Honors & Awards

  • Innovation Award, Damon Runyon Cancer Research Foundation (1/1/11-12/31/13)
  • Medical Research Program, W. M. Keck Foundation (7/1/2013 - 6/30/2016)

Professional Education

  • Ph.D., Harvard University, Cell Biology (2006)
  • B.S., Cornell University, Biology, Genetics (1998)

Research & Scholarship

Current Research and Scholarly Interests

The Elias Lab seeks to develop and apply methods for large scale proteome characterization to solve fundamental problems in cell biology and disease. The growing field of proteomics has the lofty goal of characterizing the proteins in any isolated complex, subcellular compartment, cultured cell line or tissue. We use mass spectrometry-based approaches which can do more than simply determining the identity of a protein isolated in a polyacrylamide gel -- rapidly advancing technologies are allowing us to measure dynamic changes in protein abundances, post-translational modification states, splice isoforms, interaction partners, and localization across multiple cell states. The combination of liquid chromatography with tandem mass spectrometry (LC-MS/MS) has emerged as the most robust technology for making proteome-scale discoveries. Although the tools of proteomics have greatly advanced in recent years, many challenges lie ahead. Our lab focuses on developing new methods in protein fractionation, instrumentation, and data analysis to meet these challenges, and then applies them to studying important biomedical paradigms, including antigen presentation in cancer, and monitoring host responses to the gut microbiome..


2018-19 Courses

Stanford Advisees

Graduate and Fellowship Programs


All Publications

  • TagGraph reveals vast protein modification landscapes from large tandem mass spectrometry datasets. Nature biotechnology Devabhaktuni, A., Lin, S., Zhang, L., Swaminathan, K., Gonzalez, C. G., Olsson, N., Pearlman, S. M., Rawson, K., Elias, J. E. 2019; 37 (4): 469–79


    Although mass spectrometry is well suited to identifying thousands of potential protein post-translational modifications (PTMs), it has historically been biased towards just a few. To measure the entire set of PTMs across diverse proteomes, software must overcome the dual challenges of covering enormous search spaces and distinguishing correct from incorrect spectrum interpretations. Here, we describe TagGraph, a computational tool that overcomes both challenges with an unrestricted string-based search method that is as much as 350-fold faster than existing approaches, and a probabilistic validation model that we optimized for PTM assignments. We applied TagGraph to a published human proteomic dataset of 25million mass spectra and tripled confident spectrum identifications compared to its original analysis. We identified thousands of modification types on almost 1million sites in the proteome. We show alternative contexts for highly abundant yet understudied PTMs such as proline hydroxylation, and its unexpected association with cancer mutations. By enabling broad characterization of PTMs, TagGraph informs as to how their functions and regulation intersect.

    View details for DOI 10.1038/s41587-019-0067-5

    View details for PubMedID 30936560

  • B cell lymphomas present immunoglobulin neoantigens. Blood Khodadoust, M. S., Olsson, N., Chen, B., Sworder, B., Shree, T., Liu, C. L., Zhang, L., Czerwinski, D. K., Davis, M. M., Levy, R., Elias, J. E., Alizadeh, A. A. 2018

    View details for DOI 10.1182/blood-2018-06-845156

    View details for PubMedID 30545830

  • T-cell immunopeptidomes reveal cell subtype surface markers derived from intracellular proteins. Proteomics Olsson, N., Schultz, L. M., Zhang, L., Khodadoust, M. S., Narayan, R., Czerwinski, D. K., Levy, R., Elias, J. E. 2018


    Immunopeptidomes promise novel surface markers as ideal immunotherapy targets, but their characterization by mass spectrometry (MS) remains challenging. Until recently, cell numbers exceeding 109were needed to survey thousands of HLA ligands. Such limited analytical sensitivity has historically constrained the types of clinical specimens that can be evaluated to cell cultures or bulk tissues. Measuring immunopeptidomes from purified cell subpopulations would be preferable for many applications, particularly those evaluating rare, primary hematopoietic cell lineages. Here, we test the feasibility of immunopeptidome profiling from limited numbers of primary purified human regulatory T cells (TReg), conventional T cells (Tconv) and activated T cells. The combined T-cell immunopeptide dataset reported here contains 13,804 unique HLA ligands derived from 5,049 proteins. Of these, more than 700 HLA ligands were derived from 82 proteins that we exclusively identified from TReg-enriched cells. This study 1) demonstrates that primary, lineage-enriched T cell supbopulations recovered from single donors are compatible with immunopeptidome analysis; 2) presents new TReg-biased ligand candidates; and 3) supports immunopeptidome surveys value for revealing T cell biology that may not be apparent from expression data alone. Taken together, these findings open up new avenues for targeting TRegand abrogating their suppressive functions to treat cancer. This article is protected by copyright. All rights reserved.

    View details for DOI 10.1002/pmic.201700410

    View details for PubMedID 29493099

  • Antigen presentation profiling reveals recognition of lymphoma immunoglobulin neoantigens NATURE Khodadoust, M. S., Olsson, N., Wagar, L. E., Haabeth, O. A., Chen, B., Swaminathan, K., Rawson, K., Liu, C. L., Steiner, D., Lund, P., Rao, S., Zhang, L., Marceau, C., Stehr, H., Newman, A. M., Czerwinski, D. K., Carlton, V. E., Moorhead, M., Faham, M., Kohrt, H. E., Carette, J., Green, M. R., Davis, M. M., Levy, R., Elias, J. E., Alizadeh, A. A. 2017; 543 (7647): 723-?


    Cancer somatic mutations can generate neoantigens that distinguish malignant from normal cells. However, the personalized identification and validation of neoantigens remains a major challenge. Here we discover neoantigens in human mantle-cell lymphomas by using an integrated genomic and proteomic strategy that interrogates tumour antigen peptides presented by major histocompatibility complex (MHC) class I and class II molecules. We applied this approach to systematically characterize MHC ligands from 17 patients. Remarkably, all discovered neoantigenic peptides were exclusively derived from the lymphoma immunoglobulin heavy- or light-chain variable regions. Although we identified MHC presentation of private polymorphic germline alleles, no mutated peptides were recovered from non-immunoglobulin somatically mutated genes. Somatic mutations within the immunoglobulin variable region were almost exclusively presented by MHC class II. We isolated circulating CD4(+) T cells specific for immunoglobulin-derived neoantigens and found these cells could mediate killing of autologous lymphoma cells. These results demonstrate that an integrative approach combining MHC isolation, peptide identification, and exome sequencing is an effective platform to uncover tumour neoantigens. Application of this strategy to human lymphoma implicates immunoglobulin neoantigens as targets for lymphoma immunotherapy.

    View details for DOI 10.1038/nature21433

    View details for Web of Science ID 000397619700057

    View details for PubMedID 28329770

  • Application of de Novo Sequencing to Large-Scale Complex Proteomics Data Sets. Journal of proteome research Devabhaktuni, A., Elias, J. E. 2016; 15 (3): 732-742


    Dependent on concise, predefined protein sequence databases, traditional search algorithms perform poorly when analyzing mass spectra derived from wholly uncharacterized protein products. Conversely, de novo peptide sequencing algorithms can interpret mass spectra without relying on reference databases. However, such algorithms have been difficult to apply to complex protein mixtures, in part due to a lack of methods for automatically validating de novo sequencing results. Here, we present novel metrics for benchmarking de novo sequencing algorithm performance on large-scale proteomics data sets and present a method for accurately calibrating false discovery rates on de novo results. We also present a novel algorithm (LADS) that leverages experimentally disambiguated fragmentation spectra to boost sequencing accuracy and sensitivity. LADS improves sequencing accuracy on longer peptides relative to that of other algorithms and improves discriminability of correct and incorrect sequences. Using these advancements, we demonstrate accurate de novo identification of peptide sequences not identifiable using database search-based approaches.

    View details for DOI 10.1021/acs.jproteome.5b00861

    View details for PubMedID 26743026

  • Host-Microbiota Interactions in the Pathogenesis of Antibiotic-Associated Diseases CELL REPORTS Lichtman, J. S., Ferreyra, J. A., Ng, K. M., Smits, S. A., Sonnenburg, J. L., Elias, J. E. 2016; 14 (5): 1049-1061


    Improved understanding of the interplay between host and microbes stands to illuminate new avenues for disease diagnosis, treatment, and prevention. Here, we provide a high-resolution view of the dynamics between host and gut microbiota during antibiotic-induced intestinal microbiota depletion, opportunistic Salmonella typhimurium and Clostridium difficile pathogenesis, and recovery from these perturbed states in a mouse model. Host-centric proteome and microbial community profiles provide a nuanced longitudinal view, revealing the interdependence between host and microbiota in evolving dysbioses. Time- and condition-specific molecular and microbial signatures are evident and clearly distinguished from pathogen-independent inflammatory fingerprints. Our data reveal that mice recovering from antibiotic treatment or C. difficile infection retain lingering signatures of inflammation, despite compositional normalization of the microbiota, and host responses could be rapidly and durably relieved through fecal transplant. These experiments demonstrate insights that emerge from the combination of these orthogonal, untargeted approaches to the gastrointestinal ecosystem.

    View details for DOI 10.1016/j.celrep.2016.01.009

    View details for Web of Science ID 000369616100009

    View details for PubMedID 26832403

  • Monitoring host responses to the gut microbiota ISME JOURNAL Lichtman, J. S., Sonnenburg, J. L., Elias, J. E. 2015; 9 (9): 1908-1915


    The gastrointestinal (GI) ecosystem is increasingly understood to be a fundamental component of health, and has been identified as a new focal point for diagnosing, correcting and preventing countless disorders. Shotgun DNA sequencing has emerged as the dominant technology for determining the genetic and microbial composition of the gut microbiota. This technology has linked microbiota dysbioses to numerous GI diseases including inflammatory bowel disease, obesity and allergy, and to non-GI diseases like autism and depression. The importance of establishing causality in the deterioration of the host-microbiota relationship is well appreciated; however, discovery of candidate molecules and pathways that underlie mechanisms remains a major challenge. Targeted approaches, transcriptional assays, cytokine panels and imaging analyses, applied to animals, have yielded important insight into host responses to the microbiota. However, non-invasive, hypothesis-independent means of measuring host responses in humans are necessary to keep pace with similarly unbiased sequencing efforts that monitor microbes. Mass spectrometry-based proteomics has served this purpose in many other fields, but stool proteins exist in such diversity and dynamic range as to overwhelm conventional proteomics technologies. Focused analysis of host protein secretion into the gut lumen and monitoring proteome-level dynamics in stool provides a tractable route toward non-invasively evaluating dietary, microbial, surgical or pharmacological intervention efficacies. This review is intended to guide GI biologists and clinicians through the methods currently used to elucidate host responses in the gut, with a specific focus on mass spectrometry-based shotgun proteomics applied to the study of host protein dynamics within the GI ecosystem.The ISME Journal advance online publication, 9 June 2015; doi:10.1038/ismej.2015.93.

