over 70% of cases. Despite significant amount of accumulating genomic and transcriptomic data, the fundamental mechanistic differences of gliomagenesis in these two types of high-grade astrocytoma remain poorly understood. Only a few studies have attempted to investigate the proteome, phosphorylation signaling, and epigenetic regulation in astrocytoma. In the present study, we applied quantitative phosphoproteomics to identify the main signaling differences between oncogenic HRAS and mutant IDH1-driven glioma …

Biological and translational cancer proteomics

lmu, 2018

Sophia Doll

Cancer is the second leading cause of death worldwide and many cancer subtypes remain poorly understood. Most conventional chemotherapeutic treatments are still associated with life-threatening toxic side effects that primarily result from a lack of specificity directed towards cancer cells. Recent breakthroughs in genomic and transcriptomic sequencing technologies have allowed the molecular profiling of thousands of tumors in different cancer types. It has become evident that cancer cannot be considered a singular disease and that its manifestations cannot exclusively be explained by the accumulation of genetic mutations. Instead, epigenetic and proteomic changes as well as posttranslational modifications (PTMs) of proteins are crucial drivers of oncogenesis. In this thesis, I investigated systemwide alterations in cancer at several biological and cellular levels using mass spectrometry (MS). Starting from the nucleus of the cell, I explored the epigenetic changes in lymphoma at the biotechnology company Genentech Inc. We found that the methyltransferase EZH2 is the most significantly over-expressed epigenetic regulator in cancer, and is co-regulated with a cell cycle network. Zooming out from the nuclear level, I analyzed phosphorylation-signaling alterations in primary and secondary glioblastoma cell line models at the University of California, San Francisco (UCSF). Here, I focused on the interplay between the MAPK and PI3K signaling cascades. At the Max Planck Institute (MPI) of Biochemistry, I moved on to translational proteomics, working with human cancer tissues. I optimized an MS-based proteomic workflow for the rapid screening …

Ethical principles, constraints, and opportunities in clinical proteomics

Molecular & Cellular Proteomics 20, 2021

Sebastian Porsdam Mann, Peter V Treit, Philipp E Geyer, Gilbert S Omenn, Matthias Mann

Recent advances in MS-based proteomics have vastly increased the quality and scope of biological information that can be derived from human samples. These advances have rendered current workflows increasingly applicable in biomedical and clinical contexts. As proteomics is poised to take an important role in the clinic, associated ethical responsibilities increase in tandem with impacts on the health, privacy, and well-being of individuals. We conducted and here report a systematic literature review of ethical issues in clinical proteomics. We add our perspectives from a background of bioethics, the results of our accompanying article extracting individual-sensitive results from patient samples, and the literature addressing similar issues in genomics. The spectrum of potential issues ranges from patient reidentification to incidental findings of clinical significance. The latter can be divided into actionable and …

Accurate MS-based Rab10 phosphorylation stoichiometry determination as readout for LRRK2 activity in Parkinson's disease

Molecular & Cellular Proteomics 19 (9), 1546-1560, 2020

Özge Karayel, Francesca Tonelli, Sebastian Virreira Winter, Phillip E Geyer, Ying Fan, Esther M Sammler, Dario R Alessi, Martin Steger, Matthias Mann

Pathogenic mutations in the Leucine-rich repeat kinase 2 (LRRK2) are the predominant genetic cause of Parkinson's disease (PD). They increase its activity, resulting in augmented Rab10-Thr73 phosphorylation and conversely, LRRK2 inhibition decreases pRab10 levels. Currently, there is no assay to quantify pRab10 levels for drug target engagement or patient stratification. To meet this challenge, we developed an high accuracy and sensitivity targeted mass spectrometry (MS)-based assay for determining Rab10-Thr73 phosphorylation stoichiometry in human samples. It uses synthetic stable isotope-labeled (SIL) analogues for both phosphorylated and nonphosphorylated tryptic peptides surrounding Rab10-Thr73 to directly derive the percentage of Rab10 phosphorylation from attomole amounts of the endogenous phosphopeptide. The SIL and the endogenous phosphopeptides are separately admitted into …

Proteome profiling of cerebrospinal fluid reveals novel biomarker candidates for parkinson’s Disease

bioRxiv, 2021.07. 22.453322, 2021

Ozge Karayel, Sebastian Virreira Winter, Shalini Padmanabhan, Yuliya I Kuras, Duc Tung Vu, Idil Tuncali, Kalpana Merchant, Anne-Marie Wills, Clemens R Scherzer, Matthias Mann

Parkinson’s disease (PD) is a growing burden worldwide, and despite ongoing efforts to find reliable biomarkers for early and differential diagnosis, prognosis and disease monitoring, there is no biofluid biomarker used in clinical routine to date. Cerebrospinal fluid (CSF) is collected often and should closely reflect structural and functional alterations in PD patients’ brains. Here we describe a scalable and sensitive mass spectrometry (MS)-based proteomics workflow for CSF proteome profiling to find specific biomarkers and identify disease-related changes in CSF protein levels in PD. From two independent cohorts consisting of more than 200 individuals, our workflow reproducibly quantified over 1,700 proteins from minimal sample amounts. Combined with machine learning, this identified a group of several proteins, including OMD, CD44, VGF, PRL, and MAN2B1 that were altered in PD patients or significantly correlate with clinical scores, indicative of disease progression. Interestingly, we uncovered signatures of enhanced neuroinflammation in patients with familial PD (LRRK2 G2019S carriers) as indicated by increased levels of CTSS, PLD4, HLA-DRA, HLA-DRB1, and HLA-DPA1. A comparison with urinary proteome changes in PD patients revealed a large overlap in protein composition PD-associated changes in these body fluids, including lysosomal factors like CTSS. Our results validate MS-based proteomics of CSF as a valuable strategy for biomarker discovery and patient stratification in a neurodegenerative disease like PD. Consistent proteomic signatures across two independent CSF cohorts and previously acquired urinary …

A novel LC system embeds analytes in pre-formed gradients for rapid, ultra-robust proteomics

Molecular & Cellular Proteomics 17 (11), 2284-2296, 2018

Nicolai Bache, Philipp E Geyer, Dorte B Bekker-Jensen, Ole Hoerning, Lasse Falkenby, Peter V Treit, Sophia Doll, Igor Paron, Johannes B Müller, Florian Meier, Jesper V Olsen, Ole Vorm, Matthias Mann

To further integrate mass spectrometry (MS)-based proteomics into biomedical research and especially into clinical settings, high throughput and robustness are essential requirements. They are largely met in high-flow rate chromatographic systems for small molecules but these are not sufficiently sensitive for proteomics applications. Here we describe a new concept that delivers on these requirements while maintaining the sensitivity of current nano-flow LC systems. Low-pressure pumps elute the sample from a disposable trap column, simultaneously forming a chromatographic gradient that is stored in a long storage loop. An auxiliary gradient creates an offset, ensuring the re-focusing of the peptides before the separation on the analytical column by a single high-pressure pump. This simplified design enables robust operation over thousands of sample injections. Furthermore, the steps between injections are …

Proteomic profiling of colorectal adenomas identifies a predictive risk signature for development of metachronous advanced colorectal neoplasia

Gastroenterology 165 (1), 121-132. e5, 2023

Jacob Mathias Bech, Thilde Terkelsen, Annette Snejbjerg Bartels, Fabian Coscia, Sophia Doll, Siqi Zhao, Zhaojun Zhang, Nils Brünner, Jan Lindebjerg, Gunvor Iben Madsen, Xiangdong Fang, Matthias Mann, José Manuel Afonso Moreira

Colonic adenomatous polyps, or adenomas, are frequent precancerous lesions and the origin of most cases of colorectal adenocarcinoma. However, we know from epidemiologic studies that although most colorectal cancers (CRCs) originate from adenomas, only a small fraction of adenomas (3%–5%) ever progress to cancer. At present, there are no molecular markers to guide follow-up surveillance programs.We profiled, by mass spectrometry–based proteomics combined with machine learning analysis, a selected cohort of formalin-fixed, paraffin-embedded high-grade (HG) adenomas with long clinical follow-up, collected as part of the Danish national screening program. We grouped subjects in the cohort according to their subsequent history of findings: a nonmetachronous advanced neoplasia group (G0), with no new HG adenomas or CRCs up to 10 years after polypectomy, and …

Proteomics for blood biomarker exploration of severe mental illness: pitfalls of the past and potential for the future

Translational psychiatry 8 (1), 160, 2018

Ashley L Comes, Sergi Papiol, Thorsten Mueller, Philipp E Geyer, Matthias Mann, Thomas G Schulze

Recent improvements in high-throughput proteomic approaches are likely to constitute an essential advance in biomarker discovery, holding promise for improved personalized care and drug development. These methodologies have been applied to study multivariate protein patterns and provide valuable data of peripheral tissues. To highlight findings of the last decade for three of the most common psychiatric disorders, namely schizophrenia (SZ), bipolar disorder (BD), and major depressive disorder (MDD), we queried PubMed. Here we delve into the findings from thirty studies, which used proteomics and multiplex immunoassay approaches for peripheral blood biomarker exploration. In an explorative approach, we ran enrichment analyses in peripheral blood according to these results and ascertained the overlap between proteomic findings and genetic loci identified in genome-wide association studies …

OutKnocker: a web tool for rapid and simple genotyping of designer nuclease edited cell lines

Genome research 24 (10), 1719-1723, 2014

Jonathan L Schmid-Burgk, Tobias Schmidt, Moritz M Gaidt, Karin Pelka, Eicke Latz, Thomas S Ebert, Veit Hornung

The application of designer nucleases allows the induction of DNA double-strand breaks (DSBs) at user-defined genomic loci. Due to imperfect DNA repair mechanisms, DSBs can lead to alterations in the genomic architecture, such as the disruption of the reading frame of a critical exon. This can be exploited to generate somatic knockout cell lines. While high genome editing activities can be achieved in various cellular systems, obtaining cell clones that contain all-allelic frameshift mutations at the target locus of interest remains a laborious task. To this end, we have developed an easy-to-follow deep sequencing workflow and the evaluation tool OutKnocker (www.OutKnocker.org), which allows convenient, reliable, and cost-effective identification of knockout cell lines.

