Human SRMAtlas: A Resource of Targeted Assays to Quantify the Complete Human Proteome.

The ability to reliably and reproducibly measure any protein of the human proteome in any tissue or cell type would be transformative for understanding systems-level properties as well as specific pathways in physiology and disease. Here, we describe the generation and verification of a compendium of highly specific assays that enable quantification of 99.7% of the 20,277 annotated human proteins by the widely accessible, sensitive, and robust targeted mass spectrometric method selected reaction monitoring, SRM. This human SRMAtlas provides definitive coordinates that conclusively identify the respective peptide in biological samples. We report data on 166,174 proteotypic peptides providing multiple, independent assays to quantify any human protein and numerous spliced variants, non-synonymous mutations, and post-translational modifications. The data are freely accessible as a resource at http://www.srmatlas.org/, and we demonstrate its utility by examining the network response to inhibition of cholesterol synthesis in liver cells and to docetaxel in prostate cancer lines.

Detection and Quantification of Drug-Protein Adducts in Human Liver.

Covalent protein adducts formed by drugs or their reactive metabolites are risk factors for adverse reactions, and inactivation of cytochrome P450 (CYP) enzymes. Characterization of drug-protein adducts is limited due to lack of methods identifying and quantifying covalent adducts in complex matrices. This study presents a workflow that combines data-dependent and data-independent acquisition (DDA and DIA) based liquid chromatography with tandem mass spectrometry (LC-MS/MS) to detect very low abundance adducts resulting from CYP mediated drug metabolism in human liver microsomes (HLMs). HLMs were incubated with raloxifene as a model compound and adducts were detected in 78 proteins, including CYP3A and CYP2C family enzymes. Experiments with recombinant CYP3A and CYP2C enzymes confirmed adduct formation in all CYPs tested, including CYPs not subject to time-dependent inhibition by raloxifene. These data suggest adducts can be benign. DIA analysis showed variable adduct abundance in many peptides between livers, but no concomitant decrease of unadducted peptides. This study sets a new standard for adduct detection in complex samples, offering insights into the human adductome resulting from reactive metabolite exposure. The methodology presented will aid mechanistic studies to identify, quantify and differentiate between adducts that result in adverse drug reactions and those that are benign.

Trans-Proteomic Pipeline: Robust Mass Spectrometry-Based Proteomics Data Analysis Suite.

The Trans-Proteomic Pipeline (TPP) mass spectrometry data analysis suite has been in continual development and refinement since its first tools, PeptideProphet and ProteinProphet, were published 20 years ago. The current release provides a large complement of tools for spectrum processing, spectrum searching, search validation, abundance computation, protein inference, and more. Many of the tools include machine-learning modeling to extract the most information from data sets and build robust statistical models to compute the probabilities that derived information is correct. Here we present the latest information on the many TPP tools, and how TPP can be deployed on various platforms from personal Windows laptops to Linux clusters and expansive cloud computing environments. We describe tutorials on how to use TPP in a variety of ways and describe synergistic projects that leverage TPP. We conclude with plans for continued development of TPP.

Improved Analysis of Cross-Linking Mass Spectrometry Data with Kojak 2.0, Advanced by Integration into the Trans-Proteomic Pipeline.

Fragmentation ion spectral analysis of chemically cross-linked proteins is an established technology in the proteomics research repertoire for determining protein interactions, spatial orientation, and structure. Here we present Kojak version 2.0, a major update to the original Kojak algorithm, which was developed to identify cross-linked peptides from fragment ion spectra using a database search approach. A substantially improved algorithm with updated scoring metrics, support for cleavable cross-linkers, and identification of cross-links between 15N-labeled homomultimers are among the newest features of Kojak 2.0 presented here. Kojak 2.0 is now integrated into the Trans-Proteomic Pipeline, enabling access to dozens of additional tools within that suite. In particular, the PeptideProphet and iProphet tools for validation of cross-links improve the sensitivity and accuracy of correct cross-link identifications at user-defined thresholds. These new features improve the versatility of the algorithm, enabling its use in a wider range of experimental designs and analysis pipelines. Kojak 2.0 remains open-source and multiplatform.

Insights from the First Phosphopeptide Challenge of the MS Resource Pillar of the HUPO Human Proteome Project.