    View details for DOI 10.1038/ismej.2015.93

    View details for Web of Science ID 000360019500002

  • Host-centric Proteomics of Stool: A Novel Strategy Focused on intestinal Responses to the Gut Microbiota. Molecular & cellular proteomics Lichtman, J. S., Marcobal, A., Sonnenburg, J. L., Elias, J. E. 2013; 12 (11): 3310-3318


    The diverse community of microbes that inhabits the human bowel is vitally important to human health. Host-expressed proteins are essential for maintaining this mutualistic relationship and serve as reporters on the status of host-microbiota interaction. Therefore, unbiased and sensitive methods focused on host proteome characterization are needed. Herein we describe a novel method for applying shotgun proteomics to the analysis of feces, focusing on the secreted host proteome. We have conducted the most complete analysis of the extracellular mouse gut proteome to date by employing a gnotobiotic mouse model. Using mice colonized with defined microbial communities of increasing complexity or a complete human microbiota ('humanized'), we show that the complexity of the host stool proteome mirrors the complexity of microbiota composition. We further show that host responses exhibit signatures specific to the different colonization states. We demonstrate feasibility of this approach in human stool samples and provide evidence for a "core" stool proteome as well as personalized host response features. Our method provides a new avenue for noninvasive monitoring of host-microbiota interaction dynamics via host-produced proteins in stool.

    View details for DOI 10.1074/mcp.M113.029967

    View details for PubMedID 23982161

    View details for PubMedCentralID PMC3820941

  • The Phosphoproteomes of Plasmodium falciparum and Toxoplasma gondii Reveal Unusual Adaptations Within and Beyond the Parasites' Boundaries CELL HOST & MICROBE Treeck, M., Sanders, J. L., Elias, J. E., Boothroyd, J. C. 2011; 10 (4): 410-419


    Plasmodium falciparum and Toxoplasma gondii are obligate intracellular apicomplexan parasites that rapidly invade and extensively modify host cells. Protein phosphorylation is one mechanism by which these parasites can control such processes. Here we present a phosphoproteome analysis of peptides enriched from schizont stage P. falciparum and T. gondii tachyzoites that are either "intracellular" or purified away from host material. Using liquid chromatography-tandem mass spectrometry, we identified over 5,000 and 10,000 previously unknown phosphorylation sites in P. falciparum and T. gondii, respectively, revealing that protein phosphorylation is an extensively used regulation mechanism both within and beyond parasite boundaries. Unexpectedly, both parasites have phosphorylated tyrosines, and P. falciparum has unusual phosphorylation motifs that are apparently shaped by its A:T-rich genome. This data set provides important information on the role of phosphorylation in the host-pathogen interaction and clues to the evolutionary forces operating on protein phosphorylation motifs in both parasites.

    View details for DOI 10.1016/j.chom.2011.09.004

    View details for Web of Science ID 000296600700016

    View details for PubMedID 22018241

    View details for PubMedCentralID PMC3254672

  • A function for cyclin D1 in DNA repair uncovered by protein interactome analyses in human cancers NATURE Jirawatnotai, S., Hu, Y., Michowski, W., Elias, J. E., Becks, L., Bienvenu, F., Zagozdzon, A., Goswami, T., Wang, Y. E., Clark, A. B., Kunkel, T. A., van Harn, T., Xia, B., Correll, M., Quackenbush, J., Livingston, D. M., Gygi, S. P., Sicinski, P. 2011; 474 (7350): 230-234


    Cyclin D1 is a component of the core cell cycle machinery. Abnormally high levels of cyclin D1 are detected in many human cancer types. To elucidate the molecular functions of cyclin D1 in human cancers, we performed a proteomic screen for cyclin D1 protein partners in several types of human tumours. Analyses of cyclin D1 interactors revealed a network of DNA repair proteins, including RAD51, a recombinase that drives the homologous recombination process. We found that cyclin D1 directly binds RAD51, and that cyclin D1-RAD51 interaction is induced by radiation. Like RAD51, cyclin D1 is recruited to DNA damage sites in a BRCA2-dependent fashion. Reduction of cyclin D1 levels in human cancer cells impaired recruitment of RAD51 to damaged DNA, impeded the homologous recombination-mediated DNA repair, and increased sensitivity of cells to radiation in vitro and in vivo. This effect was seen in cancer cells lacking the retinoblastoma protein, which do not require D-cyclins for proliferation. These findings reveal an unexpected function of a core cell cycle protein in DNA repair and suggest that targeting cyclin D1 may be beneficial also in retinoblastoma-negative cancers which are currently thought to be unaffected by cyclin D1 inhibition.

    View details for DOI 10.1038/nature10155

    View details for Web of Science ID 000291397800054

    View details for PubMedID 21654808

  • The dynamic state of protein turnover: It's about time TRENDS IN CELL BIOLOGY Hinkson, I. V., Elias, J. E. 2011; 21 (5): 293-303


    The continual destruction and renewal of proteins that maintain cellular homeostasis has been rigorously studied since the late 1930s. Experimental techniques for measuring protein turnover have evolved to measure the dynamic regulation of key proteins and now, entire proteomes. In the past decade, the proteomics field has aimed to discover how cells adjust their proteomes to execute numerous regulatory programs in response to specific cellular and environmental cues. By combining classical biochemical techniques with modern, high-throughput technologies, researchers have begun to reveal the synthesis and degradation mechanisms that shape protein turnover on a global scale. This review examines several recent developments in protein turnover research, emphasizing the combination of metabolic labeling and mass spectrometry.

    View details for DOI 10.1016/j.tcb.2011.02.002

    View details for Web of Science ID 000291136100005

    View details for PubMedID 21474317

  • A Tissue-Specific Atlas of Mouse Protein Phosphorylation and Expression CELL Huttlin, E. L., Jedrychowski, M. P., Elias, J. E., Goswami, T., Rad, R., Beausoleil, S. A., Villen, J., Haas, W., Sowa, M. E., Gygi, S. P. 2010; 143 (7): 1174-1189


    Although most tissues in an organism are genetically identical, the biochemistry of each is optimized to fulfill its unique physiological roles, with important consequences for human health and disease. Each tissue's unique physiology requires tightly regulated gene and protein expression coordinated by specialized, phosphorylation-dependent intracellular signaling. To better understand the role of phosphorylation in maintenance of physiological differences among tissues, we performed proteomic and phosphoproteomic characterizations of nine mouse tissues. We identified 12,039 proteins, including 6296 phosphoproteins harboring nearly 36,000 phosphorylation sites. Comparing protein abundances and phosphorylation levels revealed specialized, interconnected phosphorylation networks within each tissue while suggesting that many proteins are regulated by phosphorylation independently of their expression. Our data suggest that the "typical" phosphoprotein is widely expressed yet displays variable, often tissue-specific phosphorylation that tunes protein activity to the specific needs of each tissue. We offer this dataset as an online resource for the biological research community.

    View details for DOI 10.1016/j.cell.2010.12.001

    View details for Web of Science ID 000285625400014

    View details for PubMedID 21183079

  • Target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry NATURE METHODS Elias, J. E., Gygi, S. P. 2007; 4 (3): 207-214


    Liquid chromatography and tandem mass spectrometry (LC-MS/MS) has become the preferred method for conducting large-scale surveys of proteomes. Automated interpretation of tandem mass spectrometry (MS/MS) spectra can be problematic, however, for a variety of reasons. As most sequence search engines return results even for 'unmatchable' spectra, proteome researchers must devise ways to distinguish correct from incorrect peptide identifications. The target-decoy search strategy represents a straightforward and effective way to manage this effort. Despite the apparent simplicity of this method, some controversy surrounds its successful application. Here we clarify our preferred methodology by addressing four issues based on observed decoy hit frequencies: (i) the major assumptions made with this database search strategy are reasonable; (ii) concatenated target-decoy database searches are preferable to separate target and decoy database searches; (iii) the theoretical error associated with target-decoy false positive (FP) rate measurements can be estimated; and (iv) alternate methods for constructing decoy databases are similarly effective once certain considerations are taken into account.

    View details for DOI 10.1038/nmeth1019

    View details for Web of Science ID 000244715100013

    View details for PubMedID 17327847

  • Comparative evaluation of mass spectrometry platforms used in large-scale proteomics investigations NATURE METHODS ELIAS, J. E., HAAS, W., Faherty, B. K., Gygi, S. P. 2005; 2 (9): 667-675


    Researchers have several options when designing proteomics experiments. Primary among these are choices of experimental method, instrumentation and spectral interpretation software. To evaluate these choices on a proteome scale, we compared triplicate measurements of the yeast proteome by liquid chromatography tandem mass spectrometry (LC-MS/MS) using linear ion trap (LTQ) and hybrid quadrupole time-of-flight (QqTOF; QSTAR) mass spectrometers. Acquired MS/MS spectra were interpreted with Mascot and SEQUEST algorithms with and without the requirement that all returned peptides be tryptic. Using a composite target decoy database strategy, we selected scoring criteria yielding 1% estimated false positive identifications at maximum sensitivity for all data sets, allowing reasonable comparisons between them. These comparisons indicate that Mascot and SEQUEST yield similar results for LTQ-acquired spectra but less so for QSTAR spectra. Furthermore, low reproducibility between replicate data acquisitions made on one or both instrument platforms can be exploited to increase sensitivity and confidence in large-scale protein identifications.

    View details for DOI 10.1038/NMETH785

    View details for Web of Science ID 000235261900019

    View details for PubMedID 16118637

  • Ribosomal protein RPL26 is the principal target of UFMylation. Proceedings of the National Academy of Sciences of the United States of America Walczak, C. P., Leto, D. E., Zhang, L., Riepe, C., Muller, R. Y., DaRosa, P. A., Ingolia, N. T., Elias, J. E., Kopito, R. R. 2019


    Ubiquitin fold modifier 1 (UFM1) is a small, metazoan-specific, ubiquitin-like protein modifier that is essential for embryonic development. Although loss-of-function mutations in UFM1 conjugation are linked to endoplasmic reticulum (ER) stress, neither the biological function nor the relevant cellular targets of this protein modifier are known. Here, we show that a largely uncharacterized ribosomal protein, RPL26, is the principal target of UFM1 conjugation. RPL26 UFMylation and de-UFMylation is catalyzed by enzyme complexes tethered to the cytoplasmic surface of the ER and UFMylated RPL26 is highly enriched on ER membrane-bound ribosomes and polysomes. Biochemical analysis and structural modeling establish that UFMylated RPL26 and the UFMylation machinery are in close proximity to the SEC61 translocon, suggesting that this modification plays a direct role in cotranslational protein translocation into the ER. These data suggest that UFMylation is a ribosomal modification specialized to facilitate metazoan-specific protein biogenesis at the ER.