AFA-sonication Followed by Modified Protein Aggregation Capture (APAC) Enables Direct, Reproducible and Non-toxic Sample Preparation of FFPE Tissue for Mass Spectrometrybased Proteomics

Covaris Appl. Note-M020141. Available online: https://d24ci5y4j5ezt1 …, 2020

Lisa Schweizer, Fabian Coscia, Johannes Müller, Sophia Doll, Michael Wierer, Matthias Mann

The preparation of formalin-fixed and paraffin-embedded (FFPE) tissue for mass spectrometry-based (MS) proteomics relies on efficient removal of paraffin, which commonly involves toxic xylol as gold standard. Besides its toxicity, paraffin removal by xylol typically comes at the cost of reproducibility and sample throughput. Here, we developed an alternative, non-toxic MS based proteomics workflow based on Adaptive Focused Acoustics®(AFA®) sonication and a modified Protein Aggregation Capture (PAC) method (APAC). Considering depth of proteome analysis, de-crosslinking efficiency and dynamic range of protein abundance, our method completely removed any disadvantages to existing xylol-based deparaffinization protocols, while also increasing reproducibility. The protocol can be easily adjusted to large or small sample amounts and, based on Covaris 96-well plates, applied to cohorts in a clinical cancer setting. Thus, our protocol represents a nontoxic, reproducible and high-throughput FFPE tissue analysis method by mass spectrometry-based proteomics.

Prolonged IKKβ inhibition improves ongoing CTL antitumor responses by incapacitating regulatory T cells

Cell reports 21 (3), 578-586, 2017

Christoph Heuser, Janine Gotot, Eveline Christina Piotrowski, Marie-Sophie Philipp, Christina Johanna Felicia Courreges, Martin Sylvester Otte, Linlin Guo, Jonathan Leo Schmid-Burgk, Veit Hornung, Annkristin Heine, Percy Alexander Knolle, Natalio Garbi, Edgar Serfling, Cesar Evaristo, Friedrich Thaiss, Christian Kurts

Regulatory T cells (Tregs) prevent autoimmunity but limit antitumor immunity. The canonical NF-κB signaling pathway both activates immunity and promotes thymic Treg development. Here, we report that mature Tregs continue to require NF-κB signaling through IκB-kinase β (IKKβ) after thymic egress. Mice lacking IKKβ in mature Tregs developed scurfy-like immunopathology due to death of peripheral FoxP3+ Tregs. Also, pharmacological IKKβ inhibition reduced Treg numbers in the circulation by ∼50% and downregulated FoxP3 and CD25 expression and STAT5 phosphorylation. In contrast, activated cytotoxic T lymphocytes (CTLs) were resistant to IKKβ inhibition because other pathways, in particular nuclear factor of activated T cells (NFATc1) signaling, sustained their survival and expansion. In a melanoma mouse model, IKKβ inhibition after CTL cross-priming improved the antitumor response and delayed …

Mid-infrared spectroscopy and mass spectrometry combined to build a comprehensive lung cancer fingerprint of blood plasma

European Quantum Electronics Conference, jsii_p_5, 2019

Liudmila Voronina, Marinus Huber, Philipp Geyer, Johannes Muller, Cristina Leonardo, Michael Trubetskov, Kosmas V Kepesidis, Matthias Mann, Ferenc Krausz, Mihaela Zigman

Infrared (IR) spectroscopy of liquid biopsies shows high potential to become a non-invasive, cost-efficient and fast diagnostic tool for several types of cancers, acute myocardial infarction, Alzheimer’s disease as well as possibly other pathologies [1]. However, interpretation of the disease-induced changes in an IR absorption spectrum remains challenging due to high molecular complexity of the samples. Here we perform for the first time Fourier-Transform IR (FTIR) absorption and non-targeted mass spectrometry (MS) based proteomic measurements of the very same set of human blood plasma samples, collected from lung cancer patients and a control group. This combination shows that the IR spectroscopic fingerprint of lung cancer is caused by differential regulation of a number of plasma proteins. Generally, quantitative analysis of cancer-induced changes in blood composition is of paramount importance for …

Dynamic human liver proteome atlas reveals functional insights into disease pathways

Molecular Systems Biology 18 (5), e10947, 2022

Lili Niu, Philipp E Geyer, Rajat Gupta, Alberto Santos, Florian Meier, Sophia Doll, Nicolai J Wewer Albrechtsen, Sabine Klein, Cristina Ortiz, Frank E Uschner, Robert Schierwagen, Jonel Trebicka, Matthias Mann

Deeper understanding of liver pathophysiology would benefit from a comprehensive quantitative proteome resource at cell type resolution to predict outcome and design therapy. Here, we quantify more than 150,000 sequence‐unique peptides aggregated into 10,000 proteins across total liver, the major liver cell types, time course of primary cell cultures, and liver disease states. Bioinformatic analysis reveals that half of hepatocyte protein mass is comprised of enzymes and 23% of mitochondrial proteins, twice the proportion of other liver cell types. Using primary cell cultures, we capture dynamic proteome remodeling from tissue states to cell line states, providing useful information for biological or pharmaceutical research. Our extensive data serve as spectral library to characterize a human cohort of non‐alcoholic steatohepatitis and cirrhosis. Dramatic proteome changes in liver tissue include signatures of hepatic …

Sympathetic cardiac function in early sepsis: Noninvasive evaluation with [123I]-meta-iodobenzylguanidine (123I-MIBG) in vivo SPECT imaging

Journal of Nuclear Cardiology 25, 483-491, 2018

Romain Clerc, Sophia Doll, Laurent M Riou, Pascale Perret, Alexis Broisat, Audrey Soubies, Marie-Dominique Desruet, Daniel Fagret, Carole Schwebel, Catherine Ghezzi

Sympathetic system abnormalities have been reported in sepsis-related cardiac dysfunction. The present study aimed at evaluating the potential of the norepinephrine radiolabeled analogue [123I]-meta-iodobenzylguanidine (123I-MIBG) for the noninvasive assessment of modifications in cardiac sympathetic activity occurring in lipopolysaccharide (LPS)-induced experimental acute sepsis by single-photon emission computed tomographic imaging (SPECT).Sepsis was induced in male Wistar rats by intraperitoneal injection of 10 mg·kg−1 lipopolysaccharide (n = 16), whereas control animals (n = 7) were injected with vehicle (NaCl 0.9%). Echocardiography in LPS-injected animals (n = 8) demonstrated systolic and diastolic cardiac dysfunction. 123I-MIBG was injected 1 hour after LPS or vehicle administration (n = 8 and …

Cohort profile: the MUNICH Preterm and Term Clinical study (MUNICH-PreTCl), a neonatal birth cohort with focus on prenatal and postnatal determinants of infant and childhood morbidity

BMJ open 11 (6), e050652, 2021

Susanne Pangratz-Fuehrer, Orsolya Genzel-Boroviczény, Wolfgang Bodensohn, Robin Eisenburger, Janne Scharpenack, Philipp E Geyer, Johannes B Müller-Reif, Nadja van Hagen, Alina M Müller, Majken Karoline Jensen, Christoph Klein, Matthias Mann, Claudia Nussbaum

The MUNICH Preterm and Term Clinical (MUNICH-PreTCl) birth cohort was established to uncover pathological processes contributing to infant/childhood morbidity and mortality. We collected comprehensive medical information of healthy and sick newborns and their families, together with infant blood samples for proteomic analysis. MUNICH-PreTCl aims to identify mechanism-based biomarkers in infant health and disease to deliver more precise diagnostic and predictive information for disease prevention. We particularly focused on risk factors for pregnancy complications, family history of genetically influenced health conditions such as diabetes and paediatric long-term health—all to be further monitored and correlated with proteomics data in the future.Newborns and their parents were recruited from the Perinatal Center at the LMU University Hospital, Munich, between February 2017 and …

Ultra-deep and quantitative saliva proteome reveals dynamics of the oral microbiome

Genome medicine 8, 1-13, 2016

Niklas Grassl, Nils Alexander Kulak, Garwin Pichler, Philipp Emanuel Geyer, Jette Jung, Sören Schubert, Pavel Sinitcyn, Juergen Cox, Matthias Mann