Mass spectrometry has greatly improved the analysis of phosphorylation events in complex biological systems and on a large scale. Despite considerable progress, the correct identification of phosphorylated sites, their quantification, and their interpretation regarding physiological relevance remain challenging. The MS Resource Pillar of the Human Proteome Organization (HUPO) Human Proteome Project (HPP) initiated the Phosphopeptide Challenge as a resource to help the community evaluate methods, learn procedures and data analysis routines, and establish their own workflows by comparing results obtained from a standard set of 94 phosphopeptides (serine, threonine, tyrosine) and their nonphosphorylated counterparts mixed at different ratios in a neat sample and a yeast background. Participants analyzed both samples with their method(s) of choice to report the identification and site localization of these peptides, determine their relative abundances, and enrich for the phosphorylated peptides in the yeast background. We discuss the results from 22 laboratories that used a range of different methods, instruments, and analysis software. We reanalyzed submitted data with a single software pipeline and highlight the successes and challenges in correct phosphosite localization. All of the data from this collaborative endeavor are shared as a resource to encourage the development of even better methods and tools for diverse phosphoproteomic applications. All submitted data and search results were uploaded to MassIVE (https://massive.ucsd.edu/) as data set MSV000085932 with ProteomeXchange identifier PXD020801.

A Tandem Mass Spectrometry Sequence Database Search Method for Identification of O-Fucosylated Proteins by Mass Spectrometry.

Thrombospondin type 1 repeats (TSRs), small adhesive protein domains with a wide range of functions, are usually modified with O-linked fucose, which may be extended to O-fucose-ß1,3-glucose. Collision-induced dissociation (CID) spectra of O-fucosylated peptides cannot be sequenced by standard tandem mass spectrometry (MS/MS) sequence database search engines because O-linked glycans are highly labile in the gas phase and are effectively absent from the CID peptide fragment spectra, resulting in a large mass error. Electron transfer dissociation (ETD) preserves O-linked glycans on peptide fragments, but only a subset of tryptic peptides with low m/ z can be reliably sequenced from ETD spectra compared to CID. Accordingly, studies to date that have used MS to identify O-fucosylated TSRs have required manual interpretation of CID mass spectra even when ETD was also employed. In order to facilitate high-throughput, automatic identification of O-fucosylated peptides from CID spectra, we re-engineered the MS/MS sequence database search engine Comet and the MS data analysis suite Trans-Proteomic Pipeline to enable automated sequencing of peptides exhibiting the neutral losses characteristic of labile O-linked glycans. We used our approach to reanalyze published proteomics data from Plasmodium parasites and identified multiple glycoforms of TSR-containing proteins.

PTMProphet: Fast and Accurate Mass Modification Localization for the Trans-Proteomic Pipeline.

Spectral matching sequence database search engines commonly used on mass spectrometry-based proteomics experiments excel at identifying peptide sequence ions, and in addition, possible sequence ions carrying post-translational modifications (PTMs), but most do not provide confidence metrics for the exact localization of those PTMs when several possible sites are available. Localization is absolutely required for downstream molecular cell biology analysis of PTM function in vitro and in vivo. Therefore, we developed PTMProphet, a free and open-source software tool integrated into the Trans-Proteomic Pipeline, which reanalyzes identified spectra from any search engine for which pepXML output is available to provide localization confidence to enable appropriate further characterization of biologic events. Localization of any type of mass modification (e.g., phosphorylation) is supported. PTMProphet applies Bayesian mixture models to compute probabilities for each site/peptide spectrum match where a PTM has been identified. These probabilities can be combined to compute a global false localization rate at any threshold to guide downstream analysis. We describe the PTMProphet tool, its underlying algorithms, and demonstrate its performance on ground-truth synthetic peptide reference data sets, one previously published small data set, one new larger data set, and also on a previously published phosphoenriched data set where the correct sites of modification are unknown. Data have been deposited to ProteomeXchange with identifier PXD013210.

Flexible and Fast Mapping of Peptides to a Proteome with ProteoMapper.