    View details for DOI 10.1073/pnas.1816202116

    View details for PubMedID 30626644

  • METTL13 Methylation of eEF1A Increases Translational Output to Promote Tumorigenesis. Cell Liu, S., Hausmann, S., Carlson, S. M., Fuentes, M. E., Francis, J. W., Pillai, R., Lofgren, S. M., Hulea, L., Tandoc, K., Lu, J., Li, A., Nguyen, N. D., Caporicci, M., Kim, M. P., Maitra, A., Wang, H., Wistuba, I. I., Porco, J. A., Bassik, M. C., Elias, J. E., Song, J., Topisirovic, I., Van Rechem, C., Mazur, P. K., Gozani, O. 2018


    Increased protein synthesis plays an etiologic role in diverse cancers. Here, we demonstrate that METTL13 (methyltransferase-like 13) dimethylation of eEF1A (eukaryotic elongation factor 1A) lysine 55 (eEF1AK55me2) is utilized by Ras-driven cancers to increase translational output and promote tumorigenesis invivo. METTL13-catalyzed eEF1A methylation increases eEF1A's intrinsic GTPase activity invitro and protein production in cells. METTL13 and eEF1AK55me2 levels are upregulated in cancer and negatively correlate with pancreatic and lung cancer patient survival. METTL13 deletion and eEF1AK55me2 loss dramatically reduce Ras-driven neoplastic growth in mouse models and in patient-derived xenografts (PDXs) from primary pancreatic and lung tumors. Finally, METTL13 depletion renders PDX tumors hypersensitive to drugs thattarget growth-signaling pathways. Together, our work uncovers a mechanism by which lethal cancers become dependent on the METTL13-eEF1AK55me2 axis to meet their elevated protein synthesis requirement and suggests that METTL13 inhibition may constitute a targetable vulnerability of tumors driven by aberrant Ras signaling.

    View details for DOI 10.1016/j.cell.2018.11.038

    View details for PubMedID 30612740

  • Genome-wide CRISPR Analysis Identifies Substrate-Specific Conjugation Modules in ER-Associated Degradation. Molecular cell Leto, D. E., Morgens, D. W., Zhang, L., Walczak, C. P., Elias, J. E., Bassik, M. C., Kopito, R. R. 2018


    The ubiquitin proteasome system (UPS) maintains the integrity of the proteome by selectively degrading misfolded or mis-assembled proteins, but the rules that govern how conformationally defective proteins in the secretory pathway are selected from the structurally and topologically diverse constellation of correctly folded membrane and secretory proteins for efficient degradation by cytosolic proteasomes is not well understood. Here, we combine parallel pooled genome-wide CRISPR-Cas9 forward genetic screening with a highly quantitative and sensitive protein turnover assay to discover a previously undescribed collaboration between membrane-embedded cytoplasmic ubiquitin E3 ligases to conjugate heterotypic branched or mixed ubiquitin (Ub) chains on substrates of endoplasmic-reticulum-associated degradation (ERAD). These findings demonstrate that parallel CRISPR analysis can be used to deconvolve highly complex cell biological processes and identify new biochemical pathways in protein quality control.

    View details for DOI 10.1016/j.molcel.2018.11.015

    View details for PubMedID 30581143

  • Integrative proteomics and bioinformatic prediction enable a high-confidence apicoplast proteome in malaria parasites. PLoS biology Boucher, M. J., Ghosh, S., Zhang, L., Lal, A., Jang, S. W., Ju, A., Zhang, S., Wang, X., Ralph, S. A., Zou, J., Elias, J. E., Yeh, E. 2018; 16 (9): e2005895


    Malaria parasites (Plasmodium spp.) and related apicomplexan pathogens contain a nonphotosynthetic plastid called the apicoplast. Derived from an unusual secondary eukaryote-eukaryote endosymbiosis, the apicoplast is a fascinating organelle whose function and biogenesis rely on a complex amalgamation of bacterial and algal pathways. Because these pathways are distinct from the human host, the apicoplast is an excellent source of novel antimalarial targets. Despite its biomedical importance and evolutionary significance, the absence of a reliable apicoplast proteome has limited most studies to the handful of pathways identified by homology to bacteria or primary chloroplasts, precluding our ability to study the most novel apicoplast pathways. Here, we combine proximity biotinylation-based proteomics (BioID) and a new machine learning algorithm to generate a high-confidence apicoplast proteome consisting of 346 proteins. Critically, the high accuracy of this proteome significantly outperforms previous prediction-based methods and extends beyond other BioID studies of unique parasite compartments. Half of identified proteins have unknown function, and 77% are predicted to be important for normal blood-stage growth. We validate the apicoplast localization of a subset of novel proteins and show that an ATP-binding cassette protein ABCF1 is essential for blood-stage survival and plays a previously unknown role in apicoplast biogenesis. These findings indicate critical organellar functions for newly discovered apicoplast proteins. The apicoplast proteome will be an important resource for elucidating unique pathways derived from secondary endosymbiosis and prioritizing antimalarial drug targets.

    View details for DOI 10.1371/journal.pbio.2005895

    View details for PubMedID 30212465

  • Developmental phosphoproteomics identifies the kinase CK2 as a driver of Hedgehog signaling and a therapeutic target in medulloblastoma. Science signaling Purzner, T., Purzner, J., Buckstaff, T., Cozza, G., Gholamin, S., Rusert, J. M., Hartl, T. A., Sanders, J., Conley, N., Ge, X., Langan, M., Ramaswamy, V., Ellis, L., Litzenburger, U., Bolin, S., Theruvath, J., Nitta, R., Qi, L., Li, X., Li, G., Taylor, M. D., Wechsler-Reya, R. J., Pinna, L. A., Cho, Y., Fuller, M. T., Elias, J. E., Scott, M. P. 2018; 11 (547)


    A major limitation of targeted cancer therapy is the rapid emergence of drug resistance, which often arises through mutations at or downstream of the drug target or through intrinsic resistance of subpopulations of tumor cells. Medulloblastoma (MB), the most common pediatric brain tumor, is no exception, and MBs that are driven by sonic hedgehog (SHH) signaling are particularly aggressive and drug-resistant. To find new drug targets and therapeutics for MB that may be less susceptible to common resistance mechanisms, we used a developmental phosphoproteomics approach in murine granule neuron precursors (GNPs), the developmental cell of origin of MB. The protein kinase CK2 emerged as a driver of hundreds of phosphorylation events during the proliferative, MB-like stage of GNP growth, including the phosphorylation of three of the eight proteins commonly amplified in MB. CK2 was critical to the stabilization and activity of the transcription factor GLI2, a late downstream effector in SHH signaling. CK2 inhibitors decreased the viability of primary SHH-type MB patient cells in culture and blocked the growth of murine MB tumors that were resistant to currently available Hh inhibitors, thereby extending the survival of tumor-bearing mice. Because of structural interactions, one CK2 inhibitor (CX-4945) inhibited both wild-type and mutant CK2, indicating that this drug may avoid at least one common mode of acquired resistance. These findings suggest that CK2 inhibitors may be effective for treating patients with MB and show how phosphoproteomics may be used to gain insight into developmental biology and pathology.

    View details for DOI 10.1126/scisignal.aau5147

    View details for PubMedID 30206138

  • Proteomic analysis of monolayer-integrated proteins on lipid droplets identifies amphipathic interfacial alpha-helical membrane anchors. Proceedings of the National Academy of Sciences of the United States of America Pataki, C. I., Rodrigues, J., Zhang, L., Qian, J., Efron, B., Hastie, T., Elias, J. E., Levitt, M., Kopito, R. R. 2018


    Despite not spanning phospholipid bilayers, monotopic integral proteins (MIPs) play critical roles in organizing biochemical reactions on membrane surfaces. Defining the structural basis by which these proteins are anchored to membranes has been hampered by the paucity of unambiguously identified MIPs and a lack of computational tools that accurately distinguish monolayer-integrating motifs from bilayer-spanning transmembrane domains (TMDs). We used quantitative proteomics and statistical modeling to identify 87 high-confidence candidate MIPs in lipid droplets, including 21 proteins with predicted TMDs that cannot be accommodated in these monolayer-enveloped organelles. Systematic cysteine-scanning mutagenesis showed the predicted TMD of one candidate MIP, DHRS3, to be a partially buried amphipathic alpha-helix in both lipid droplet monolayers and the cytoplasmic leaflet of endoplasmic reticulum membrane bilayers. Coarse-grained molecular dynamics simulations support these observations, suggesting that this helix is most stable at the solvent-membrane interface. The simulations also predicted similar interfacial amphipathic helices when applied to seven additional MIPs from our dataset. Our findings suggest that interfacial helices may be a common motif by which MIPs are integrated into membranes, and provide high-throughput methods to identify and study MIPs.

    View details for DOI 10.1073/pnas.1807981115

    View details for PubMedID 30104359

  • Transient Osmotic Perturbation Causes Long-Term Alteration to the Gut Microbiota. Cell Tropini, C., Moss, E. L., Merrill, B. D., Ng, K. M., Higginbottom, S. K., Casavant, E. P., Gonzalez, C. G., Fremin, B., Bouley, D. M., Elias, J. E., Bhatt, A. S., Huang, K. C., Sonnenburg, J. L. 2018; 173 (7): 1742


    Osmotic diarrhea is a prevalent condition in humans caused by food intolerance, malabsorption, and widespread laxative use. Here, we assess the resilience of the gut ecosystem to osmotic perturbation at multiple length and timescales using mice as model hosts. Osmotic stress caused reproducible extinction of highly abundant taxa and expansion of less prevalent members in human and mouse microbiotas. Quantitative imaging revealed decimation of the mucus barrier during osmotic perturbation, followed by recovery. The immune system exhibited temporary changes in cytokine levels and a lasting IgG response against commensal bacteria. Increased osmolality prevented growth of commensal strains invitro, revealing one mechanism contributing to extinction. Environmental availability of microbiota members mitigated extinction events, demonstrating how species reintroduction can affect community resilience. Our findings (1) demonstrate that even mild osmotic diarrhea can cause lasting changes to the microbiota and host and (2) lay the foundation for interventions that increase system-wide resilience.