The oral cavity is home to one of the most diverse microbial communities of the human body and a major entry portal for pathogens. Its homeostasis is maintained by saliva, which fulfills key functions including lubrication of food, pre-digestion, and bacterial defense. Consequently, disruptions in saliva secretion and changes in the oral microbiome contribute to conditions such as tooth decay and respiratory tract infections. Here we set out to quantitatively map the saliva proteome in great depth with a rapid and in-depth mass spectrometry-based proteomics workflow.We used recent improvements in mass spectrometry (MS)-based proteomics to develop a rapid workflow for mapping the saliva proteome quantitatively and at great depth. Standard clinical cotton swabs were used to collect saliva form eight healthy individuals at two …

Sequential defects in cardiac lineage commitment and maturation cause hypoplastic left heart syndrome

Circulation 144 (17), 1409-1428, 2021

Markus Krane, Martina Dreßen, Gianluca Santamaria, Ilaria My, Christine M Schneider, Tatjana Dorn, Svenja Laue, Elisa Mastantuono, Riccardo Berutti, Hilansi Rawat, Ralf Gilsbach, Pedro Schneider, Harald Lahm, Sascha Schwarz, Stefanie A Doppler, Sharon Paige, Nazan Puluca, Sophia Doll, Irina Neb, Thomas Brade, Zhong Zhang, Claudia Abou-Ajram, Bernd Northoff, Lesca M Holdt, Stefanie Sudhop, Makoto Sahara, Alexander Goedel, Andreas Dendorfer, Fleur VY Tjong, Maria E Rijlaarsdam, Julie Cleuziou, Nora Lang, Christian Kupatt, Connie Bezzina, Rüdiger Lange, Neil E Bowles, Matthias Mann, Bruce D Gelb, Lia Crotti, Lutz Hein, Thomas Meitinger, Sean Wu, Daniel Sinnecker, Peter J Gruber, Karl-Ludwig Laugwitz, Alessandra Moretti

Complex molecular programs in specific cell lineages govern human heart development. Hypoplastic left heart syndrome (HLHS) is the most common and severe manifestation within the spectrum of left ventricular outflow tract obstruction defects occurring in association with ventricular hypoplasia. The pathogenesis of HLHS is unknown, but hemodynamic disturbances are assumed to play a prominent role.To identify perturbations in gene programs controlling ventricular muscle lineage development in HLHS, we performed whole-exome sequencing of 87 HLHS parent–offspring trios, nuclear transcriptomics of cardiomyocytes from ventricles of 4 patients with HLHS and 15 controls at different stages of heart development, single cell RNA sequencing, and 3D modeling in induced pluripotent stem cells from 3 patients with HLHS and 3 controls.Gene set enrichment and protein …

Integrated phospho-proteogenomic and single-cell transcriptomic analysis of meningiomas establishes robust subtyping and reveals subtype-specific immune invasion

BioRxiv, 2021.05. 11.443369, 2021

Christina Blume, Helin Dogan, Lisa Schweizer, Matthieu Peyre, Sophia Doll, Daniel Picard, Roman Sankowski, Volker Hovestadt, Konstantin Okonechnikov, Philipp Sievers, Areeba Patel, David Reuss, Mirco Friedrich, Damian Stichel, Daniel Schrimpf, Katja Beck, Hans-Georg Wirsching, Gerhard Jungwirth, C Oliver Hanemann, Katrin Lamszus, Manfred Westphal, Nima Etminan, Andreas Unterberg, Christian Mawrin, Marc Remke, Olivier Ayrault, Peter Lichter, Stefan M Pfister, Guido Reifenberger, Michael Platten, Till Milde, David TW Jones, Rachel Grossmann, Zvi Ram, Miriam Ratliff, Christel Herold-Mende, Jan-Philipp Mallm, Marian C Neidert, Wolfgang Wick, Marco Prinz, Michael Weller, Matthias Mann, Michel Kalamarides, Andreas von Deimling, Matthias Schlesner, Felix Sahm

Meningiomas are the most frequent primary intracranial tumors. They can follow a wide clinical spectrum from benign to highly aggressive clinical course. No specific therapy exists for refractory cases or cases not amenable to resection and radiotherapy. Identification of risk of recurrence and malignant transformation for the individual patients is challenging. However, promising molecular markers and prognostic subgrouping by DNA methylation are emerging. Still, the biological underpinnings of these diagnostic subgroups are elusive, and, consequently, no novel therapeutic options arise thereof. Here we establish robust subgroups across the full landscape of meningiomas, consistent through DNA methylation, mutations, the transcriptomic, proteomic and phospho-proteomic level. Pronounced proliferative stress and DNA damage repair signals in malignant cells and in clusters exclusive to recurrent tumors are in line with their higher mitotic activity, but also provide an explanation for the accumulation of genomic instability in anaplastic meningiomas. Although homozygous deletion of CDKN2A/B is a diagnostic marker of high-grade meningioma, the expression of its gene product increased from low to non-deleted high-grade cases. Differences between subgroups in lymphocyte and myeloid cell infiltration, representing a majority of tumor mass in low-grade NF2 tumors, could be assigned to cluster-specific interaction with tumor cells. Activation to a more proinflammatory phenotype and decreased infiltration of myeloid cells in high-grade cases correlated with lower expression of CSF1, located on chromosome arm 1p, whose deletion is known …

Immobility-associated thromboprotection is conserved across mammalian species from bear to human

Science 380 (6641), 178-187, 2023

Manuela Thienel, Johannes B Müller-Reif, Zhe Zhang, Vincent Ehreiser, Judith Huth, Khrystyna Shchurovska, Badr Kilani, Lisa Schweizer, Philipp E Geyer, Maximilian Zwiebel, Julia Novotny, Enzo Lüsebrink, Gemma Little, Martin Orban, Leo Nicolai, Shaza El Nemr, Anna Titova, Michael Spannagl, Jonas Kindberg, Alina L Evans, Orpheus Mach, Matthias Vogel, Steffen Tiedt, Steffen Ormanns, Barbara Kessler, Anne Dueck, Andrea Friebe, Peter Godsk Jørgensen, Monir Majzoub-Altweck, Andreas Blutke, Amin Polzin, Konstantin Stark, Stefan Kääb, Doris Maier, Jonathan M Gibbins, Ulrich Limper, Ole Frobert, Matthias Mann, Steffen Massberg, Tobias Petzold

Venous thromboembolism (VTE) comprising deep venous thrombosis and pulmonary embolism is a major cause of morbidity and mortality. Short-term immobility-related conditions are a major risk factor for the development of VTE. Paradoxically, long-term immobilized free-ranging hibernating brown bears and paralyzed spinal cord injury (SCI) patients are protected from VTE. We aimed to identify mechanisms of immobility-associated VTE protection in a cross-species approach. Mass spectrometry–based proteomics revealed an antithrombotic signature in platelets of hibernating brown bears with heat shock protein 47 (HSP47) as the most substantially reduced protein. HSP47 down-regulation or ablation attenuated immune cell activation and neutrophil extracellular trap formation, contributing to thromboprotection in bears, SCI patients, and mice. This cross-species conserved platelet signature may give rise to …

Hierarchical ligation-independent assembly of PCR fragments

DNA Cloning and Assembly Methods, 49-58, 2014

Jonathan L Schmid-Burgk, Zhen Xie, Yaakov Benenson

The emerging field of synthetic biology requires novel cloning techniques that allow the rapid assembly of multiple expression units to build artificial genetic circuits. Here, we describe a rapid, flexible, and cost-efficient cloning method that requires only standard laboratory equipment and skills. Our technique relies on the 3′–5′ exonuclease activity of T4 DNA polymerase to generate 20 nt single-stranded DNA overhangs that allow annealing and ligation-independent cloning (LIC) of four DNA fragments in one tube. The resulting intermediate-size constructs can be reused to hierarchically assemble constructs of more than 24 kb by the same method.

Phosphoproteome analysis of the MAPK pathway reveals previously undetected feedback mechanisms

Proteomics 16 (14), 1998-2004, 2016

Florian Gnad, Sophia Doll, Kyung Song, Matthew P Stokes, John Moffat, Bonnie Liu, David Arnott, Jeffrey Wallin, Lori S Friedman, Georgia Hatzivassiliou, Marcia Belvin

The RAS‐RAF‐MEK‐ERK (MAPK) pathway is prevalently perturbed in cancer. Recent large‐scale sequencing initiatives profiled thousands of tumors providing insight into alterations at the DNA and RNA levels. These efforts confirmed that key nodes of the MAPK pathway, in particular KRAS and BRAF, are among the most frequently altered proteins in cancer. The establishment of targeted therapies, however, has proven difficult. To decipher the underlying challenges, it is essential to decrypt the phosphorylation network spanned by the MAPK core axis. Using mass spectrometry we identified 2241 phosphorylation sites on 1020 proteins, and measured their responses to inhibition of MEK or ERK. Multiple phosphorylation patterns revealed previously undetected feedback, as upstream signaling nodes, including receptor kinases, showed changes at the phosphorylation level. We provide a dataset rich in potential …

Diverse enzymatic activities mediate antiviral immunity in prokaryotes

Science 369 (6507), 1077-1084, 2020

Linyi Gao, Han Altae-Tran, Francisca Böhning, Kira S Makarova, Michael Segel, Jonathan L Schmid-Burgk, Jeremy Koob, Yuri I Wolf, Eugene V Koonin, Feng Zhang