Bottom-up proteomics relies on the proteolytic or chemical cleavage of proteins into peptides, the identification of those peptides via mass spectrometry, and the mapping of the identified peptides back to the reference proteome to infer which possible proteins are identified. Reliable mapping of peptides to proteins still poses substantial challenges when considering similar proteins, protein families, splice isoforms, sequence variation, and possible residue mass modifications, combined with an imperfect and incomplete understanding of the proteome. The ProteoMapper tool enables a comprehensive and rapid mapping of peptides to a reference proteome. The indexer component creates a segmented index for an input proteome from a FASTA or PEFF file. The ProMaST component provides ultrafast mapping of one or more input peptides against the index. ProteoMapper allows searches that take into account known sequence variation encoded in PEFF files. It also enables fuzzy searches to find highly similar peptides with residue order changes or other isobaric or near-isobaric substitutions within a specified mass tolerance. We demonstrate an example of a one-hit-wonder identification in PeptideAtlas that may be better explained by a combination of catalogued and uncatalogued sequence variation in another highly observed protein. ProteoMapper is a free and open source, available for local use after downloading, embedding in other applications, as an online web tool at http://www.peptideatlas.org/map , and as a web service.

Opening a SWATH Window on Posttranslational Modifications: Automated Pursuit of Modified Peptides.

Posttranslational modifications of proteins play an important role in biology. For example, phosphorylation is a key component in signal transduction in all three domains of life, and histones can be modified in such a variety of ways that a histone code for gene regulation has been proposed. Shotgun proteomics is commonly used to identify posttranslational modifications as well as chemical modifications from sample processing. However, it favors the detection of abundant peptides over the repertoire presented, and the data analysis usually requires advance specification of modification masses and target amino acids, their number constrained by available computational resources. Recent advances in data independent acquisition mass spectrometry technologies such as SWATH-MS enable a deeper recording of the peptide contents of samples, including peptides with modifications. Here, we present a novel approach that applies the power of SWATH-MS analysis to the automated pursuit of modified peptides. With the new SWATHProphet(PTM) functionality added to the open source SWATHProphet software, precursor ions consistent with a modification are identified along with the mass and localization of the modification in the peptide sequence in a sensitive and unrestricted manner without the need to anticipate the modifications in advance. Using this method, we demonstrate the detection of a wide assortment of modified peptides, many unanticipated, in samples containing unpurified synthetic peptides and human urine, as well as in phospho-enriched human tissue culture cell samples.

Processing shotgun proteomics data on the Amazon cloud with the trans-proteomic pipeline.

Cloud computing, where scalable, on-demand compute cycles and storage are available as a service, has the potential to accelerate mass spectrometry-based proteomics research by providing simple, expandable, and affordable large-scale computing to all laboratories regardless of location or information technology expertise. We present new cloud computing functionality for the Trans-Proteomic Pipeline, a free and open-source suite of tools for the processing and analysis of tandem mass spectrometry datasets. Enabled with Amazon Web Services cloud computing, the Trans-Proteomic Pipeline now accesses large scale computing resources, limited only by the available Amazon Web Services infrastructure, for all users. The Trans-Proteomic Pipeline runs in an environment fully hosted on Amazon Web Services, where all software and data reside on cloud resources to tackle large search studies. In addition, it can also be run on a local computer with computationally intensive tasks launched onto the Amazon Elastic Compute Cloud service to greatly decrease analysis times. We describe the new Trans-Proteomic Pipeline cloud service components, compare the relative performance and costs of various Elastic Compute Cloud service instance types, and present on-line tutorials that enable users to learn how to deploy cloud computing technology rapidly with the Trans-Proteomic Pipeline. We provide tools for estimating the necessary computing resources and costs given the scale of a job and demonstrate the use of cloud enabled Trans-Proteomic Pipeline by performing over 1100 tandem mass spectrometry files through four proteomic search engines in 9 h and at a very low cost.

Automated Validation of Results and Removal of Fragment Ion Interferences in Targeted Analysis of Data-independent Acquisition Mass Spectrometry (MS) using SWATHProphet.

Proteomics by mass spectrometry technology is widely used for identifying and quantifying peptides and proteins. The breadth and sensitivity of peptide detection have been advanced by the advent of data-independent acquisition mass spectrometry. Analysis of such data, however, is challenging due to the complexity of fragment ion spectra that have contributions from multiple co-eluting precursor ions. We present SWATHProphet software that identifies and quantifies peptide fragment ion traces in data-independent acquisition data, provides accurate probabilities to ensure results are correct, and automatically detects and removes contributions to quantitation originating from interfering precursor ions. Integration in the widely used open source Trans-Proteomic Pipeline facilitates subsequent analyses such as combining results of multiple data sets together for improved discrimination using iProphet and inferring sample proteins using ProteinProphet. This novel development should greatly help make data-independent acquisition mass spectrometry accessible to large numbers of users.

Tiered Human Integrated Sequence Search Databases for Shotgun Proteomics.