    View details for DOI 10.1016/j.cell.2018.05.008

    View details for PubMedID 29906449

  • Multiple Click-Selective tRNA Synthetases Expand Mammalian Cell-Specific Proteomics JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Yang, A. C., Du Bois, H., Olsson, N., Gate, D., Lehallier, B., Berdnik, D., Brewer, K. D., Bertozzi, C. R., Elias, J. E., Wyss-Coray, T. 2018; 140 (23): 7046–51


    Bioorthogonal tools enable cell-type-specific proteomics, a prerequisite to understanding biological processes in multicellular organisms. Here we report two engineered aminoacyl-tRNA synthetases for mammalian bioorthogonal labeling: a tyrosyl ( ScTyrY43G) and a phenylalanyl ( MmPheT413G) tRNA synthetase that incorporate azide-bearing noncanonical amino acids specifically into the nascent proteomes of host cells. Azide-labeled proteins are chemoselectively tagged via azide-alkyne cycloadditions with fluorophores for imaging or affinity resins for mass spectrometric characterization. Both mutant synthetases label human, hamster, and mouse cell line proteins and selectively activate their azido-bearing amino acids over 10-fold above the canonical. ScTyrY43G and MmPheT413G label overlapping but distinct proteomes in human cell lines, with broader proteome coverage upon their coexpression. In mice, ScTyrY43G and MmPheT413G label the melanoma tumor proteome and plasma secretome. This work furnishes new tools for mammalian residue-specific bioorthogonal chemistry, and enables more robust and comprehensive cell-type-specific proteomics in live mammals.

    View details for DOI 10.1021/jacs.8b03074

    View details for Web of Science ID 000435525500001

    View details for PubMedID 29775058

  • Minimal Information About an Immuno-Peptidomics Experiment (MIAIPE) PROTEOMICS Lill, J. R., van Veelen, P. A., Tenzer, S., Admon, A., Caron, E., Elias, J. E., Heck, A. R., Marcilla, M., Marino, F., Mueller, M., Peters, B., Purcell, A., Sette, A., Sturm, T., Ternette, N., Vizcaino, J., Bassani-Sternberg, M. 2018; 18 (12): e1800110


    Minimal information about an immuno-peptidomics experiment (MIAIPE) is an initiative of the members of the Human Immuno-Peptidome Project (HIPP), an international program organized by the Human Proteome Organization (HUPO). The aim of the MIAIPE guidelines is to deliver technical guidelines representing the minimal information required to sufficiently support the evaluation and interpretation of immunopeptidomics experiments. The MIAIPE document has been designed to report essential information about sample preparation, mass spectrometric measurement, and associated mass spectrometry (MS)-related bioinformatics aspects that are unique to immunopeptidomics and may not be covered by the general proteomics MIAPE (minimal information about a proteomics experiment) guidelines.

    View details for DOI 10.1002/pmic.201800110

    View details for Web of Science ID 000436407900012

    View details for PubMedID 29791771

  • Disseminating Metaproteomic Informatics Capabilities and Knowledge Using the Galaxy-P Framework PROTEOMES Blank, C., Easterly, C., Gruening, B., Johnson, J., Kolmeder, C. A., Kumar, P., May, D., Mehta, S., Mesuere, B., Brown, Z., Elias, J. E., Hervey, W., McGowan, T., Muth, T., Nunn, B. L., Rudney, J., Tanca, A., Griffin, T. J., Jagtap, P. D. 2018; 6 (1)


    The impact of microbial communities, also known as the microbiome, on human health and the environment is receiving increased attention. Studying translated gene products (proteins) and comparing metaproteomic profiles may elucidate how microbiomes respond to specific environmental stimuli, and interact with host organisms. Characterizing proteins expressed by a complex microbiome and interpreting their functional signature requires sophisticated informatics tools and workflows tailored to metaproteomics. Additionally, there is a need to disseminate these informatics resources to researchers undertaking metaproteomic studies, who could use them to make new and important discoveries in microbiome research. The Galaxy for proteomics platform (Galaxy-P) offers an open source, web-based bioinformatics platform for disseminating metaproteomics software and workflows. Within this platform, we have developed easily-accessible and documented metaproteomic software tools and workflows aimed at training researchers in their operation and disseminating the tools for more widespread use. The modular workflows encompass the core requirements of metaproteomic informatics: (a) database generation; (b) peptide spectral matching; (c) taxonomic analysis and (d) functional analysis. Much of the software available via the Galaxy-P platform was selected, packaged and deployed through an online metaproteomics "Contribution Fest" undertaken by a unique consortium of expert software developers and users from the metaproteomics research community, who have co-authored this manuscript. These resources are documented on GitHub and freely available through the Galaxy Toolshed, as well as a publicly accessible metaproteomics gateway Galaxy instance. These documented workflows are well suited for the training of novice metaproteomics researchers, through online resources such as the Galaxy Training Network, as well as hands-on training workshops. Here, we describe the metaproteomics tools available within these Galaxy-based resources, as well as the process by which they were selected and implemented in our community-based work. We hope this description will increase access to and utilization of metaproteomics tools, as well as offer a framework for continued community-based development and dissemination of cutting edge metaproteomics software.

    View details for DOI 10.3390/proteomes6010007

    View details for Web of Science ID 000428563100006

    View details for PubMedID 29385081

    View details for PubMedCentralID PMC5874766

  • SETD3 is an actin histidine methyltransferase that prevents primary dystocia. Nature Wilkinson, A. W., Diep, J., Dai, S., Liu, S., Ooi, Y. S., Song, D., Li, T. M., Horton, J. R., Zhang, X., Liu, C., Trivedi, D. V., Ruppel, K. M., Vilches-Moure, J. G., Casey, K. M., Mak, J., Cowan, T., Elias, J. E., Nagamine, C. M., Spudich, J. A., Cheng, X., Carette, J. E., Gozani, O. 2018


    For more than 50 years, the methylation of mammalian actin at histidine 73 has been known to occur1. Despite the pervasiveness of His73 methylation, which we find is conserved in several model animals and plants, its function remains unclear and the enzyme that generates this modification is unknown. Here we identify SET domain protein 3 (SETD3) as the physiological actin His73 methyltransferase. Structural studies reveal that an extensive network of interactions clamps the actin peptide onto the surface of SETD3 to orient His73 correctly within the catalytic pocket and to facilitate methyl transfer. His73 methylation reduces the nucleotide-exchange rate on actin monomers and modestly accelerates the assembly of actin filaments. Mice that lack SETD3 show complete loss of actin His73 methylation in several tissues, and quantitative proteomics analysis shows that actin His73 methylation is the only detectable physiological substrate of SETD3. SETD3-deficient female mice have severely decreased litter sizes owing to primary maternal dystocia that is refractory to ecbolic induction agents. Furthermore, depletion of SETD3 impairs signal-induced contraction in primary human uterine smooth muscle cells. Together, our results identify a mammalian histidine methyltransferase and uncover a pivotal role for SETD3 and actin His73 methylation in the regulation of smooth muscle contractility. Our data also support the broader hypothesis that protein histidine methylation acts as a common regulatory mechanism.

    View details for DOI 10.1038/s41586-018-0821-8

    View details for PubMedID 30626964

  • RBM25 is a global splicing factor promoting inclusion of alternatively spliced exons and is itself regulated by lysine mono-methylation JOURNAL OF BIOLOGICAL CHEMISTRY Carlson, S. M., Soulette, C. M., Yang, Z., Elias, J. E., Brooks, A. N., Gozani, O. 2017; 292 (32): 13381–90


    In eukaryotes, precursor mRNA (pre-mRNA) splicing removes non-coding intron sequences to produce mature mRNA. This removal is controlled in part by RNA-binding proteins that regulate alternative splicing decisions through interactions with the splicing machinery. RNA binding motif protein 25 (RBM25) is a putative splicing factor strongly conserved across eukaryotic lineages. However, the role of RBM25 in global splicing regulation and its cellular functions are unknown. Here we show that RBM25 is required for the viability of multiple human cell lines, suggesting that it could play a key role in pre-mRNA splicing. Indeed, transcriptome-wide analysis of splicing events demonstrated that RBM25 regulates a large fraction of alternatively spliced exons throughout the human genome. Moreover, proteomic analysis indicated that RBM25 interacts with components of the early spliceosome and regulators of alternative splicing. Previously, we identified an RBM25 species that is mono-methylated at lysine 77 (RBM25K77me1), and here we used quantitative mass spectrometry to show that RBM25K77me1 is abundant in multiple human cell lines. We also identified a region of RBM25 spanning Lys-77 that binds with high affinity to serine- and arginine-rich splicing factor 2 (SRSF2), a crucial protein in exon definition, but only when Lys-77 is unmethylated. Together, our findings uncover a pivotal role for RBM25 as an essential regulator of alternative splicing and reveal a new potential mechanism for regulation of pre-mRNA splicing by lysine methylation of a splicing factor.

    View details for DOI 10.1074/jbc.M117.784371

    View details for Web of Science ID 000407465300024

    View details for PubMedID 28655759

    View details for PubMedCentralID PMC5555197

  • On the hunt for cancer neoantigens: is mass spectrometry the solution? Elias, J., Olsson, N., Khodadoust, M., Wagar, L., Swaminathan, K., Green, M. R., Davis, M. M., Levy, R., Alizadeh, A. A. AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC. 2017: S65
  • Characterization of protein complexes of the endoplasmic reticulum-associated degradation E3 ubiquitin ligase Hrd1. journal of biological chemistry Hwang, J., Walczak, C. P., Shaler, T. A., Olzmann, J. A., Zhang, L., Elias, J. E., Kopito, R. R. 2017; 292 (22): 9104-9116


    Hrd1 is the core structural component of a large endoplasmic reticulum membrane-embedded protein complex that coordinates the destruction of folding-defective proteins in the early secretory pathway. Defining the composition, dynamics, and ultimately, the structure of the Hrd1 complex is a crucial step in understanding the molecular basis of glycoprotein quality control but has been hampered by the lack of suitable techniques to interrogate this complex under native conditions. In this study we used genome editing to generate clonal HEK293 (Hrd1.KI) cells harboring a homozygous insertion of a small tandem affinity tag knocked into the endogenous Hrd1 locus. We found that steady-state levels of tagged Hrd1 in these cells are indistinguishable from those of Hrd1 in unmodified cells and that the tagged variant is functional in supporting the degradation of well characterized luminal and membrane substrates. Analysis of detergent-solubilized Hrd1.KI cells indicates that the composition and stoichiometry of Hrd1 complexes are strongly influenced by Hrd1 expression levels. Analysis of affinity-captured Hrd1 complexes from these cells by size-exclusion chromatography, immunodepletion, and absolute quantification mass spectrometry identified two major high-molecular-mass complexes with distinct sets of interacting proteins and variable stoichiometries, suggesting a hitherto unrecognized heterogeneity in the functional units of Hrd1-mediated protein degradation.