Bacteria and archaea are frequently attacked by viruses and other mobile genetic elements and rely on dedicated antiviral defense systems, such as restriction endonucleases and CRISPR, to survive. The enormous diversity of viruses suggests that more types of defense systems exist than are currently known. By systematic defense gene prediction and heterologous reconstitution, here we discover 29 widespread antiviral gene cassettes, collectively present in 32% of all sequenced bacterial and archaeal genomes, that mediate protection against specific bacteriophages. These systems incorporate enzymatic activities not previously implicated in antiviral defense, including RNA editing and retron satellite DNA synthesis. In addition, we computationally predict a diverse set of other putative defense genes that remain to be characterized. These results highlight an immense array of molecular functions that microbes …

Novel approaches to unravel risk factors and mechanisms of venous thrombosis

Thrombosis and Haemostasis 120 (03), 372-372, 2020

Inga Scheller, Bernhard Nieswandt

Venous thrombosis (VT) is a leading cause of mortality and morbidity in industrialized countries. However, the precise mechanisms that trigger clotting in large veins are not fully understood. Many genetic and acquired risk factors have been identified for VT that alter blood flow, activate the endothelium, and alter the activity of coagulation factors. 1 Many of these clotting factors are localized in the plasma, making analysis of this blood compartment highly interesting for the understanding of VT. As an alternative to activity-or antibody-based plasma protein assays, which provide only limited information on single proteins in a sample, mass spectrometry (MS)-based proteomics allows high-throughput, quantitative analysis of biomolecules. Indeed, quantitative MS has been successfully applied to understand the pathomechanisms of multiple diseases. 2 In this issue of Thrombosis and Haemostasis, Tilburg et al attempt …

Sensing endogenous RNA in living human cells using a CRISPR-activated protease

bioRxiv, 2024.07. 17.603943, 2024

Katja Blumenstock, Alexander Hoch, Leo D Hinterlang, Caroline I Fandrey, Niels Schneberger, Gregor Hagelueken, Jonathan L Schmid-Burgk

Most techniques used to detect specific mRNAs in eukaryotic cells require to extract nucleic acids and thereby kill the cells. A programmable sensor for monitoring endogenous transcripts in living cells, in contrast, would enable to enrich living cells based on a specific transcription or splicing event, and studying these cells by live microscopy or sequencing methods requiring intact cells. We have engineered CRISPR-READ, a live cell RNA detector based on the CRISPR-associated Lon protease CalpL and a cA4-producing Type III CRISPR system. Upon RNA-programmable RNA sensing, CRISPR-READ produces an orthogonal second messenger, which leads to the cleavage of a dual FRET / localization reporter compatible with FACS sorting and live microscopy. Using this genetically encoded sensing circuit as a readout for a genome-wide CRISPR perturbation screen, we identified an extended Type-I interferon signaling cascade; RNA-Seq on sensor-sorted cells enabled unbiased identification of correlated stochasticity in gene expression across single cells.

The second-generation exportin-1 inhibitor KPT-8602 demonstrates potent activity against acute lymphoblastic leukemia

Clinical Cancer Research 23 (10), 2528-2541, 2017

Thomas Vercruysse, Jolien De Bie, Jasper E Neggers, Maarten Jacquemyn, Els Vanstreels, Jonathan L Schmid-Burgk, Veit Hornung, Erkan Baloglu, Yosef Landesman, William Senapedis, Sharon Shacham, Antonis Dagklis, Jan Cools, Dirk Daelemans

Purpose: Human exportin-1 (XPO1) is the key nuclear-cytoplasmic transport protein that exports different cargo proteins out of the nucleus. Inducing nuclear accumulation of these proteins by inhibiting XPO1 causes cancer cell death. First clinical validation of pharmacological inhibition of XPO1 was obtained with the Selective Inhibitor of Nuclear Export (SINE) compound selinexor (KPT-330) demonstrating activity in phase-II/IIb clinical trials when dosed 1 to 3 times weekly. The second-generation SINE compound KPT-8602 shows improved tolerability and can be dosed daily. Here, we investigate and validate the drug–target interaction of KPT-8602 and explore its activity against acute lymphoblastic leukemia (ALL). Experimental Design: We examined the effect of KPT-8602 on XPO1 function and XPO1-cargo as well as on a panel of leukemia cell lines. Mutant XPO1 leukemia cells were …

Limited environmental serine confers sensitivity to PHGDH inhibition in brain metastasis

bioRxiv, 2020.03. 03.974980, 2020

Bryan Ngo, Eugenie Kim, Victoria Osorio-Vasquez, Sophia Doll, Sophia Bustraan, Alba Luengo, Shawn M Davidson, Ahmed Ali, Gino D Ferraro, Diane Kang, Jing Ni, Roger Liang, Ariana Plasger, Edward R Kastenhuber, Roozbeh Eskandari, Sarah Bacha, Roshan K Siriam, Samuel F Bakhoum, Edouard Mullarky, Matija Snuderl, Paolo Cotzia, Nello Mainolfi, Vipin Suri, Adam Friedman, Mark Manfredi, David M Sabatini, Drew Jones, Min Yu, Jean J Zhao, Rakesh K Jain, Matthew G Vander Heiden, Eva Hernando, Matthias Mann, Lewis C Cantley, Michael E Pacold

A hallmark of metastasis is the adaptation of tumor cells to new environments. Although it is well established that the metabolic milieu of the brain is severely deprived of nutrients, particularly the amino acids serine and its catabolite glycine, how brain metastases rewire their metabolism to survive in the nutrient-limited environment of the brain is poorly understood. Here we demonstrate that cell-intrinsic de novo serine synthesis is a major determinant of brain metastasis. Whole proteome comparison of triple-negative breast cancer (TNBC) cells that differ in their capacity to colonize the brain reveals that 3-phosphoglycerate dehydrogenase (PHGDH), which catalyzes the rate-limiting step of glucose-derived serine synthesis, is the most significantly upregulated protein in cells that efficiently metastasize to the brain. Genetic silencing or pharmacological inhibition of PHGDH attenuated brain metastasis and improved overall survival in mice, whereas expression of catalytically active PHGDH in a non-brain trophic cell line promoted brain metastasis. Collectively, these findings indicate that nutrient availability determines serine synthesis pathway dependence in brain metastasis, and suggest that PHGDH inhibitors may be useful in the treatment of patients with cancers that have spread to the brain.Our study highlights how limited serine and glycine availability within the brain microenvironment potentiates tumor cell sensitivity to serine synthesis inhibition. This finding underscores the importance of studying cancer metabolism in physiologically-relevant contexts, and provides a rationale for using PHGDH inhibitors to treat brain …

Highly parallel profiling of Cas9 variant specificity

Molecular cell 78 (4), 794-800. e8, 2020

Jonathan L Schmid-Burgk, Linyi Gao, David Li, Zachary Gardner, Jonathan Strecker, Blake Lash, Feng Zhang

Determining the off-target cleavage profile of programmable nucleases is an important consideration for any genome editing experiment, and a number of Cas9 variants have been reported that improve specificity. We describe here tagmentation-based tag integration site sequencing (TTISS), an efficient, scalable method for analyzing double-strand breaks (DSBs) that we apply in parallel to eight Cas9 variants across 59 targets. Additionally, we generated thousands of other Cas9 variants and screened for variants with enhanced specificity and activity, identifying LZ3 Cas9, a high specificity variant with a unique +1 insertion profile. This comprehensive comparison reveals a general trade-off between Cas9 activity and specificity and provides information about the frequency of generation of +1 insertions, which has implications for correcting frameshift mutations.

Myosin binding protein H-like (MYBPHL): a promising biomarker to predict atrial damage

Scientific Reports 9 (1), 9986, 2019

Harald Lahm, Martina Dreßen, Nicole Beck, Stefanie Doppler, Marcus-André Deutsch, Shunsuke Matsushima, Irina Neb, Karl Christian König, Konstantinos Sideris, Stefanie Voss, Lena Eschenbach, Nazan Puluca, Isabel Deisenhofer, Sophia Doll, Stefan Holdenrieder, Matthias Mann, Rüdiger Lange, Markus Krane

Myosin binding protein H-like (MYBPHL) is a protein associated with myofilament structures in atrial tissue. The protein exists in two isoforms that share an identical amino acid sequence except for a deletion of 23 amino acids in isoform 2. In this study, MYBPHL was found to be expressed preferentially in atrial tissue. The expression of isoform 2 was almost exclusively restricted to the atria and barely detectable in the ventricle, arteria mammaria interna, and skeletal muscle. After atrial damage induced by cryo- or radiofrequency ablation, MYBPHL was rapidly and specifically released into the peripheral circulation in a time-dependent manner. The plasma MYBPHL concentration remained substantially elevated up to 24 hours after the arrival of patients at the intensive care unit. In addition, the recorded MYBPHL values were strongly correlated with those of the established biomarker CK-MB. In contrast, an increase …

Fate of antibody-drug conjugates in cancer cells

Journal of Experimental & Clinical Cancer Research 37, 1-12, 2018

Cécile Chalouni, Sophia Doll

Antibody-Drug Conjugates (ADCs) are a class of cancer therapeutics that combines antigen specificity and potent cytotoxicity in a single molecule as they are comprised of an engineered antibody linked chemically to a cytotoxic drug. Four ADCs have received approval by the Food and Drug Administration (FDA) and the European Medicine Agency (EMA) and can be prescribed for metastatic conditions while around 60 ADCs are currently enrolled in clinical trials. The efficacy of an ADC greatly relies on its intracellular trafficking and processing of its components to trigger tumor cell death. A limited number of studies have addressed these critical processes that both challenge and help foster the design of ADCs. This review highlights those mechanisms and their relevance for future development of ADCs as cancer therapeutics.

