Mass spectrometry-based draft of the mouse proteome.

The laboratory mouse ranks among the most important experimental systems for biomedical research and molecular reference maps of such models are essential informational tools. Here, we present a quantitative draft of the mouse proteome and phosphoproteome constructed from 41 healthy tissues and several lines of analyses exemplify which insights can be gleaned from the data. For instance, tissue- and cell-type resolved profiles provide protein evidence for the expression of 17,000 genes, thousands of isoforms and 50,000 phosphorylation sites in vivo. Proteogenomic comparison of mouse, human and Arabidopsis reveal common and distinct mechanisms of gene expression regulation and, despite many similarities, numerous differentially abundant orthologs that likely serve species-specific functions. We leverage the mouse proteome by integrating phenotypic drug (n > 400) and radiation response data with the proteomes of 66 pancreatic ductal adenocarcinoma (PDAC) cell lines to reveal molecular markers for sensitivity and resistance. This unique atlas complements other molecular resources for the mouse and can be explored online via ProteomicsDB and PACiFIC.

Illuminating phenotypic drug responses of sarcoma cells to kinase inhibitors by phosphoproteomics.

Kinase inhibitors (KIs) are important cancer drugs but often feature polypharmacology that is molecularly not understood. This disconnect is particularly apparent in cancer entities such as sarcomas for which the oncogenic drivers are often not clear. To investigate more systematically how the cellular proteotypes of sarcoma cells shape their response to molecularly targeted drugs, we profiled the proteomes and phosphoproteomes of 17 sarcoma cell lines and screened the same against 150 cancer drugs. The resulting 2550 phenotypic profiles revealed distinct drug responses and the cellular activity landscapes derived from deep (phospho)proteomes (9-10,000 proteins and 10-27,000 phosphorylation sites per cell line) enabled several lines of analysis. For instance, connecting the (phospho)proteomic data with drug responses revealed known and novel mechanisms of action (MoAs) of KIs and identified markers of drug sensitivity or resistance. All data is publicly accessible via an interactive web application that enables exploration of this rich molecular resource for a better understanding of active signalling pathways in sarcoma cells, identifying treatment response predictors and revealing novel MoA of clinical KIs.

Unified Workflow for the Rapid and In-Depth Characterization of Bacterial Proteomes.

Bacteria are the most abundant and diverse organisms among the kingdoms of life. Due to this excessive variance, finding a unified, comprehensive, and safe workflow for quantitative bacterial proteomics is challenging. In this study, we have systematically evaluated and optimized sample preparation, mass spectrometric data acquisition, and data analysis strategies in bacterial proteomics. We investigated workflow performances on six representative species with highly different physiologic properties to mimic bacterial diversity. The best sample preparation strategy was a cell lysis protocol in 100% trifluoroacetic acid followed by an in-solution digest. Peptides were separated on a 30-min linear microflow liquid chromatography gradient and analyzed in data-independent acquisition mode. Data analysis was performed with DIA-NN using a predicted spectral library. Performance was evaluated according to the number of identified proteins, quantitative precision, throughput, costs, and biological safety. With this rapid workflow, over 40% of all encoded genes were detected per bacterial species. We demonstrated the general applicability of our workflow on a set of 23 taxonomically and physiologically diverse bacterial species. We could confidently identify over 45,000 proteins in the combined dataset, of which 30,000 have not been experimentally validated before. Our work thereby provides a valuable resource for the microbial scientific community. Finally, we grew Escherichia coli and Bacillus cereus in replicates under 12 different cultivation conditions to demonstrate the high-throughput suitability of the workflow. The proteomic workflow we present in this manuscript does not require any specialized equipment or commercial software and can be easily applied by other laboratories to support and accelerate the proteomic exploration of the bacterial kingdom.

Defining the carrier proteome limit for single-cell proteomics.