The results of analysis of shotgun proteomics mass spectrometry data can be greatly affected by the selection of the reference protein sequence database against which the spectra are matched. For many species there are multiple sources from which somewhat different sequence sets can be obtained. This can lead to confusion about which database is best in which circumstances-a problem especially acute in human sample analysis. All sequence databases are genome-based, with sequences for the predicted gene and their protein translation products compiled. Our goal is to create a set of primary sequence databases that comprise the union of sequences from many of the different available sources and make the result easily available to the community. We have compiled a set of four sequence databases of varying sizes, from a small database consisting of only the ∼20,000 primary isoforms plus contaminants to a very large database that includes almost all nonredundant protein sequences from several sources. This set of tiered, increasingly complete human protein sequence databases suitable for mass spectrometry proteomics sequence database searching is called the Tiered Human Integrated Search Proteome set. In order to evaluate the utility of these databases, we have analyzed two different data sets, one from the HeLa cell line and the other from normal human liver tissue, with each of the four tiers of database complexity. The result is that approximately 0.8%, 1.1%, and 1.5% additional peptides can be identified for Tiers 2, 3, and 4, respectively, as compared with the Tier 1 database, at substantially increasing computational cost. This increase in computational cost may be worth bearing if the identification of sequence variants or the discovery of sequences that are not present in the reviewed knowledge base entries is an important goal of the study. We find that it is useful to search a data set against a simpler database, and then check the uniqueness of the discovered peptides against a more complex database. We have set up an automated system that downloads all the source databases on the first of each month and automatically generates a new set of search databases and makes them available for download at http://www.peptideatlas.org/thisp/ .

State of the Human Proteome in 2014/2015 As Viewed through PeptideAtlas: Enhancing Accuracy and Coverage through the AtlasProphet.

The Human PeptideAtlas is a compendium of the highest quality peptide identifications from over 1000 shotgun mass spectrometry proteomics experiments collected from many different laboratories, all reanalyzed through a uniform processing pipeline. The latest 2015-03 build contains substantially more input data than past releases, is mapped to a recent version of our merged reference proteome, and uses improved informatics processing and the development of the AtlasProphet to provide the highest quality results. Within the set of ∼20,000 neXtProt primary entries, 14,070 (70%) are confidently detected in the latest build, 5% are ambiguous, 9% are redundant, leaving the total percentage of proteins for which there are no mapping detections at just 16% (3166), all derived from over 133 million peptide-spectrum matches identifying more than 1 million distinct peptides using AtlasProphet to characterize and classify the protein matches. Improved handling for detection and presentation of single amino-acid variants (SAAVs) reveals the detection of 5326 uniquely mapping SAAVs across 2794 proteins. With such a large amount of data, the control of false positives is a challenge. We present the methodology and results for maintaining rigorous quality along with a discussion of the implications of the remaining sources of errors in the build.

The microvesicle component of HIV-1 inocula modulates dendritic cell infection and maturation and enhances adhesion to and activation of T lymphocytes.

HIV-1 is taken up by immature monocyte derived dendritic cells (iMDDCs) into tetraspanin rich caves from which the virus can either be transferred to T lymphocytes or enter into endosomes resulting in degradation. HIV-1 binding and fusion with the DC membrane results in low level de novo infection that can also be transferred to T lymphocytes at a later stage. We have previously reported that HIV-1 can induce partial maturation of iMDDCs at both stages of trafficking. Here we show that CD45⁺ microvesicles (MV) which contaminate purified HIV-1 inocula due to similar size and density, affect DC maturation, de novo HIV-1 infection and transfer to T lymphocytes. Comparing iMDDCs infected with CD45-depleted HIV-1BaL or matched non-depleted preparations, the presence of CD45⁺ MVs was shown to enhance DC maturation and ICAM-1 (CD54) expression, which is involved in DC∶T lymphocyte interactions, while restricting HIV-1 infection of MDDCs. Furthermore, in the DC culture HIV-1 infected (p24⁺) MDDCs were more mature than bystander cells. Depletion of MVs from the HIV-1 inoculum markedly inhibited DC∶T lymphocyte clustering and the induction of alloproliferation as well as limiting HIV-1 transfer from DCs to T lymphocytes. The effects of MV depletion on these functions were reversed by the re-addition of purified MVs from activated but not non-activated SUPT1.CCR5-CL.30 or primary T cells. Analysis of the protein complement of these MVs and of these HIV-1 inocula before and after MV depletion showed that Heat Shock Proteins (HSPs) and nef were the likely DC maturation candidates. Recombinant HSP90α and ß and nef all induced DC maturation and ICAM-1 expression, greater when combined. These results suggest that MVs contaminating HIV-1 released from infected T lymphocytes may be biologically important, especially in enhancing T cell activation, during uptake by DCs in vitro and in vivo, particularly as MVs have been detected in the circulation of HIV-1 infected subjects.