Pipeline for proteomic in silico cell-type deconvolution

EUROPEAN JOURNAL OF IMMUNOLOGY 53, 145-145, 2023

T Tumurbaatar, H Pelin, DE ANDRADE E SOUSA L BARROS, N CHINTALAGIRI, J MÜLLERREIF, P GEYER, O GENZEL-BOROVICZÉNY, M MANN, M PIRAUD, C KLEIN, C NUSSBAUM, S PANGRATZ-FUEHRER, S KIM-HELLMUTH

Objective: Analysing the human blood proteome grants us an in-depth look into the health status of an individual and opens many avenues for the development of novel diagnostic and therapeutic methods. However, affinity-based techniques such as ELISA and immunoblotting, which are commonly used for single protein quantifications, are increasingly being replaced by mass spectrometry (MS)-based methods that can provide comprehensive results in even small sample volumes. Despite the attractiveness of MS-based workflows, there are prominent issues that still need to be addressed, most notably, the abundance of missingness in MS datasets as well as the lack of cell type resolution in bulk tissues MS. Therefore, our goal is to develop a pipeline that is able to impute these missing values and perform cell type deconvolution from MS blood proteomics data.Methods and results: Based on pre-existing MS …

GRADIENT OFF-SET FOCUSING HPLC INSTRUMENT FOR ROBUST AND HIGH THROUGHPUT CLINICAL PROTEOMICS

Philipp Geyer, Nicolai Bache, Sophia Doll, Peter Treit, Florian Meier, Matthias Mann

Mass spectrometry-based proteomics and metabolomics are fast growing and powerful technologies, with the potential to revolutionize health care and precision medicine. Human blood plasma and serum are already the most established samples for clinical analysis and are analyzed today with antibody-based assays. However, immunoassays have some inherent limitations that could be overcome by MS-based proteomics, which should be the optimal technology to investigate changes in the human plasma proteome in a specific and unbiased manner. Recently, we developed an automated, rapid and robust shotgun proteomics workflow that allows us to analyse hundreds of plasma proteins. We call the workflow ‘plasma proteome profiling’(Geyer et al., Cell Syst., 2016) and we have already applied it to several clinical studies, comprising up to 1,300 plasma proteomes (Geyer et al., Mol. Syst. Biol., 2016). However, until now, available separation technology has severely limited throughput and robustness and thereby prevented proteomics (and metabolomics) technologies from being fully integrated and routinely used in a clinical setting.

Human GBP1 does not localize to pathogen vacuoles but restricts Toxoplasma gondii

Cellular microbiology 18 (8), 1056-1064, 2016

Ashleigh C Johnston, Anthony Piro, Barbara Clough, Malvin Siew, Sebastian Virreira Winter, Jörn Coers, Eva‐Maria Frickel

Guanylate binding proteins (GBPs) are a family of large interferon‐inducible GTPases that are transcriptionally upregulated upon infection with intracellular pathogens. Murine GBPs (mGBPs) including mGBP1 and 2 localize to and disrupt pathogen‐containing vacuoles (PVs) resulting in the cell‐autonomous clearing or innate immune detection of PV‐resident pathogens. Human GBPs (hGBPs) are known to exert antiviral host defense and activate the NLRP3 inflammasome, but it is unclear whether hGBPs can directly recognize and control intravacuolar pathogens. Here, we report that endogenous or ectopically expressed hGBP1 fails to associate with PVs formed in human cells by the bacterial pathogens Chlamydia trachomatis or Salmonella typhimurium or the protozoan pathogen Toxoplasma gondii. While we find that hGBP1 expression has no discernible effect on intracellular replication of C. trachomatis …

Region and Cell-type Specific Proteomic Map of the Human Heart

The Thoracic and Cardiovascular Surgeon 66 (S 01), DGTHG-V311, 2018

M Krane, S Doll, M Dreßen, P Geyer, D Itzhak, C Braun, S Doppler, F Meyer, MA Deutsch, H Lahm, R Lange, M Mann

Objectives: The aim of the conducted study was the generation of a spatial and cell-type resolved proteomic map of the healthy human heart. A global protein expression “footprint” of the healthy heart can be used as a reference library to compare against diseased hearts in the search for biomarkers, therapeutic targets or disease signatures.Methods: The human heart proteome was measured in 16 anatomical regions from 3 different healthy subjects and in three major cardiac cell types (endothelial cells, fibroblasts and smooth muscle cells) by high-resolution mass spectrometry-based proteomics.Results: The MaxLFQ algorithm quantified a total of 11,163 proteins, 10,751 in the 16 heart regions and 10,447 in the non-cardiomyocyte cell types. For overall assessment of proteomics similarities and differences of the 16 heart regions, we employed principal component analysis (PCA). The major groups cavities …

Multiparametric assays for accelerating early drug discovery

Trends in Pharmacological Sciences 41 (5), 318-335, 2020

Alexander Herholt, Sabrina Galinski, Philipp E Geyer, Moritz J Rossner, Michael C Wehr

Drug discovery campaigns are hampered by substantial attrition rates largely due to a lack of efficacy and safety reasons associated with candidate drugs. This is true in particular for genetically complex diseases, where insufficient knowledge of the modulatory actions of candidate drugs on targets and entire target pathways further adds to the problem of attrition. To better profile compound actions on targets, potential off-targets, and disease-linked pathways, new innovative technologies need to be developed that can elucidate the complex cellular signaling networks in health and disease. Here, we discuss progress in genetically encoded multiparametric assays and mass spectrometry (MS)-based proteomics, which both represent promising toolkits to profile multifactorial actions of drug candidates in disease-relevant cellular systems to promote drug discovery and personalized medicine.

CRISPaint allows modular base-specific gene tagging using a ligase-4-dependent mechanism

Nature communications 7 (1), 12338, 2016

Jonathan L Schmid-Burgk, Klara Höning, Thomas S Ebert, Veit Hornung

The site-specific insertion of heterologous genetic material into genomes provides a powerful means to study gene function. Here we describe a modular system entitled CRISPaint (CRISPR-assisted insertion tagging) that allows precise and efficient integration of large heterologous DNA cassettes into eukaryotic genomes. CRISPaint makes use of the CRISPR-Cas9 system to introduce a double-strand break (DSB) at a user-defined genomic location. A universal donor DNA, optionally provided as minicircle DNA, is cleaved simultaneously to be integrated at the genomic DSB, while processing the donor plasmid at three possible positions allows flexible reading-frame selection. Applying this system allows to create C-terminal tag fusions of endogenously encoded proteins in human cells with high efficiencies. Knocking out known DSB repair components reveals that site-specific insertion is completely dependent on …

TREX1 deficiency triggers cell-autonomous immunity in a cGAS-dependent manner

The Journal of Immunology 192 (12), 5993-5997, 2014

Andrea Ablasser, Inga Hemmerling, Jonathan L Schmid-Burgk, Rayk Behrendt, Axel Roers, Veit Hornung

Cytosolic detection of DNA is crucial for the initiation of antiviral immunity but can also cause autoimmunity in the context of endogenous nucleic acids being sensed. Mutations in the human 3′ repair exonuclease 1 (TREX1) have been linked to the type I IFN–associated autoimmune disease Aicardi–Goutières syndrome. The exact mechanisms driving unabated type I IFN responses in the absence of TREX1 are only partly understood, but it appears likely that accumulation of endogenous DNA species triggers a cell-autonomous immune response by activating a cytosolic DNA receptor. In this article, we demonstrate that knocking out the DNA sensor cyclic GMP–AMP synthase completely abrogates spontaneous induction of IFN-stimulated genes in TREX1-deficient cells. These findings indicate a key role of cyclic GMP–AMP synthase for the initiation of self-DNA–induced autoimmune disorders, thus providing …

Metallopeptidase inhibitor 1 (TIMP‐1) promotes receptor tyrosine kinase c‐Kit signaling in colorectal cancer

Molecular Oncology 13 (12), 2646-2662, 2019

Cathrine Nordgaard, Sophia Doll, Ana Laura de Souza Almeida Matos, Mikkel Høeberg, Julhash Uddin Kazi, Stine Friis, Jan Stenvang, Lars Rönnstrand, Matthias Mann, José Manuel Afonso Moreira

Colorectal cancer (CRC) is the third most prevalent cancer worldwide causing an estimated 700 000 deaths annually. Different types of treatment are available for patients with advanced metastatic colorectal cancer, including targeted biological agents, such as cetuximab, a monoclonal antibody that targets EGFR. We have previously reported a study indicating multiple levels of interaction between metallopeptidase inhibitor 1 (TIMP‐1) and the epidermal growth factor (EGF) signaling axis, which could explain how TIMP‐1 levels can affect the antitumor effects of EGFR inhibitors. We also reported an association between TIMP‐1‐mediated cell invasive behavior and KRAS status. To gain insight into the molecular mechanisms underlying the effects of TIMP‐1 in CRC, we examined by transcriptomics, proteomics, and kinase activity profiling a matched pair of isogenic human CRC isogenic DLD‐1 CRC cell clones …

Myoglobin regulates fatty acid trafficking and lipid metabolism in mammary epithelial cells