Single-cell proteomics by mass spectrometry (SCoPE-MS) is a recently introduced method to quantify multiplexed single-cell proteomes. While this technique has generated great excitement, the underlying technologies (isobaric labeling and mass spectrometry (MS)) have technical limitations with the potential to affect data quality and biological interpretation. These limitations are particularly relevant when a carrier proteome, a sample added at 25-500× the amount of a single-cell proteome, is used to enable peptide identifications. Here we perform controlled experiments with increasing carrier proteome amounts and evaluate quantitative accuracy, as it relates to mass analyzer dynamic range, multiplexing level and number of ions sampled. We demonstrate that an increase in carrier proteome level requires a concomitant increase in the number of ions sampled to maintain quantitative accuracy. Lastly, we introduce Single-Cell Proteomics Companion (SCPCompanion), a software tool that enables rapid evaluation of single-cell proteomic data and recommends instrument and data analysis parameters for improved data quality.

SIMSI-Transfer: Software-Assisted Reduction of Missing Values in Phosphoproteomic and Proteomic Isobaric Labeling Data Using Tandem Mass Spectrum Clustering.

Isobaric stable isotope labeling techniques such as tandem mass tags (TMTs) have become popular in proteomics because they enable the relative quantification of proteins with high precision from up to 18 samples in a single experiment. While missing values in peptide quantification are rare in a single TMT experiment, they rapidly increase when combining multiple TMT experiments. As the field moves toward analyzing ever higher numbers of samples, tools that reduce missing values also become more important for analyzing TMT datasets. To this end, we developed SIMSI-Transfer (Similarity-based Isobaric Mass Spectra 2 [MS2] Identification Transfer), a software tool that extends our previously developed software MaRaCluster (© Matthew The) by clustering similar tandem MS2 from multiple TMT experiments. SIMSI-Transfer is based on the assumption that similarity-clustered MS2 spectra represent the same peptide. Therefore, peptide identifications made by database searching in one TMT batch can be transferred to another TMT batch in which the same peptide was fragmented but not identified. To assess the validity of this approach, we tested SIMSI-Transfer on masked search engine identification results and recovered >80% of the masked identifications while controlling errors in the transfer procedure to below 1% false discovery rate. Applying SIMSI-Transfer to six published full proteome and phosphoproteome datasets from the Clinical Proteomic Tumor Analysis Consortium led to an increase of 26 to 45% of identified MS2 spectra with TMT quantifications. This significantly decreased the number of missing values across batches and, in turn, increased the number of peptides and proteins identified in all TMT batches by 43 to 56% and 13 to 16%, respectively.

Prosit-TMT: Deep Learning Boosts Identification of TMT-Labeled Peptides.

The prediction of fragment ion intensities and retention time of peptides has gained significant attention over the past few years. However, the progress shown in the accurate prediction of such properties focused primarily on unlabeled peptides. Tandem mass tags (TMT) are chemical peptide labels that are coupled to free amine groups usually after protein digestion to enable the multiplexed analysis of multiple samples in bottom-up mass spectrometry. It is a standard workflow in proteomics ranging from single-cell to high-throughput proteomics. Particularly for TMT, increasing the number of confidently identified spectra is highly desirable as it provides identification and quantification information with every spectrum. Here, we report on the generation of an extensive resource of synthetic TMT-labeled peptides as part of the ProteomeTools project and present the extension of the deep learning model Prosit to accurately predict the retention time and fragment ion intensities of TMT-labeled peptides with high accuracy. Prosit-TMT supports CID and HCD fragmentation and ion trap and Orbitrap mass analyzers in a single model. Reanalysis of published TMT data sets show that this single model extracts substantial additional information. Applying Prosit-TMT, we discovered that the expression of many proteins in human breast milk follows a distinct daily cycle which may prime the newborn for nutritional or environmental cues.

Data, Reagents, Assays and Merits of Proteomics for SARS-CoV-2 Research and Testing.