    View details for DOI 10.1074/jbc.M117.785055

    View details for PubMedID 28411238

  • From mystery to mechanism: can proteomics build systems-level understanding of our gut microbes? Expert review of proteomics Gonzalez, C. G., Zhang, L., Elias, J. E. 2017: 1-4

    View details for DOI 10.1080/14789450.2017.1311211

    View details for PubMedID 28335651

  • The Prenylated Proteome of Plasmodium falciparum Reveals Pathogen-specific Prenylation Activity and Drug Mechanism-of-action MOLECULAR & CELLULAR PROTEOMICS Gisselberg, J. E., Zhang, L., Elias, J. E., Yeh, E. 2017; 16 (4): S54-S64


    Plasmodium parasites contain several unique membrane compartments in which prenylated proteins may play important roles in pathogenesis. Protein prenylation has also been proposed as an antimalarial drug target because farnesyltransferase inhibitors cause potent growth inhibition of blood-stage Plasmodium However, the specific prenylated proteins that mediate antimalarial activity have yet to be identified. Given the potential for new parasite biology and elucidating drug mechanism-of-action, we performed a large-scale identification of the prenylated proteome in blood-stage P. falciparum parasites using an alkyne-labeled prenyl analog to specifically enrich parasite prenylated proteins. Twenty high-confidence candidates were identified, including several examples of pathogen-specific prenylation activity. One unique parasite prenylated protein was FYVE-containing coiled-coil protein (FCP), which is only conserved in Plasmodium and related Apicomplexan parasites and localizes to the parasite food vacuole. Targeting of FCP to this parasite-specific compartment was dependent on prenylation of its CaaX motif, as mutation of the prenylation site caused cytosolic mislocalization. We also showed that PfRab5b, which lacks C-terminal cysteines that are the only known site of Rab GTPase modification, is prenylated. Finally, we show that the THQ class of farnesyltransferase inhibitors abolishes FCP prenylation and causes its mislocalization, providing the first demonstration of a specific prenylated protein disrupted by antimalarial farnesyl transferase inhibitors. Altogether, these findings identify prenylated proteins that reveal unique parasite biology and are useful for evaluating prenyltransferase inhibitors for antimalarial drug development.

    View details for DOI 10.1074/mcp.M116.064550

    View details for Web of Science ID 000398812800006

    View details for PubMedCentralID PMC5393391

  • Seasonal cycling in the gut microbiome of the Hadza hunter-gatherers of Tanzania. Science (New York, N.Y.) Smits, S. A., Leach, J., Sonnenburg, E. D., Gonzalez, C. G., Lichtman, J. S., Reid, G., Knight, R., Manjurano, A., Changalucha, J., Elias, J. E., Dominguez-Bello, M. G., Sonnenburg, J. L. 2017; 357 (6353): 802–6


    Although humans have cospeciated with their gut-resident microbes, it is difficult to infer features of our ancestral microbiome. Here, we examine the microbiome profile of 350 stool samples collected longitudinally for more than a year from the Hadza hunter-gatherers of Tanzania. The data reveal annual cyclic reconfiguration of the microbiome, in which some taxa become undetectable only to reappear in a subsequent season. Comparison of the Hadza data set with data collected from 18 populations in 16 countries with varying lifestyles reveals that gut community membership corresponds to modernization: Notably, the taxa within the Hadza that are the most seasonally volatile similarly differentiate industrialized and traditional populations. These data indicate that some dynamic lineages of microbes have decreased in prevalence and abundance in modernized populations.

    View details for DOI 10.1126/science.aan4834

    View details for PubMedID 28839072

  • Isotope-targeted glycoproteomics (IsoTaG) analysis of sialylated N- and O-glycopeptides on an Orbitrap Fusion Tribrid using azido and alkynyl sugars ANALYTICAL AND BIOANALYTICAL CHEMISTRY Woo, C. M., Felix, A., Zhang, L., Elias, J. E., Bertozzi, C. R. 2017; 409 (2): 579-588


    Protein glycosylation is a post-translational modification (PTM) responsible for many aspects of proteomic diversity and biological regulation. Assignment of intact glycan structures to specific protein attachment sites is a critical step towards elucidating the function encoded in the glycome. Previously, we developed isotope-targeted glycoproteomics (IsoTaG) as a mass-independent mass spectrometry method to characterize azide-labeled intact glycopeptides from complex proteomes. Here, we extend the IsoTaG approach with the use of alkynyl sugars as metabolic labels and employ new probes in analysis of the sialylated glycoproteome from PC-3 cells. Using an Orbitrap Fusion Tribrid mass spectrometer, we identified 699 intact glycopeptides from 192 glycoproteins. These intact glycopeptides represent a total of eight sialylated glycan structures across 126 N- and 576 O-glycopeptides. IsoTaG is therefore an effective platform for identification of intact glycopeptides labeled by alkynyl or azido sugars and will facilitate further studies of the glycoproteome.

    View details for DOI 10.1007/s00216-016-9934-9

    View details for Web of Science ID 000391364200021

  • Building proteomic tool boxes to monitor MHC class I and class II peptides PROTEOMICS Schumacher, F., Delamarre, L., Jhunjhunwala, S., Modrusan, Z., Phung, Q. T., Elias, J. E., Lill, J. R. 2017; 17 (1-2)


    Major histocompatibility complex Class I (MHCI) and Class II (MHCII) presented peptides powerfully modulate T cell immunity and play a vital role in generating effective anti-tumor and anti-viral immune responses in mammals. Characterizing these MHCI or MHCII presented peptides can help generate therapeutic treatments, afford information on T cell mediated biomarkers, provide insight into disease progression, and reduce adverse anti-drug side effects from engineered biotherapeutics. Here, we explore the tools and techniques commonly employed to discover both MHCI- and MHCII-presented peptides. We describe complementary strategies that enhance the characterization of these peptides and the informatics tools employed for both predicting and characterizing MHCI- and MHCII-presented epitopes. The evolution of methodologies for isolating MHC-presented peptides is discussed, as are the mass spectrometric workflows that can be employed for their characterization. We provide a perspective on where this field is headed, and how these tools may be applicable to the discovery and monitoring of epitopes in a variety of scenarios.

    View details for DOI 10.1002/pmic.201600061

    View details for Web of Science ID 000394432900015

    View details for PubMedID 27928884

  • Relative Protein Quantification Using Tandem Mass Tag Mass Spectrometry. Methods in molecular biology (Clifton, N.J.) Zhang, L., Elias, J. E. 2017; 1550: 185-198


    Measuring protein changes over time or following stimuli is one of the important tasks of proteomics. In the past decade, several strategies have been developed for the relative quantification of proteins using mass spectrometry (MS). Isobaric labeling strategies for relative quantitative proteomics allow for parallel multiplexing of quantitative experiments. With this technique, multiple peptide samples are chemically labeled with isobaric chemical tag variants and each variant has the same molecular structure and mass. Each variant, however, is designed to produce a unique "reporter ion" when fragmented inside a mass spectrometer. Once peptide samples are labeled, combined, and analyzed using MS, differentially labeled peptides are indistinguishable in a first, MS spectrum of intact peptides. However, since each tag variant contains a labile component with different mass, "reporter ions" can be generated and recorded in a subsequent MS(2) spectrum. Intensities from each variant are recorded to represent the relative abundances of the peptide in each sample. Isobaric tags for relative and absolute quantitation (iTRAQ) and tandem mass tags (TMT) are commercially available reagents for performing this technique. Here, we describe the general workflow of relative quantification of proteins using TMT by MS(2), or an additional MS(3) spectrum.

    View details for DOI 10.1007/978-1-4939-6747-6_14

    View details for PubMedID 28188531

  • The prenylated proteome of Plasmodium falciparum reveals pathogen-specific prenylation activity and drug mechanism-of-action. Molecular & cellular proteomics Gisselberg, J. E., Zhang, L., Elias, J. E., Yeh, E. 2016


    Plasmodium parasites contain several unique membrane compartments in which prenylated proteins may play important roles in pathogenesis. Protein prenylation has also been proposed as an antimalarial drug target because farnesyltransferase inhibitors cause potent growth inhibition of blood-stage Plasmodium However, the specific prenylated proteins that mediate antimalarial activity have yet to be identified. Given the potential for new parasite biology and elucidating drug mechanism-of-action, we performed a large-scale identification of the prenylated proteome in blood-stage P. falciparum parasites using an alkyne-labeled prenyl analog to specifically enrich parasite prenylated proteins. Twenty high-confidence candidates were identified, including several examples of pathogen-specific prenylation activity. One unique parasite prenylated protein was FYVE-containing coiled-coil protein (FCP), which is only conserved in Plasmodium and related Apicomplexan parasites and localizes to the parasite food vacuole. Targeting of FCP to this parasite-specific compartment was dependent on prenylation of its CaaX motif, as mutation of the prenylation site caused cytosolic mislocalization. We also showed that PfRab5b, which lacks C-terminal cysteines that are the only known site of Rab GTPase modification, is prenylated. Finally, we show that the THQ class of farnesyltransferase inhibitors abolishes FCP prenylation and causes its mislocalization, providing the first demonstration of a specific prenylated protein disrupted by antimalarial farnesyl transferase inhibitors. Altogether, these findings identify prenylated proteins that reveal unique parasite biology and are useful for evaluating prenyltransferase inhibitors for antimalarial drug development.

    View details for DOI 10.1074/mcp.M116.064550

    View details for PubMedID 28040698

    View details for PubMedCentralID PMC5393391

  • Antigen Presentation Profiling Reveals T-Cell Recognition of Lymphoma Immunoglobulin Neoantigens Khodadoust, M., Olsson, N., Wagar, L., Chen, B., Rawson, K., Liu, C., Steiner, D., Rao, S., Zhang, L., Stehr, H., Newman, A. M., Czerwinski, D. K., Carlton, V., Moorhead, M., Faham, M., Kohrt, H., Green, M. R., Davis, M. M., Levy, R., Elias, J., Alizadeh, A. A. AMER SOC HEMATOLOGY. 2016
  • Global Analysis of O-GlcNAc Glycoproteins in Activated Human T Cells. Journal of immunology Lund, P. J., Elias, J. E., Davis, M. M. 2016; 197 (8): 3086-3098


    T cell activation in response to Ag is largely regulated by protein posttranslational modifications. Although phosphorylation has been extensively characterized in T cells, much less is known about the glycosylation of serine/threonine residues by O-linked N-acetylglucosamine (O-GlcNAc). Given that O-GlcNAc appears to regulate cell signaling pathways and protein activity similarly to phosphorylation, we performed a comprehensive analysis of O-GlcNAc during T cell activation to address the functional importance of this modification and to identify the modified proteins. Activation of T cells through the TCR resulted in a global elevation of O-GlcNAc levels and in the absence of O-GlcNAc, IL-2 production and proliferation were compromised. T cell activation also led to changes in the relative expression of O-GlcNAc transferase (OGT) isoforms and accumulation of OGT at the immunological synapse of murine T cells. Using a glycoproteomics approach, we identified >200 O-GlcNAc proteins in human T cells. Many of the identified proteins had a functional relationship to RNA metabolism, and consistent with a connection between O-GlcNAc and RNA, inhibition of OGT impaired nascent RNA synthesis upon T cell activation. Overall, our studies provide a global analysis of O-GlcNAc dynamics during T cell activation and the first characterization, to our knowledge, of the O-GlcNAc glycoproteome in human T cells.