Plos one 17 (10), e0275725, 2022

Julia Armbruster, Mostafa A Aboouf, Max Gassmann, Angela Egert, Hubert Schorle, Veit Hornung, Tobias Schmidt, Jonathan L Schmid-Burgk, Glen Kristiansen, Anne Bicker, Thomas Hankeln, Hao Zhu, Thomas A Gorr

Myoglobin (MB) is known to bind and deliver oxygen in striated muscles at high expression levels. MB is also expressed at much reduced levels in mammary epithelial cells, where the protein´s function is unclear. In this study, we aim to determine whether MB impacts fatty acid trafficking and facilitates aerobic fatty acid ß-oxidation in mammary epithelial cells. We utilized MB-wildtype versus MB-knockout mice and human breast cancer cells to examine the impact of MB and its oxygenation status on fatty acid metabolism in mouse milk and mammary epithelia. MB deficient cells were generated through CRISPR/Cas9 and TALEN approaches and exposed to various oxygen tensions. Fatty acid profiling of milk and cell extracts were performed along with cell labelling and immunocytochemistry. Our findings show that MB expression in mammary epithelial cells promoted fatty acid oxidation while reducing stearyl-CoA desaturase activity for lipogenesis. In cells and milk product, presence of oxygenated MB significantly elevated indices of limited fatty acid ß-oxidation, i.e., the organelle-bound removal of a C2 moiety from long-chain saturated or monounsaturated fatty acids, thus shifting the composition toward more saturated and shorter fatty acid species. Presence of the globin also increased cytoplasmic fatty acid solubility under normoxia and fatty acid deposition to lipid droplets under severe hypoxia. We conclude that MB can function in mammary epithelia as intracellular O2-dependent shuttle of oxidizable fatty acid substrates. MB’s impact on limited oxidation of fatty acids could generate inflammatory mediator lipokines, such as 7-hexadecenoate …

Profiling the human intestinal environment under physiological conditions

Nature 617 (7961), 581-591, 2023

Dari Shalon, Rebecca Neal Culver, Jessica A Grembi, Jacob Folz, Peter V Treit, Handuo Shi, Florian A Rosenberger, Les Dethlefsen, Xiandong Meng, Eitan Yaffe, Andrés Aranda-Díaz, Philipp E Geyer, Johannes B Mueller-Reif, Sean Spencer, Andrew D Patterson, George Triadafilopoulos, Susan P Holmes, Matthias Mann, Oliver Fiehn, David A Relman, Kerwyn Casey Huang

The spatiotemporal structure of the human microbiome,, proteome and metabolome, reflects and determines regional intestinal physiology and may have implications for disease. Yet, little is known about the distribution of microorganisms, their environment and their biochemical activity in the gut because of reliance on stool samples and limited access to only some regions of the gut using endoscopy in fasting or sedated individuals. To address these deficiencies, we developed an ingestible device that collects samples from multiple regions of the human intestinal tract during normal digestion. Collection of 240 intestinal samples from 15 healthy individuals using the device and subsequent multi-omics analyses identified significant differences between bacteria, phages, host proteins and metabolites in the intestines versus stool. Certain microbial taxa were differentially enriched and prophage induction was more …

The DNA inflammasome in human myeloid cells is initiated by a STING-cell death program upstream of NLRP3

Cell 171 (5), 1110-1124. e18, 2017

Moritz M Gaidt, Thomas S Ebert, Dhruv Chauhan, Katharina Ramshorn, Francesca Pinci, Sarah Zuber, Fionan O’Duill, Jonathan L Schmid-Burgk, Florian Hoss, Raymund Buhmann, Georg Wittmann, Eicke Latz, Marion Subklewe, Veit Hornung

Detection of cytosolic DNA constitutes a central event in the context of numerous infectious and sterile inflammatory conditions. Recent studies have uncovered a bipartite mode of cytosolic DNA recognition, in which the cGAS-STING axis triggers antiviral immunity, whereas AIM2 triggers inflammasome activation. Here, we show that AIM2 is dispensable for DNA-mediated inflammasome activation in human myeloid cells. Instead, detection of cytosolic DNA by the cGAS-STING axis induces a cell death program initiating potassium efflux upstream of NLRP3. Forward genetics identified regulators of lysosomal trafficking to modulate this cell death program, and subsequent studies revealed that activated STING traffics to the lysosome, where it triggers membrane permeabilization and thus lysosomal cell death (LCD). Importantly, the cGAS-STING-NLRP3 pathway constitutes the default inflammasome response during …

Integrative analysis of cell state changes in lung fibrosis with peripheral protein biomarkers

EMBO molecular medicine 13 (4), e12871, 2021

Christoph H Mayr, Lukas M Simon, Gabriela Leuschner, Meshal Ansari, Janine Schniering, Philipp E Geyer, Ilias Angelidis, Maximilian Strunz, Pawandeep Singh, Nikolaus Kneidinger, Frank Reichenberger, Edith Silbernagel, Stephan Böhm, Heiko Adler, Michael Lindner, Britta Maurer, Anne Hilgendorff, Antje Prasse, Jürgen Behr, Matthias Mann, Oliver Eickelberg, Fabian J Theis, Herbert B Schiller

The correspondence of cell state changes in diseased organs to peripheral protein signatures is currently unknown. Here, we generated and integrated single‐cell transcriptomic and proteomic data from multiple large pulmonary fibrosis patient cohorts. Integration of 233,638 single‐cell transcriptomes (n = 61) across three independent cohorts enabled us to derive shifts in cell type proportions and a robust core set of genes altered in lung fibrosis for 45 cell types. Mass spectrometry analysis of lung lavage fluid (n = 124) and plasma (n = 141) proteomes identified distinct protein signatures correlated with diagnosis, lung function, and injury status. A novel SSTR2+ pericyte state correlated with disease severity and was reflected in lavage fluid by increased levels of the complement regulatory factor CFHR1. We further discovered CRTAC1 as a biomarker of alveolar type‐2 epithelial cell health status in lavage fluid and …

A ligation-independent cloning technique for high-throughput assembly of transcription activator–like effector genes

Nature biotechnology 31 (1), 76-81, 2013

Jonathan L Schmid-Burgk, Tobias Schmidt, Vera Kaiser, Klara Höning, Veit Hornung

Transcription activator–like (TAL) effector proteins derived from Xanthomonas species have emerged as versatile scaffolds for engineering DNA-binding proteins of user-defined specificity and functionality. Here we describe a rapid, simple, ligation-independent cloning (LIC) technique for synthesis of TAL effector genes. Our approach is based on a library of DNA constructs encoding individual TAL effector repeat unit combinations that can be processed to contain long, unique single-stranded DNA overhangs suitable for LIC. Assembly of TAL effector arrays requires only the combinatorial mixing of fluids and has exceptional fidelity. TAL effector nucleases (TALENs) produced by this method had high genome-editing activity at endogenous loci in HEK 293T cells (64% were active). To maximize throughput, we generated a comprehensive 5-mer TAL effector repeat unit fragment library that allows automated assembly …

The bacterial pigment pyocyanin inhibits the NLRP3 inflammasome through intracellular reactive oxygen and nitrogen species

Journal of Biological Chemistry 293 (13), 4893-4900, 2018

Sebastian Virreira Winter, Arturo Zychlinsky

Inflammasomes are cytosolic complexes that mature and secrete the inflammatory cytokines interleukin 1β (IL-1β) and IL-18 and induce pyroptosis. The NLRP3 (NACHT, LRR, and PYD domains–containing protein 3) inflammasome detects many pathogen- and danger-associated molecular patterns, and reactive oxygen species (ROS)/reactive nitrogen species (RNS) have been implicated in its activation. The phenazine pyocyanin (PCN) is a virulence factor of Pseudomonas aeruginosa and generates superoxide in cells. Here we report that PCN inhibits IL-1β and IL-18 release and pyroptosis upon NLRP3 inflammasome activation in macrophages by preventing speck formation and Caspase-1 maturation. Of note, PCN did not regulate the AIM2 (absent in melanoma 2) or NLRC4 inflammasomes or tumor necrosis factor (TNF) secretion. Imaging of the fluorescent glutathione redox potential sensor Grx1-roGFP2 …

Design principles for cyclin K molecular glue degraders

Nature chemical biology 20 (1), 93-102, 2024

Zuzanna Kozicka, Dakota J Suchyta, Vivian Focht, Georg Kempf, Georg Petzold, Marius Jentzsch, Charles Zou, Cristina Di Genua, Katherine A Donovan, Seemon Coomar, Marko Cigler, Cristina Mayor-Ruiz, Jonathan L Schmid-Burgk, Daniel Häussinger, Georg E Winter, Eric S Fischer, Mikołaj Słabicki, Dennis Gillingham, Benjamin L Ebert, Nicolas H Thomä

Molecular glue degraders are an effective therapeutic modality, but their design principles are not well understood. Recently, several unexpectedly diverse compounds were reported to deplete cyclin K by linking CDK12–cyclin K to the DDB1–CUL4–RBX1 E3 ligase. Here, to investigate how chemically dissimilar small molecules trigger cyclin K degradation, we evaluated 91 candidate degraders in structural, biophysical and cellular studies and reveal all compounds acquire glue activity via simultaneous CDK12 binding and engagement of DDB1 interfacial residues, in particular Arg928. While we identify multiple published kinase inhibitors as cryptic degraders, we also show that these glues do not require pronounced inhibitory properties for activity and that the relative degree of CDK12 inhibition versus cyclin K degradation is tuneable. We further demonstrate cyclin K degraders have transcriptional signatures …