Combining results of multiple search engines in proteomics.

A crucial component of the analysis of shotgun proteomics datasets is the search engine, an algorithm that attempts to identify the peptide sequence from the parent molecular ion that produced each fragment ion spectrum in the dataset. There are many different search engines, both commercial and open source, each employing a somewhat different technique for spectrum identification. The set of high-scoring peptide-spectrum matches for a defined set of input spectra differs markedly among the various search engine results; individual engines each provide unique correct identifications among a core set of correlative identifications. This has led to the approach of combining the results from multiple search engines to achieve improved analysis of each dataset. Here we review the techniques and available software for combining the results of multiple search engines and briefly compare the relative performance of these techniques.

Oncogenic H-ras reprograms Madin-Darby canine kidney (MDCK) cell-derived exosomal proteins following epithelial-mesenchymal transition.

Epithelial-mesenchymal transition (EMT) is a highly conserved morphogenic process defined by the loss of epithelial characteristics and the acquisition of a mesenchymal phenotype. EMT is associated with increased aggressiveness, invasiveness, and metastatic potential in carcinoma cells. To assess the contribution of extracellular vesicles following EMT, we conducted a proteomic analysis of exosomes released from Madin-Darby canine kidney (MDCK) cells, and MDCK cells transformed with oncogenic H-Ras (21D1 cells). Exosomes are 40-100 nm membranous vesicles originating from the inward budding of late endosomes and multivesicular bodies and are released from cells on fusion of multivesicular bodies with the plasma membrane. Exosomes from MDCK cells (MDCK-Exos) and 21D1 cells (21D1-Exos) were purified from cell culture media using density gradient centrifugation (OptiPrep™), and protein content identified by GeLC-MS/MS proteomic profiling. Both MDCK- and 21D1-Exos populations were morphologically similar by cryo-electron microscopy and contained stereotypical exosome marker proteins such as TSG101, Alix, and CD63. In this study we show that the expression levels of typical EMT hallmark proteins seen in whole cells correlate with those observed in MDCK- and 21D1-Exos, i.e. reduction of characteristic inhibitor of angiogenesis, thrombospondin-1, and epithelial markers E-cadherin, and EpCAM, with a concomitant up-regulation of mesenchymal makers such as vimentin. Further, we reveal that 21D1-Exos are enriched with several proteases (e.g. MMP-1, -14, -19, ADAM-10, and ADAMTS1), and integrins (e.g. ITGB1, ITGA3, and ITGA6) that have been recently implicated in regulating the tumor microenvironment to promote metastatic progression. A salient finding of this study was the unique presence of key transcriptional regulators (e.g. the master transcriptional regulator YBX1) and core splicing complex components (e.g. SF3B1, SF3B3, and SFRS1) in mesenchymal 21D1-Exos. Taken together, our findings reveal that exosomes from Ras-transformed MDCK cells are reprogrammed with factors which may be capable of inducing EMT in recipient cells.

State of the human proteome in 2013 as viewed through PeptideAtlas: comparing the kidney, urine, and plasma proteomes for the biology- and disease-driven Human Proteome Project.

The kidney, urine, and plasma proteomes are intimately related: proteins and metabolic waste products are filtered from the plasma by the kidney and excreted via the urine, while kidney proteins may be secreted into the circulation or released into the urine. Shotgun proteomics data sets derived from human kidney, urine, and plasma samples were collated and processed using a uniform software pipeline, and relative protein abundances were estimated by spectral counting. The resulting PeptideAtlas builds yielded 4005, 2491, and 3553 nonredundant proteins at 1% FDR for the kidney, urine, and plasma proteomes, respectively - for kidney and plasma, the largest high-confidence protein sets to date. The same pipeline applied to all available human data yielded a 2013 Human PeptideAtlas build containing 12,644 nonredundant proteins and at least one peptide for each of ∼14,000 Swiss-Prot entries, an increase over 2012 of ∼7.5% of the predicted human proteome. We demonstrate that abundances are correlated between plasma and urine, examine the most abundant urine proteins not derived from either plasma or kidney, and consider the biomarker potential of proteins associated with renal decline. This analysis forms part of the Biology and Disease-driven Human Proteome Project (B/D-HPP) and is a contribution to the Chromosome-centric Human Proteome Project (C-HPP) special issue.

iProphet: multi-level integrative analysis of shotgun proteomic data improves peptide and protein identification rates and error estimates.