    View details for PubMedID 27655845

    View details for PubMedCentralID PMC5055199

  • Immuno-proteomic interrogation of antigen presentation during dengue infection reveals novel and HLA haplotype-specific MHC-I antigens Swaminathan, K., Lund, P., Olsson, N., Marceau, C., Carette, J., Davis, M., Elias, J. WILEY-BLACKWELL. 2016: 889
  • Genetic dissection of Flaviviridae host factors through genome-scale CRISPR screens NATURE Marceau, C. D., Puschnik, A. S., Majzoub, K., Ooi, Y. S., Brewer, S. M., Fuchs, G., Swaminathan, K., Mata, M. A., Elias, J. E., Sarnow, P., Carette, J. E. 2016; 535 (7610): 159-?


    The Flaviviridae are a family of viruses that cause severe human diseases. For example, dengue virus (DENV) is a rapidly emerging pathogen causing an estimated 100 million symptomatic infections annually worldwide. No approved antivirals are available to date and clinical trials with a tetravalent dengue vaccine showed disappointingly low protection rates. Hepatitis C virus (HCV) also remains a major medical problem, with 160 million chronically infected patients worldwide and only expensive treatments available. Despite distinct differences in their pathogenesis and modes of transmission, the two viruses share common replication strategies. A detailed understanding of the host functions that determine viral infection is lacking. Here we use a pooled CRISPR genetic screening strategy to comprehensively dissect host factors required for these two highly important Flaviviridae members. For DENV, we identified endoplasmic-reticulum (ER)-associated multi-protein complexes involved in signal sequence recognition, N-linked glycosylation and ER-associated degradation. DENV replication was nearly completely abrogated in cells deficient in the oligosaccharyltransferase (OST) complex. Mechanistic studies pinpointed viral RNA replication and not entry or translation as the crucial step requiring the OST complex. Moreover, we show that viral non-structural proteins bind to the OST complex. The identified ER-associated protein complexes were also important for infection by other mosquito-borne flaviviruses including Zika virus, an emerging pathogen causing severe birth defects. By contrast, the most significant genes identified in the HCV screen were distinct and included viral receptors, RNA-binding proteins and enzymes involved in metabolism. We found an unexpected link between intracellular flavin adenine dinucleotide (FAD) levels and HCV replication. This study shows notable divergence in host-depenency factors between DENV and HCV, and illuminates new host targets for antiviral therapy.

    View details for DOI 10.1038/nature18631

    View details for Web of Science ID 000379015600044

    View details for PubMedID 27383987

    View details for PubMedCentralID PMC4964798

  • The effect of microbial colonization on the host proteome varies by gastrointestinal location ISME JOURNAL Lichtman, J. S., Alsentzer, E., Jaffe, M., Sprockett, D., Masutani, E., Ikwa, E., Fragiadakis, G. K., Clifford, D., Huang, B. E., Sonnenburg, J. L., Huang, K. C., Elias, J. E. 2016; 10 (5): 1170-1181


    Endogenous intestinal microbiota have wide-ranging and largely uncharacterized effects on host physiology. Here, we used reverse-phase liquid chromatography-coupled tandem mass spectrometry to define the mouse intestinal proteome in the stomach, jejunum, ileum, cecum and proximal colon under three colonization states: germ-free (GF), monocolonized with Bacteroides thetaiotaomicron and conventionally raised (CR). Our analysis revealed distinct proteomic abundance profiles along the gastrointestinal (GI) tract. Unsupervised clustering showed that host protein abundance primarily depended on GI location rather than colonization state and specific proteins and functions that defined these locations were identified by random forest classifications. K-means clustering of protein abundance across locations revealed substantial differences in host protein production between CR mice relative to GF and monocolonized mice. Finally, comparison with fecal proteomic data sets suggested that the identities of stool proteins are not biased to any region of the GI tract, but are substantially impacted by the microbiota in the distal colon.

    View details for DOI 10.1038/ismej.2015.187

    View details for Web of Science ID 000374377200014

    View details for PubMedID 26574685

    View details for PubMedCentralID PMC5029216

  • A PWWP Domain of Histone-Lysine N-Methyltransferase NSD2 Binds to Dimethylated Lys-36 of Histone H3 and Regulates NSD2 Function at Chromatin JOURNAL OF BIOLOGICAL CHEMISTRY Sankaran, S. M., Wilkinson, A. W., Elias, J. E., Gozani, O. 2016; 291 (16): 8465-8474


    The readout of histone modifications plays a critical role in chromatin-regulated processes. Dimethylation at Lys-36 on histone H3 (H3K36me2) is associated with actively transcribed genes, and global up-regulation of this modification is associated with several cancers. However, the molecular mechanism by which H3K36me2 is sensed and transduced to downstream biological outcomes remains unclear. Here we identify a PWWP domain within the histone lysine methyltransferase and oncoprotein NSD2 that preferentially binds to nucleosomes containing H3K36me2. In cells, the NSD2 PWWP domain interaction with H3K36me2 plays a role in stabilizing NSD2 at chromatin. Furthermore, NSD2's ability to induce global increases in H3K36me2 via its enzymatic activity, and consequently promote cellular proliferation, is compromised by mutations within the PWWP domain that specifically abrogate H3K36me2-recognition. Together, our results identify a pivotal role for NSD2 binding to its catalytic product in regulating its cellular functions, and suggest a model for how this interaction may facilitate epigenetic spreading and propagation of H3K36me2.

    View details for DOI 10.1074/jbc.M116.720748

    View details for Web of Science ID 000374773200013

    View details for PubMedID 26912663

    View details for PubMedCentralID PMC4861420

  • Reproductive Aging Drives Protein Accumulation in the Uterus and Limits Lifespan in C. elegans PLOS GENETICS Zimmerman, S. M., Hinkson, I. V., Elias, J. E., Kim, S. K. 2015; 11 (12)


    Aging in Caenorhabditis elegans is characterized by widespread physiological and molecular changes, but the mechanisms that determine the rate at which these changes occur are not well understood. In this study, we identify a novel link between reproductive aging and somatic aging in C. elegans. By measuring global age-related changes in the proteome, we identify a previously uncharacterized group of secreted proteins in the adult uterus that dramatically increase in abundance with age. This accumulation is blunted in animals with an extended reproductive period and accelerated in sterile animals lacking a germline. Uterine proteins are not removed in old post-reproductive animals or in young vulvaless worms, indicating that egg-laying is necessary for their rapid removal in wild-type young animals. Together, these results suggest that age-induced infertility contributes to extracellular protein accumulation in the uterus with age. Finally, we show that knocking down multiple age-increased proteins simultaneously extends lifespan. These results provide a mechanistic example of how the cessation of reproduction contributes to detrimental changes in the soma, and demonstrate how the timing of reproductive decline can influence the rate of aging.

    View details for DOI 10.1371/journal.pgen.1005725

    View details for Web of Science ID 000368518400054

    View details for PubMedCentralID PMC4676719

  • Unbiased proteomic-genomic investigation of MHC class I peptide ligands reveals unconventional translation origins Scharf, A. D., Flockhart, R. J., Olsson, N. E., Devabhaktuni, A., Zhang, J., Zhao, X., Khavari, P., Elias, J. E. PERGAMON-ELSEVIER SCIENCE LTD. 2015: 135–36
  • Quantitative Imaging of Gut Microbiota Spatial Organization CELL HOST & MICROBE Earle, K. A., Billings, G., Sigal, M., Lichtman, J. S., Hansson, G. C., Elias, J. E., Amieva, M. R., Huang, K. C., Sonnenburg, J. L. 2015; 18 (4): 478-488


    Genomic technologies have significantly advanced our understanding of the composition and diversity of host-associated microbial populations. However, their spatial organization and functional interactions relative to the host have been more challenging to study. Here we present a pipeline for the assessment of intestinal microbiota localization within immunofluorescence images of fixed gut cross-sections that includes a flexible software package, BacSpace, for high-throughput quantification of microbial organization. Applying this pipeline to gnotobiotic and human microbiota-colonized mice, we demonstrate that elimination of microbiota-accessible carbohydrates (MACs) from the diet results in thinner mucus in the distal colon, increased proximity of microbes to the epithelium, and heightened expression of the inflammatory marker REG3β. Measurements of microbe-microbe proximity reveal that a MAC-deficient diet alters monophyletic spatial clustering. Furthermore, we quantify the invasion of Helicobacter pylori into the glands of the mouse stomach relative to host mitotic progenitor cells, illustrating the generalizability of this approach.

    View details for DOI 10.1016/j.chom.2015.09.002

    View details for Web of Science ID 000365111600016

    View details for PubMedID 26439864

  • Metaproteomic analysis adds a functional glimpse into host-microbe succession in an infant's GI PROTEOMICS Elias, J. E. 2015; 15 (20): 3407–8


    Succession is a fundamental concept in ecology, describing the process by which pioneering species colonize a new environmental niche, paving the way for increasingly complex, yet stable ecosystems. The human gut-among the most complex ecosystems known-develops from a nearly sterile state as newborns to a thriving functional network of trillions of organisms living in concert with the host. Recent microbiome analyses have begun to characterize the identities of the microbes during this period of colonization. However, the biochemical processes that govern the stages of interorganism succession remains a poorly understood, yet exciting frontier. Toward the goal of learning how host-microbe symbiosis arises and is maintained, Young et al. (Proteomics 2015, 15, 3463-3473) present a longitudinal metaproteomic case study of an infant during her second and third weeks of life. In the first such analysis of its kind, Young et al. portray overlapping stages of protein production by both the microbiota and the host consistent with their increasing complexity, and deepening interactions.