Consistency across multi‐omics layers in a drug‐perturbed gut microbial community

Molecular Systems Biology 19 (9), e11525, 2023

Sander Wuyts, Renato Alves, Maria Zimmermann‐Kogadeeva, Suguru Nishijima, Sonja Blasche, Marja Driessen, Philipp E Geyer, Rajna Hercog, Ece Kartal, Lisa Maier, Johannes B Müller, Sarela Garcia Santamarina, Thomas Sebastian B Schmidt, Daniel C Sevin, Anja Telzerow, Peter V Treit, Tobias Wenzel, Athanasios Typas, Kiran R Patil, Matthias Mann, Michael Kuhn, Peer Bork

Multi‐omics analyses are used in microbiome studies to understand molecular changes in microbial communities exposed to different conditions. However, it is not always clear how much each omics data type contributes to our understanding and whether they are concordant with each other. Here, we map the molecular response of a synthetic community of 32 human gut bacteria to three non‐antibiotic drugs by using five omics layers (16S rRNA gene profiling, metagenomics, metatranscriptomics, metaproteomics and metabolomics). We find that all the omics methods with species resolution are highly consistent in estimating relative species abundances. Furthermore, different omics methods complement each other for capturing functional changes. For example, while nearly all the omics data types captured that the antipsychotic drug chlorpromazine selectively inhibits Bacteroidota representatives in the …

Mycobacterium tuberculosis differentially activates cGAS-and inflammasome-dependent intracellular immune responses through ESX-1

Cell host & microbe 17 (6), 799-810, 2015

Ruth Wassermann, Muhammet F Gulen, Claudia Sala, Sonia Garcia Perin, Ye Lou, Jan Rybniker, Jonathan L Schmid-Burgk, Tobias Schmidt, Veit Hornung, Stewart T Cole, Andrea Ablasser

Cytosolic detection of microbial products is essential for the initiation of an innate immune response against intracellular pathogens such as Mycobacterium tuberculosis (Mtb). During Mtb infection of macrophages, activation of cytosolic surveillance pathways is dependent on the mycobacterial ESX-1 secretion system and leads to type I interferon (IFN) and interleukin-1β (IL-1β) production. Whereas the inflammasome regulates IL-1β secretion, the receptor(s) responsible for the activation of type I IFNs has remained elusive. We demonstrate that the cytosolic DNA sensor cyclic GMP-AMP synthase (cGAS) is essential for initiating an IFN response to Mtb infection. cGAS associates with Mtb DNA in the cytosol to stimulate cyclic GAMP (cGAMP) synthesis. Notably, activation of cGAS-dependent cytosolic host responses can be uncoupled from inflammasome activation by modulating the secretion of ESX-1 substrates. Our …

Proteomics in the study of liver diseases

The Human Gut-Liver-Axis in Health and Disease, 165-193, 2019

Lili Niu, Philipp E Geyer, Matthias Mann

In this chapter, we describe the workflow of mass spectrometry (MS)-based proteomics with a focus on shotgun proteomics. We illustrate how MS-based proteomics can be applied to study liver pathophysiology using protein expression profiling, characterization of post-translational modifications (PTMs) and protein-protein interactions (PPIs). The publications on serum or plasma proteomics in the study of liver diseases during the years 2012 to 2017 are reviewed. We analyze the proportions of studies with regard to different kinds of liver disease and different proteomics workflows applied. Remarkably, outdated proteomics techniques were still being used in recent years and even account for a large proportion of the reviewed literature. The effort spent in different liver diseases is largely skewed to hepatocellular carcinoma and hepatic viral infection while a relatively small proportion focused on non-alcoholic …

Gastric Adenocarcinomas Express the Glycosphingolipid Gb3/CD77: Targeting of Gastric Cancer Cells with Shiga Toxin B-Subunit

Molecular cancer therapeutics 15 (5), 1008-1017, 2016

Philipp Emanuel Geyer, Matthias Maak, Ulrich Nitsche, Markus Perl, Alexander Novotny, Julia Slotta-Huspenina, Estelle Dransart, Anne Holtorf, Ludger Johannes, Klaus-Peter Janssen

The B-subunit of the bacterial Shiga toxin (STxB), which is nontoxic and has low immunogenicity, can be used for tumor targeting of breast, colon, and pancreatic cancer. Here, we tested whether human gastric cancers, which are among the most aggressive tumor entities, express the cellular receptor of Shiga toxin, the glycosphingolipid globotriaosylceramide (Gb3/CD77). The majority of cases showed an extensive staining for Gb3 (36/50 cases, 72%), as evidenced on tissue sections of surgically resected specimen. Gb3 expression was detected independent of type (diffuse/intestinal), and was negatively correlated to increasing tumor–node–metastasis stages (P = 0.0385), as well as with markers for senescence. Gb3 expression in nondiseased gastric mucosa was restricted to chief and parietal cells at the bottom of the gastric glands, and was not elevated in endoscopic samples of gastritis (n = 10). Gb3 …

Priming enables a NEK7-independent route of NLRP3 activation

bioRxiv, 799320, 2019

Niklas A Schmacke, Moritz M Gaidt, Inga Szymanska, Fionan O’Duill, Che A Stafford, Dhruv Chauhan, Adrian L Fröhlich, Dennis Nagl, Francesca Pinci, Jonathan L Schmid-Burgk, Veit Hornung

The NLRP3 inflammasome plays a central role in antimicrobial defense, as well as in sterile inflammatory conditions. NLRP3 activity is governed by two independent signals. The first signal primes NLRP3, allowing it to respond to its activation signal. In the murine system, the mitotic spindle kinase NEK7 has been identified as a crucial factor in relaying the activation signal to NLRP3. Here we show that the requirement for NEK7 can be bypassed by TAK1-dependent post-translational priming. Under pro-inflammatory conditions that activate TAK1, NEK7 was dispensable for NLRP3 inflammasome formation in human and murine cells. Intriguingly, dissecting the NEK7 requirement in iPSC-derived primary human macrophages revealed that this NEK7-independent mechanism constitutes the predominant NLRP3 priming pathway in these cells. In summary, our results suggest that NEK7 functions as an NLRP3 priming – rather than activation – factor that can work in synergy or redundancy with other priming pathways to accelerate inflammasome activation.

Cohort profile: Cohort profile: the MUNICH Preterm and Term Clinical study (MUNICH-PreTCl), a neonatal birth cohort with focus on prenatal and postnatal determinants of infant and childhood morbidity

BMJ Open 11 (6), 2021

Susanne Pangratz-Fuehrer, Orsolya Genzel-Boroviczény, Wolfgang Bodensohn, Robin Eisenburger, Janne Scharpenack, Philipp E Geyer, Johannes B Müller-Reif, Nadja van Hagen, Alina M Müller, Majken Karoline Jensen, Christoph Klein, Matthias Mann, Claudia Nussbaum

PurposeThe MUNICH Preterm and Term Clinical (MUNICH-PreTCl) birth cohort was established to uncover pathological processes contributing to infant/childhood morbidity and mortality. We collected comprehensive medical information of healthy and sick newborns and their families, together with infant blood samples for proteomic analysis. MUNICH-PreTCl aims to identify mechanism-based biomarkers in infant health and disease to deliver more precise diagnostic and predictive information for disease prevention. We particularly focused on risk factors for pregnancy complications, family history of genetically influenced health conditions such as diabetes and paediatric long-term health—all to be further monitored and correlated with proteomics data in the future.

Characterization of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection clusters based on integrated genomic surveillance, outbreak analysis and contact tracing in an urban setting

Clinical Infectious Diseases 74 (6), 1039-1046, 2022

Andreas Walker, Torsten Houwaart, Patrick Finzer, Lutz Ehlkes, Alona Tyshaieva, Maximilian Damagnez, Daniel Strelow, Ashley Duplessis, Jessica Nicolai, Tobias Wienemann, Teresa Tamayo, Malte Kohns Vasconcelos, Lisanna Hülse, Katrin Hoffmann, Nadine Lübke, Sandra Hauka, Marcel Andree, Martin P Däumer, Alexander Thielen, Susanne Kolbe-Busch, Klaus Göbels, Rainer Zotz, Klaus Pfeffer, Jörg Timm, Alexander T Dilthey

Tracing of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission chains is still a major challenge for public health authorities, when incidental contacts are not recalled or are not perceived as potential risk contacts. Viral sequencing can address key questions about SARS-CoV-2 evolution and may support reconstruction of viral transmission networks by integration of molecular epidemiology into classical contact tracing.In collaboration with local public health authorities, we set up an integrated system of genomic surveillance in an urban setting, combining a) viral surveillance sequencing, b) genetically based identification of infection clusters in the population, c) integration of public health authority contact tracing data, and d) a user-friendly dashboard application as a central data analysis platform.