The combination of tandem mass spectrometry and sequence database searching is the method of choice for the identification of peptides and the mapping of proteomes. Over the last several years, the volume of data generated in proteomic studies has increased dramatically, which challenges the computational approaches previously developed for these data. Furthermore, a multitude of search engines have been developed that identify different, overlapping subsets of the sample peptides from a particular set of tandem mass spectrometry spectra. We present iProphet, the new addition to the widely used open-source suite of proteomic data analysis tools Trans-Proteomics Pipeline. Applied in tandem with PeptideProphet, it provides more accurate representation of the multilevel nature of shotgun proteomic data. iProphet combines the evidence from multiple identifications of the same peptide sequences across different spectra, experiments, precursor ion charge states, and modified states. It also allows accurate and effective integration of the results from multiple database search engines applied to the same data. The use of iProphet in the Trans-Proteomics Pipeline increases the number of correctly identified peptides at a constant false discovery rate as compared with both PeptideProphet and another state-of-the-art tool Percolator. As the main outcome, iProphet permits the calculation of accurate posterior probabilities and false discovery rate estimates at the level of sequence identical peptide identifications, which in turn leads to more accurate probability estimates at the protein level. Fully integrated with the Trans-Proteomics Pipeline, it supports all commonly used MS instruments, search engines, and computer platforms. The performance of iProphet is demonstrated on two publicly available data sets: data from a human whole cell lysate proteome profiling experiment representative of typical proteomic data sets, and from a set of Streptococcus pyogenes experiments more representative of organism-specific composite data sets.

A comprehensive spectral assay library to quantify the Halobacterium salinarum NRC-1 proteome by DIA/SWATH-MS.

Data-Independent Acquisition (DIA) is a mass spectrometry-based method to reliably identify and reproducibly quantify large fractions of a target proteome. The peptide-centric data analysis strategy employed in DIA requires a priori generated spectral assay libraries. Such assay libraries allow to extract quantitative data in a targeted approach and have been generated for human, mouse, zebrafish, E. coli and few other organisms. However, a spectral assay library for the extreme halophilic archaeon Halobacterium salinarum NRC-1, a model organism that contributed to several notable discoveries, is not publicly available yet. Here, we report a comprehensive spectral assay library to measure 2,563 of 2,646 annotated H. salinarum NRC-1 proteins. We demonstrate the utility of this library by measuring global protein abundances over time under standard growth conditions. The H. salinarum NRC-1 library includes 21,074 distinct peptides representing 97% of the predicted proteome and provides a new, valuable resource to confidently measure and quantify any protein of this archaeon. Data and spectral assay libraries are available via ProteomeXchange (PXD042770, PXD042774) and SWATHAtlas (SAL00312-SAL00319).

Borrelia PeptideAtlas: A proteome resource of common Borrelia burgdorferi isolates for Lyme research.

Lyme disease, caused by an infection with the spirochete Borrelia burgdorferi, is the most common vector-borne disease in North America. B. burgdorferi strains harbor extensive genomic and proteomic variability and further comparison is key to understanding the spirochetes infectivity and biological impacts of identified sequence variants. To achieve this goal, both transcript and mass spectrometry (MS)-based proteomics was applied to assemble peptide datasets of laboratory strains B31, MM1, B31-ML23, infective isolates B31-5A4, B31-A3, and 297, and other public datasets, to provide a publicly available Borrelia PeptideAtlas http://www.peptideatlas.org/builds/borrelia/. Included is information on total proteome, secretome, and membrane proteome of these B. burgdorferi strains. Proteomic data collected from 35 different experiment datasets, with a total of 855 mass spectrometry runs, identified 76,936 distinct peptides at a 0.1% peptide false-discovery-rate, which map to 1,221 canonical proteins (924 core canonical and 297 noncore canonical) and covers 86% of the total base B31 proteome. The diverse proteomic information from multiple isolates with credible data presented by the Borrelia PeptideAtlas can be useful to pinpoint potential protein targets which are common to infective isolates and may be key in the infection process.