    View details for DOI 10.1002/pmic.201500359

    View details for Web of Science ID 000363090700002

    View details for PubMedID 26384154

  • A Proteomic Strategy Identifies Lysine Methylation of Splicing Factor snRNP70 by the SETMAR Enzyme JOURNAL OF BIOLOGICAL CHEMISTRY Carlson, S. M., Moore, K. E., Sankaran, S. M., Reynoird, N., Elias, J. E., Gozani, O. 2015; 290 (19): 12040-12047


    The lysine methyltransferase (KMT) SETMAR is implicated in the response to and repair of DNA damage, but its molecular function is not clear. SETMAR has been associated with dimethylation of histone H3 lysine 36 (H3K36) at sites of DNA damage. However, SETMAR does not methylate H3K36 in vitro. This and the observation that SETMAR is not active on nucleosomes suggest that H3K36 methylation is not a physiologically relevant activity. To identify potential non-histone substrates, we utilized a strategy on the basis of quantitative proteomic analysis of methylated lysine. Our approach identified lysine 130 of the mRNA splicing factor snRNP70 as a SETMAR substrate in vitro, and we show that the enzyme primarily generates monomethylation at this position. Furthermore, we show that SETMAR methylates snRNP70 Lys-130 in cells. Because snRNP70 is a key early regulator of 5' splice site selection, our results suggest a model in which methylation of snRNP70 by SETMAR regulates constitutive and/or alternative splicing. In addition, the proteomic strategy described here is broadly applicable and is a promising route for large-scale mapping of KMT substrates.

    View details for DOI 10.1074/jbc.M115.641530

    View details for Web of Science ID 000354388600019

    View details for PubMedID 25795785

    View details for PubMedCentralID PMC4424340

  • Quantifying dynamic host-microbiota signatures of antibiotic-associated GI infection: What can the host proteome tell us? Elias, J., Lichtman, J., Sonnenburg, J. FEDERATION AMER SOC EXP BIOL. 2015
  • Calcineurin regulates the yeast synaptojanin Inp53/Sjl3 during membrane stress. Molecular biology of the cell Guiney, E. L., Goldman, A. R., Elias, J. E., Cyert, M. S. 2015; 26 (4): 769-785


    During hyperosmotic shock, Saccharomyces cerevisiae adjusts to physiological challenges, including large plasma membrane invaginations generated by rapid cell shrinkage. Calcineurin, the Ca(2+)/calmodulin-dependent phosphatase, is normally cytosolic but concentrates in puncta and at sites of polarized growth during intense osmotic stress; inhibition of calcineurin-activated gene expression suggests that restricting its access to substrates tunes calcineurin signaling specificity. Hyperosmotic shock promotes calcineurin binding to and dephosphorylation of the PI(4,5)P2 phosphatase synaptojanin/Inp53/Sjl3 and causes dramatic calcineurin-dependent reorganization of PI(4,5)P2-enriched membrane domains. Inp53 normally promotes sorting at the trans-Golgi network but localizes to cortical actin patches in osmotically stressed cells. By activating Inp53, calcineurin repolarizes the actin cytoskeleton and maintains normal plasma membrane morphology in synaptojanin-limited cells. In response to hyperosmotic shock and calcineurin-dependent regulation, Inp53 shifts from associating predominantly with clathrin to interacting with endocytic proteins Sla1, Bzz1, and Bsp1, suggesting that Inp53 mediates stress-specific endocytic events. This response has physiological and molecular similarities to calcineurin-regulated activity-dependent bulk endocytosis in neurons, which retrieves a bolus of plasma membrane deposited by synaptic vesicle fusion. We propose that activation of Ca(2+)/calcineurin and PI(4,5)P2 signaling to regulate endocytosis is a fundamental and conserved response to excess membrane in eukaryotic cells.

    View details for DOI 10.1091/mbc.E14-05-1019

    View details for PubMedID 25518934

    View details for PubMedCentralID PMC4325846

  • Role of disease-associated tolerance in infectious superspreaders PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Gopinath, S., Lichtman, J. S., Bouley, D. M., Elias, J. E., Monack, D. M. 2014; 111 (44): 15780-15785


    Natural populations show striking heterogeneity in their ability to transmit disease. For example, a minority of infected individuals known as superspreaders carries out the majority of pathogen transmission events. In a mouse model of Salmonella infection, a subset of infected hosts becomes superspreaders, shedding high levels of bacteria (>10(8) cfu per g of feces) but remain asymptomatic with a dampened systemic immune state. Here we show that superspreader hosts remain asymptomatic when they are treated with oral antibiotics. In contrast, nonsuperspreader Salmonella-infected hosts that are treated with oral antibiotics rapidly shed superspreader levels of the pathogen but display signs of morbidity. This morbidity is linked to an increase in inflammatory myeloid cells in the spleen followed by increased production of acute-phase proteins and proinflammatory cytokines. The degree of colonic inflammation is similar in antibiotic-treated superspreader and nonsuperspreader hosts, indicating that the superspreader hosts are tolerant of antibiotic-mediated perturbations in the intestinal tract. Importantly, neutralization of acute-phase proinflammatory cytokines in antibiotic-induced superspreaders suppresses the expansion of inflammatory myeloid cells and reduces morbidity. We describe a unique disease-associated tolerance to oral antibiotics in superspreaders that facilitates continued transmission of the pathogen.

    View details for DOI 10.1073/pnas.1409968111

    View details for Web of Science ID 000344088100047

    View details for PubMedCentralID PMC4226084

  • Role of disease-associated tolerance in infectious superspreaders. Proceedings of the National Academy of Sciences of the United States of America Gopinath, S., Lichtman, J. S., Bouley, D. M., Elias, J. E., Monack, D. M. 2014; 111 (44): 15780-15785


    Natural populations show striking heterogeneity in their ability to transmit disease. For example, a minority of infected individuals known as superspreaders carries out the majority of pathogen transmission events. In a mouse model of Salmonella infection, a subset of infected hosts becomes superspreaders, shedding high levels of bacteria (>10(8) cfu per g of feces) but remain asymptomatic with a dampened systemic immune state. Here we show that superspreader hosts remain asymptomatic when they are treated with oral antibiotics. In contrast, nonsuperspreader Salmonella-infected hosts that are treated with oral antibiotics rapidly shed superspreader levels of the pathogen but display signs of morbidity. This morbidity is linked to an increase in inflammatory myeloid cells in the spleen followed by increased production of acute-phase proteins and proinflammatory cytokines. The degree of colonic inflammation is similar in antibiotic-treated superspreader and nonsuperspreader hosts, indicating that the superspreader hosts are tolerant of antibiotic-mediated perturbations in the intestinal tract. Importantly, neutralization of acute-phase proinflammatory cytokines in antibiotic-induced superspreaders suppresses the expansion of inflammatory myeloid cells and reduces morbidity. We describe a unique disease-associated tolerance to oral antibiotics in superspreaders that facilitates continued transmission of the pathogen.

    View details for DOI 10.1073/pnas.1409968111

    View details for PubMedID 25331868

  • The cyclin D1-CDK4 oncogenic interactome enables identification of potential novel oncogenes and clinical prognosis CELL CYCLE Jirawatnotai, S., Sharma, S., Michowski, W., Suktitipat, B., Geng, Y., Quackenbush, J., Elias, J. E., Gygi, S. P., Wang, Y. E., Sicinski, P. 2014; 13 (18): 2889-2900


    Overexpression of cyclin D1 and its catalytic partner, CDK4, is frequently seen in human cancers. We constructed cyclin D1 and CDK4 protein interaction network in a human breast cancer cell line MCF7, and identified novel CDK4 protein partners. Among CDK4 interactors we observed several proteins functioning in protein folding and in complex assembly. One of the novel partners of CDK4 is FKBP5, which we found to be required to maintain CDK4 levels in cancer cells. An integrative analysis of the extended cyclin D1 cancer interactome and somatic copy number alterations in human cancers identified BAIAPL21 as a potential novel human oncogene. We observed that in several human tumor types BAIAPL21 is expressed at higher levels as compared to normal tissue. Forced overexpression of BAIAPL21 augmented anchorage independent growth, increased colony formation by cancer cells and strongly enhanced the ability of cells to form tumors in vivo. Lastly, we derived an Aggregate Expression Score (AES), which quantifies the expression of all cyclin D1 interactors in a given tumor. We observed that AES has a prognostic value among patients with ER-positive breast cancers. These studies illustrate the utility of analyzing the interactomes of proteins involved in cancer to uncover potential oncogenes, or to allow better cancer prognosis.

    View details for DOI 10.4161/15384101.2014.946850

    View details for Web of Science ID 000348325800017

    View details for PubMedID 25486477

  • Assessing subunit dependency of the Plasmodium proteasome using small molecule inhibitors and active site probes. ACS chemical biology Li, H., van der Linden, W. A., Verdoes, M., Florea, B. I., McAllister, F. E., Govindaswamy, K., Elias, J. E., Bhanot, P., Overkleeft, H. S., Bogyo, M. 2014; 9 (8): 1869-1876


    The ubiquitin-proteasome system (UPS) is a potential pathway for therapeutic intervention for pathogens such as Plasmodium, the causative agent of malaria. However, due to the essential nature of this proteolytic pathway, proteasome inhibitors must avoid inhibition of the host enzyme complex to prevent toxic side effects. The Plasmodium proteasome is poorly characterized, making rational design of inhibitors that induce selective parasite killing difficult. In this study, we developed a chemical probe that labels all catalytic sites of the Plasmodium proteasome. Using this probe, we identified several subunit selective small molecule inhibitors of the parasite enzyme complex. Treatment with an inhibitor that is specific for the β5 subunit during blood stage schizogony led to a dramatic decrease in parasite replication while short-term inhibition of the β2 subunit did not affect viability. Interestingly, coinhibition of both the β2 and β5 catalytic subunits resulted in enhanced parasite killing at all stages of the blood stage life cycle and reduced parasite levels in vivo to barely detectable levels. Parasite killing was achieved with overall low host toxicity, something that has not been possible with existing proteasome inhibitors. Our results highlight differences in the subunit dependency of the parasite and human proteasome, thus providing a strategy for development of potent antimalarial drugs with overall low host toxicity.

    View details for DOI 10.1021/cb5001263

    View details for PubMedID 24918547

    View details for PubMedCentralID PMC4136710

  • The Calcium-Dependent Protein Kinase 3 of Toxoplasma Influences Basal Calcium Levels and Functions beyond Egress as Revealed by Quantitative Phosphoproteome Analysis PLOS PATHOGENS Treeck, M., Sanders, J. L., Gaji, R. Y., LaFavers, K. A., Child, M. A., Arrizabalaga, G., Elias, J. E., Boothroyd, J. C. 2014; 10 (6)


    Calcium-dependent protein kinases (CDPKs) are conserved in plants and apicomplexan parasites. In Toxoplasma gondii, TgCDPK3 regulates parasite egress from the host cell in the presence of a calcium-ionophore. The targets and the pathways that the kinase controls, however, are not known. To identify pathways regulated by TgCDPK3, we measured relative phosphorylation site usage in wild type and TgCDPK3 mutant and knock-out parasites by quantitative mass-spectrometry using stable isotope-labeling with amino acids in cell culture (SILAC). This revealed known and novel phosphorylation events on proteins predicted to play a role in host-cell egress, but also a novel function of TgCDPK3 as an upstream regulator of other calcium-dependent signaling pathways, as we also identified proteins that are differentially phosphorylated prior to egress, including proteins important for ion-homeostasis and metabolism. This observation is supported by the observation that basal calcium levels are increased in parasites where TgCDPK3 has been inactivated. Most of the differential phosphorylation observed in CDPK3 mutants is rescued by complementation of the mutants with a wild type copy of TgCDPK3. Lastly, the TgCDPK3 mutants showed hyperphosphorylation of two targets of a related calcium-dependent kinase (TgCDPK1), as well as TgCDPK1 itself, indicating that this latter kinase appears to play a role downstream of TgCDPK3 function. Overexpression of TgCDPK1 partially rescues the egress phenotype of the TgCDPK3 mutants, reinforcing this conclusion. These results show that TgCDPK3 plays a pivotal role in regulating tachyzoite functions including, but not limited to, egress.