IKKβ primes inflammasome formation by recruiting NLRP3 to the trans-Golgi network

Immunity 55 (12), 2271-2284. e7, 2022

Niklas A Schmacke, Fionan O’Duill, Moritz M Gaidt, Inga Szymanska, Julia M Kamper, Jonathan L Schmid-Burgk, Sophia C Mädler, Timur Mackens-Kiani, Tatsuya Kozaki, Dhruv Chauhan, Dennis Nagl, Che A Stafford, Hartmann Harz, Adrian L Fröhlich, Francesca Pinci, Florent Ginhoux, Roland Beckmann, Matthias Mann, Heinrich Leonhardt, Veit Hornung

The NLRP3 inflammasome plays a central role in antimicrobial defense as well as in the context of sterile inflammatory conditions. NLRP3 activity is governed by two independent signals: the first signal primes NLRP3, rendering it responsive to the second signal, which then triggers inflammasome formation. Our understanding of how NLRP3 priming contributes to inflammasome activation remains limited. Here, we show that IKKβ, a kinase activated during priming, induces recruitment of NLRP3 to phosphatidylinositol-4-phosphate (PI4P), a phospholipid enriched on the trans-Golgi network. NEK7, a mitotic spindle kinase that had previously been thought to be indispensable for NLRP3 activation, was redundant for inflammasome formation when IKKβ recruited NLRP3 to PI4P. Studying iPSC-derived human macrophages revealed that the IKKβ-mediated NEK7-independent pathway constitutes the predominant …

Ethical principles, opportunities and constraints in clinical proteomics.

Molecular & Cellular Proteomics: MCP, mcp. RA120. 002435-mcp. RA120. 002435, 2021

PV Treit, PE Geyer, GS Omenn, M Mann

Recent advances in MS-based proteomics have vastly increased the quality and scope of biological information that can be derived from human samples. These advances have rendered current workflows increasingly applicable in biomedical and clinical contexts. As proteomics is poised to take an important role in the clinic, associated ethical responsibilities increase in tandem with the impact on the health, privacy, and well-being of individuals. Here we conducted and report a systematic literature review of ethical issues in clinical proteomics. We add our perspectives from a background of bioethics, the results of our accompanying paper extracting individual-sensitive results from patient samples, and the literature addressing similar issues in genomics. The spectrum of potential issues ranges from patient re-identification to incidental findings of clinical significance. The latter can be divided into actionable and unactionable findings. Some of these have the potential to be employed in discriminatory or privacy-infringing ways. However, incidental findings may also have great positive potential. A plasma proteome profile, for instance, could inform on the general health or disease status of an individual regardless of the narrow diagnostic question that prompted it. We suggest that early discussion of ethical issues in clinical proteomics is important to ensure that eventual regulations reflect the considered judgment of the community as well as to anticipate opportunities and problems that may arise as the technology matures further.

A novel FRET assay for the intracellular activation of ADC linkers

Cancer Research 75 (15_Supplement), 211-211, 2015

Byoung-Chul Lee, Cecile Chalouni, Sam Nalle, Sophia Doll, Martine Darwish, Ira Mellman, Richard Vandlen

Evaluating the intracellular activation of ADC by a novel FRET assay.Despite the recent success of ADCs as cancer therapeutics, their mechanisms of action are not fully understood. In this study, we developed ADCs using a novel fluorescence resonance energy transfer (FRET) linker in order to facilitate monitoring the details of intracellular uptake, vesicular trafficking and payload release. In the FRET linker, the cathepsin-cleavable dipeptide of val-cit was inserted between a fluorescence donor Alexa Fluor 488 (or later, fluorescein) and an acceptor tetramethylrhodamine (TAMRA). Upon cleavage of the val-cit linker, a fluorescence signal from Alexa Fluor 488 or fluorescein is expected to appear as a probe for monitoring intracellular activation of ADC.We used two in-vitro human cancer cell lines, SKBR3 and PC3. SKBR3 is a Her2-positive breast cancer cell line and PC3 is a prostate cancer cell line that has been …

Molecular Origin of Blood‐Based Infrared Spectroscopic Fingerprints

Angewandte Chemie 133 (31), 17197-17206, 2021

Liudmila Voronina, Cristina Leonardo, Johannes B Mueller‐Reif, Philipp E Geyer, Marinus Huber, Michael Trubetskov, Kosmas V Kepesidis, Jürgen Behr, Matthias Mann, Ferenc Krausz, Mihaela Žigman

Infrared spectroscopy of liquid biopsies is a time‐ and cost‐effective approach that may advance biomedical diagnostics. However, the molecular nature of disease‐related changes of infrared molecular fingerprints (IMFs) remains poorly understood, impeding the method's applicability. Here we probe 148 human blood sera and reveal the origin of the variations in their IMFs. To that end, we supplemented infrared spectroscopy with biochemical fractionation and proteomic profiling, providing molecular information about serum composition. Using lung cancer as an example of a medical condition, we demonstrate that the disease‐related differences in IMFs are dominated by contributions from twelve highly abundant proteins—that, if used as a pattern, may be instrumental for detecting malignancy. Tying proteomic to spectral information and machine learning advances our understanding of the infrared spectra of …

Aggrecan: a new biomarker for acute type A aortic dissection

Scientific Reports 11 (1), 10371, 2021

Karl C König, Harald Lahm, Martina Dreßen, Stefanie A Doppler, Stefan Eichhorn, Nicole Beck, Kathrin Kraehschuetz, Sophia Doll, Stefan Holdenrieder, Adnan Kastrati, Rüdiger Lange, Markus Krane

Acute type A aortic dissection (ATAAD) constitutes a life-threatening aortic pathology with significant morbidity and mortality. Without surgical intervention the usual mortality rate averages between 1 and 2% per hour. Thus, an early diagnosis of ATAAD is of pivotal importance to direct the affected patients to the appropriate treatment. Preceding tests to find an appropriate biomarker showed among others an increased aggrecan (ACAN) mRNA expression in aortic tissue of ATAAD patients. As a consequence, we investigated whether ACAN is a potential biomarker for diagnosing ATAAD. Mean ACAN protein concentration showed a significantly higher plasma concentration in ATAAD patients (38.59 ng/mL, n = 33) compared to plasma of patients with thoracic aortic aneurysms (4.45 ng/mL, n = 13), patients with myocardial infarction (11.77 ng/mL, n = 18) and healthy volunteers (8.05 ng/mL, n = 12). Cardiac …

LSC-2023-Longitudinal proteomic profiling reveals early onset increase in collagens and (auto-) antibodies in COVID-19 patients

European Respiratory Journal 62 (suppl 67), 2023

Anna Semenova, Taylor Alexander Poor, Philipp Geyer, Ali Oender Yildirim, Theodore S Kapellos, Alexander V Misharin, Gr Scott Budinger, Herbert B Schiller

Infectious respiratory diseases comprise the 4th most fatal group of diseases worldwide. Most studies on COVID-19 lack appropriate comparison to other viral pneumonias with similar severity. Here, we leverage the SCRIPT cohort at NWU Chicago for longitudinal proteomic profiling of bronchoalveolar lavage fluid (BALF) and patient plasma to analyze pathogen-specific differences during disease progression. We used mass spectrometry to analyze BALF and matched plasma from COVID-19 (n=14), bacterial pneumonia (n=8), influenza (n=8) patients, and non-pneumonia controls (n=8) at up to five time points after intubation in the intensive care unit.BALF of COVID-19 patients was specifically enriched in immunoglobulins, blood clotting proteins, and collagens, suggesting increased fibrogenesis and B-cell immunity already at the time of intubation compared to other bacterial or viral types of pneumonia. We …

Bioinformatics analysis of thousands of TCGA tumors to determine the involvement of epigenetic regulators in human cancer

BMC genomics 16, 1-15, 2015

Florian Gnad, Sophia Doll, Gerard Manning, David Arnott, Zemin Zhang

Many cancer cells show distorted epigenetic landscapes. The Cancer Genome Atlas (TCGA) project profiles thousands of tumors, allowing the discovery of somatic alterations in the epigenetic machinery and the identification of potential cancer drivers among members of epigenetic protein families.We integrated mutation, expression, and copy number data from 5943 tumors from 13 cancer types to train a classification model that predicts the likelihood of being an oncogene (OG), tumor suppressor (TSG) or neutral gene (NG). We applied this predictor to epigenetic regulator genes (ERGs), and used differential expression and correlation network analysis to identify dysregulated ERGs along with co-expressed cancer genes. Furthermore, we quantified global proteomic changes by mass spectrometry after EZH2 inhibition …

Antiviral signalling by a cyclic nucleotide activated CRISPR protease

Nature 614 (7946), 168-174, 2023

Christophe Rouillon, Niels Schneberger, Haotian Chi, Katja Blumenstock, Stefano Da Vela, Katrin Ackermann, Jonas Moecking, Martin F Peter, Wolfgang Boenigk, Reinhard Seifert, Bela E Bode, Jonathan L Schmid-Burgk, Dmitri Svergun, Matthias Geyer, Malcolm F White, Gregor Hagelueken

CRISPR defence systems such as the well-known DNA-targeting Cas9 and the RNA-targeting type III systems are widespread in prokaryotes,. The latter orchestrates a complex antiviral response that is initiated through the synthesis of cyclic oligoadenylates after recognition of foreign RNA, –. Among the large set of proteins that are linked to type III systems and predicted to bind cyclic oligoadenylates,, a CRISPR-associated Lon protease (CalpL) stood out to us. CalpL contains a sensor domain of the SAVED family fused to a Lon protease effector domain. However, the mode of action of this effector is unknown. Here we report the structure and function of CalpL and show that this soluble protein forms a stable tripartite complex with two other proteins, CalpT and CalpS, that are encoded on the same operon. After activation by cyclic tetra-adenylate (cA4), CalpL oligomerizes and specifically cleaves the MazF …

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