As the COVID-19 pandemic continues to spread, thousands of scientists around the globe have changed research direction to understand better how the virus works and to find out how it may be tackled. The number of manuscripts on preprint servers is soaring and peer-reviewed publications using MS-based proteomics are beginning to emerge. To facilitate proteomic research on SARS-CoV-2, the virus that causes COVID-19, this report presents deep-scale proteomes (10,000 proteins; >130,000 peptides) of common cell line models, notably Vero E6, Calu-3, Caco-2, and ACE2-A549 that characterize their protein expression profiles including viral entry factors such as ACE2 or TMPRSS2. Using the 9 kDa protein SRP9 and the breast cancer oncogene BRCA1 as examples, we show how the proteome expression data can be used to refine the annotation of protein-coding regions of the African green monkey and the Vero cell line genomes. Monitoring changes of the proteome on viral infection revealed widespread expression changes including transcriptional regulators, protease inhibitors, and proteins involved in innate immunity. Based on a library of 98 stable-isotope labeled synthetic peptides representing 11 SARS-CoV-2 proteins, we developed PRM (parallel reaction monitoring) assays for nano-flow and micro-flow LC-MS/MS. We assessed the merits of these PRM assays using supernatants of virus-infected Vero E6 cells and challenged the assays by analyzing two diagnostic cohorts of 24 (+30) SARS-CoV-2 positive and 28 (+9) negative cases. In light of the results obtained and including recent publications or manuscripts on preprint servers, we critically discuss the merits of MS-based proteomics for SARS-CoV-2 research and testing.

Evaluation of Disposable Trap Column nanoLC-FAIMS-MS/MS for the Proteomic Analysis of FFPE Tissue.

Proteomic biomarker discovery using formalin-fixed paraffin-embedded (FFPE) tissue requires robust workflows to support the analysis of large cohorts of patient samples. It also requires finding a reasonable balance between achieving a high proteomic depth and limiting the overall analysis time. To this end, we evaluated the merits of online coupling of single-use disposable trap column nanoflow liquid chromatography, high-field asymmetric-waveform ion-mobility spectrometry (FAIMS), and tandem mass spectrometry (nLC-FAIMS-MS/MS). The data show that ≤600 ng of peptide digest should be loaded onto the chromatographic part of the system. Careful characterization of the FAIMS settings enabled the choice of optimal combinations of compensation voltages (CVs) as a function of the employed LC gradient time. We found nLC-FAIMS-MS/MS to be on par with StageTip-based off-line basic pH reversed-phase fractionation in terms of proteomic depth and reproducibility of protein quantification (coefficient of variation ≤15% for 90% of all proteins) but requiring 50% less sample and substantially reducing sample handling. Using FFPE materials from the lymph node, lung, and prostate tissue as examples, we show that nLC-FAIMS-MS/MS can identify 5000-6000 proteins from the respective tissue within a total of 3 h of analysis time.

Robust Microflow LC-MS/MS for Proteome Analysis: 38 000 Runs and Counting.

Microflow liquid chromatography tandem mass spectrometry (µLC-MS/MS) is becoming a viable alternative to nanoflow LC-MS/MS for the analysis of proteomes. We have recently demonstrated the potential of such a system operating with a 1 mm i.d. × 150 mm column and at a flow rate of 50 µL/min for high-throughput applications. On the basis of the analysis of ∼38 000 samples measured on two instruments over the past two years, we now show that the approach is extremely robust. Up to 1500 analyses were performed within one month, and >14 000 samples could be analyzed on a single column without loss of chromatographic performance. Samples included proteomes of cell lines, tissues, and human body fluids, which were analyzed with or without prior peptide fractionation or stable isotope labeling. We show that the µLC-MS/MS system is capable of measuring 2600 proteins from undepleted human plasma and ∼5000 proteins from crude human urine in 1 day, demonstrating its potential for in-depth as well as high-throughput clinical application.

Decrypting lysine deacetylase inhibitor action and protein modifications by dose-resolved proteomics.

Lysine deacetylase inhibitors (KDACis) are approved drugs for cutaneous T cell lymphoma (CTCL), peripheral T cell lymphoma (PTCL), and multiple myeloma, but many aspects of their cellular mechanism of action (MoA) and substantial toxicity are not well understood. To shed more light on how KDACis elicit cellular responses, we systematically measured dose-dependent changes in acetylation, phosphorylation, and protein expression in response to 21 clinical and pre-clinical KDACis. The resulting 862,000 dose-response curves revealed, for instance, limited cellular specificity of histone deacetylase (HDAC) 1, 2, 3, and 6 inhibitors; strong cross-talk between acetylation and phosphorylation pathways; localization of most drug-responsive acetylation sites to intrinsically disordered regions (IDRs); an underappreciated role of acetylation in protein structure; and a shift in EP300 protein abundance between the cytoplasm and the nucleus. This comprehensive dataset serves as a resource for the investigation of the molecular mechanisms underlying KDACi action in cells and can be interactively explored online in ProteomicsDB.

CurveCurator: a recalibrated F-statistic to assess, classify, and explore significance of dose-response curves.

Dose-response curves are key metrics in pharmacology and biology to assess phenotypic or molecular actions of bioactive compounds in a quantitative fashion. Yet, it is often unclear whether or not a measured response significantly differs from a curve without regulation, particularly in high-throughput applications or unstable assays. Treating potency and effect size estimates from random and true curves with the same level of confidence can lead to incorrect hypotheses and issues in training machine learning models. Here, we present CurveCurator, an open-source software that provides reliable dose-response characteristics by computing p-values and false discovery rates based on a recalibrated F-statistic and a target-decoy procedure that considers dataset-specific effect size distributions. The application of CurveCurator to three large-scale datasets enables a systematic drug mode of action analysis and demonstrates its scalable utility across several application areas, facilitated by a performant, interactive dashboard for fast data exploration.

Decrypting drug actions and protein modifications by dose- and time-resolved proteomics.

Although most cancer drugs modulate the activities of cellular pathways by changing posttranslational modifications (PTMs), little is known regarding the extent and the time- and dose-response characteristics of drug-regulated PTMs. In this work, we introduce a proteomic assay called decryptM that quantifies drug-PTM modulation for thousands of PTMs in cells to shed light on target engagement and drug mechanism of action. Examples range from detecting DNA damage by chemotherapeutics, to identifying drug-specific PTM signatures of kinase inhibitors, to demonstrating that rituximab kills CD20-positive B cells by overactivating B cell receptor signaling. DecryptM profiling of 31 cancer drugs in 13 cell lines demonstrates the broad applicability of the approach. The resulting 1.8 million dose-response curves are provided as an interactive molecular resource in ProteomicsDB.

Robust, reproducible and quantitative analysis of thousands of proteomes by micro-flow LC-MS/MS.

Nano-flow liquid chromatography tandem mass spectrometry (nano-flow LC-MS/MS) is the mainstay in proteome research because of its excellent sensitivity but often comes at the expense of robustness. Here we show that micro-flow LC-MS/MS using a 1 × 150 mm column shows excellent reproducibility of chromatographic retention time (<0.3% coefficient of variation, CV) and protein quantification (<7.5% CV) using data from >2000 samples of human cell lines, tissues and body fluids. Deep proteome analysis identifies >9000 proteins and >120,000 peptides in 16 h and sample multiplexing using tandem mass tags increases throughput to 11 proteomes in 16 h. The system identifies >30,000 phosphopeptides in 12 h and protein-protein or protein-drug interaction experiments can be analyzed in 20 min per sample. We show that the same column can be used to analyze >7500 samples without apparent loss of performance. This study demonstrates that micro-flow LC-MS/MS is suitable for a broad range of proteomic applications.