    View details for DOI 10.1371/journal.ppat.1004197

    View details for Web of Science ID 000338197400032

    View details for PubMedCentralID PMC4063958

  • The calcium-dependent protein kinase 3 of toxoplasma influences basal calcium levels and functions beyond egress as revealed by quantitative phosphoproteome analysis. PLoS pathogens Treeck, M., Sanders, J. L., Gaji, R. Y., LaFavers, K. A., Child, M. A., Arrizabalaga, G., Elias, J. E., Boothroyd, J. C. 2014; 10 (6)


    Calcium-dependent protein kinases (CDPKs) are conserved in plants and apicomplexan parasites. In Toxoplasma gondii, TgCDPK3 regulates parasite egress from the host cell in the presence of a calcium-ionophore. The targets and the pathways that the kinase controls, however, are not known. To identify pathways regulated by TgCDPK3, we measured relative phosphorylation site usage in wild type and TgCDPK3 mutant and knock-out parasites by quantitative mass-spectrometry using stable isotope-labeling with amino acids in cell culture (SILAC). This revealed known and novel phosphorylation events on proteins predicted to play a role in host-cell egress, but also a novel function of TgCDPK3 as an upstream regulator of other calcium-dependent signaling pathways, as we also identified proteins that are differentially phosphorylated prior to egress, including proteins important for ion-homeostasis and metabolism. This observation is supported by the observation that basal calcium levels are increased in parasites where TgCDPK3 has been inactivated. Most of the differential phosphorylation observed in CDPK3 mutants is rescued by complementation of the mutants with a wild type copy of TgCDPK3. Lastly, the TgCDPK3 mutants showed hyperphosphorylation of two targets of a related calcium-dependent kinase (TgCDPK1), as well as TgCDPK1 itself, indicating that this latter kinase appears to play a role downstream of TgCDPK3 function. Overexpression of TgCDPK1 partially rescues the egress phenotype of the TgCDPK3 mutants, reinforcing this conclusion. These results show that TgCDPK3 plays a pivotal role in regulating tachyzoite functions including, but not limited to, egress.

    View details for DOI 10.1371/journal.ppat.1004197

    View details for PubMedID 24945436

  • Modification of proteins by O-linked N-acetylglucosamine in activated T cells Lund, P., Elias, J., Davis, M. AMER ASSOC IMMUNOLOGISTS. 2014
  • A Role for the MRN Complex in ATR Activation via TOPBP1 Recruitment MOLECULAR CELL Duursma, A. M., Driscoll, R., Elias, J. E., Cimprich, K. A. 2013; 50 (1): 116-122


    The MRN (MRE11-RAD50-NBS1) complex has been implicated in many aspects of the DNA damage response. It has key roles in sensing and processing DNA double-strand breaks, as well as in activation of ATM (ataxia telangiectasia mutated). We reveal a function for MRN in ATR (ATM- and RAD3-related) activation by using defined ATR-activating DNA structures in Xenopus egg extracts. Strikingly, we demonstrate that MRN is required for recruitment of TOPBP1 to an ATR-activating structure that contains a single-stranded DNA (ssDNA) and a double-stranded DNA (dsDNA) junction and that this recruitment is necessary for phosphorylation of CHK1. We also show that the 911 (RAD9-RAD1-HUS1) complex is not required for TOPBP1 recruitment but is essential for TOPBP1 function. Thus, whereas MRN is required for TOPBP1 recruitment at an ssDNA-to-dsDNA junction, 911 is required for TOPBP1 "activation." These findings provide molecular insights into how ATR is activated.

    View details for DOI 10.1016/j.molcel.2013.03.006

    View details for Web of Science ID 000317558300012

    View details for PubMedID 23582259

    View details for PubMedCentralID PMC3669687

  • Proteomics Meets Translation Analysis: An Attempt to Balance the Protein Turnover Equation 57th Annual Meeting of the Biophysical-Society Hinkson, I. V., Bates, J. G., Elias, J. CELL PRESS. 2013: 356A–356A
  • Calcineurin regulates the yeast synaptojanin Inp53/Sjl3 to promote membrane recovery during hyperosmotic stress Guiney, E., Elias, J., Cyert, M. AMER SOC CELL BIOLOGY. 2013
  • Transcriptional role of cyclin D1 in development revealed by a genetic-proteomic screen NATURE Bienvenu, F., Jirawatnotai, S., Elias, J. E., Meyer, C. A., Mizeracka, K., Marson, A., Frampton, G. M., Cole, M. F., Odom, D. T., Odajima, J., Geng, Y., Zagozdzon, A., Jecrois, M., Young, R. A., Liu, X. S., Cepko, C. L., Gygi, S. P., Sicinski, P. 2010; 463 (7279): 374-378


    Cyclin D1 belongs to the core cell cycle machinery, and it is frequently overexpressed in human cancers. The full repertoire of cyclin D1 functions in normal development and oncogenesis is unclear at present. Here we developed Flag- and haemagglutinin-tagged cyclin D1 knock-in mouse strains that allowed a high-throughput mass spectrometry approach to search for cyclin D1-binding proteins in different mouse organs. In addition to cell cycle partners, we observed several proteins involved in transcription. Genome-wide location analyses (chromatin immunoprecipitation coupled to DNA microarray; ChIP-chip) showed that during mouse development cyclin D1 occupies promoters of abundantly expressed genes. In particular, we found that in developing mouse retinas-an organ that critically requires cyclin D1 function-cyclin D1 binds the upstream regulatory region of the Notch1 gene, where it serves to recruit CREB binding protein (CBP) histone acetyltransferase. Genetic ablation of cyclin D1 resulted in decreased CBP recruitment, decreased histone acetylation of the Notch1 promoter region, and led to decreased levels of the Notch1 transcript and protein in cyclin D1-null (Ccnd1(-/-)) retinas. Transduction of an activated allele of Notch1 into Ccnd1(-/-) retinas increased proliferation of retinal progenitor cells, indicating that upregulation of Notch1 signalling alleviates the phenotype of cyclin D1-deficiency. These studies show that in addition to its well-established cell cycle roles, cyclin D1 has an in vivo transcriptional function in mouse development. Our approach, which we term 'genetic-proteomic', can be used to study the in vivo function of essentially any protein.

    View details for DOI 10.1038/nature08684

    View details for Web of Science ID 000273748100048

    View details for PubMedID 20090754

  • Target-decoy search strategy for mass spectrometry-based proteomics. Methods in molecular biology (Clifton, N.J.) Elias, J. E., Gygi, S. P. 2010; 604: 55-71


    Accurate and precise methods for estimating incorrect peptide and protein identifications are crucial for effective large-scale proteome analyses by tandem mass spectrometry. The target-decoy search strategy has emerged as a simple, effective tool for generating such estimations. This strategy is based on the premise that obvious, necessarily incorrect "decoy" sequences added to the search space will correspond with incorrect search results that might otherwise be deemed to be correct. With this knowledge, it is possible not only to estimate how many incorrect results are in a final data set but also to use decoy hits to guide the design of filtering criteria that sensitively partition a data set into correct and incorrect identifications.

    View details for DOI 10.1007/978-1-60761-444-9_5

    View details for PubMedID 20013364

    View details for PubMedCentralID PMC2922680

  • Identification of beta-Secretase (BACE1) Substrates Using Quantitative Proteomics PLOS ONE Hemming, M. L., Elias, J. E., Gygi, S. P., Selkoe, D. J. 2009; 4 (12)


    Beta-site APP cleaving enzyme 1 (BACE1) is a transmembrane aspartyl protease with a lumenal active site that sheds the ectodomains of membrane proteins through juxtamembrane proteolysis. BACE1 has been studied principally for its role in Alzheimer's disease as the beta-secretase responsible for generating the amyloid-beta protein. Emerging evidence from mouse models has identified the importance of BACE1 in myelination and cognitive performance. However, the substrates that BACE1 processes to regulate these functions are unknown, and to date only a few beta-secretase substrates have been identified through candidate-based studies. Using an unbiased approach to substrate identification, we performed quantitative proteomic analysis of two human epithelial cell lines stably expressing BACE1 and identified 68 putative beta-secretase substrates, a number of which we validated in a cell culture system. The vast majority were of type I transmembrane topology, although one was type II and three were GPI-linked proteins. Intriguingly, a preponderance of these proteins are involved in contact-dependent intercellular communication or serve as receptors and have recognized roles in the nervous system and other organs. No consistent sequence motif predicting BACE1 cleavage was identified in substrates versus non-substrates. These findings expand our understanding of the proteins and cellular processes that BACE1 may regulate, and suggest possible mechanisms of toxicity arising from chronic BACE1 inhibition.

    View details for DOI 10.1371/journal.pone.0008477

    View details for Web of Science ID 000273105200012

    View details for PubMedID 20041192

  • The Impact of Peptide Abundance and Dynamic Range on Stable-Isotope-Based Quantitative Proteomic Analyses JOURNAL OF PROTEOME RESEARCH Bakalarski, C. E., Elias, J. E., Villen, J., Haas, W., Gerber, S. A., Everley, P. A., Gygi, S. P. 2008; 7 (11): 4756-4765


    Recently, mass spectrometry has been employed in many studies to provide unbiased, reproducible, and quantitative protein abundance information on a proteome-wide scale. However, how instruments' limited dynamic ranges impact the accuracy of such measurements has remained largely unexplored, especially in the context of complex mixtures. Here, we examined the distribution of peptide signal versus background noise (S/N) and its correlation with quantitative accuracy. With the use of metabolically labeled Jurkat cell lysate, over half of all confidently identified peptides had S/N ratios less than 10 when examined using both hybrid linear ion trap-Fourier transform ion cyclotron resonance and Orbitrap mass spectrometers. Quantification accuracy was also highly correlated with S/N. We developed a mass precision algorithm that significantly reduced measurement variance at low S/N beyond the use of highly accurate mass information alone and expanded it into a new software suite, Vista. We also evaluated the interplay between mass measurement accuracy and S/N; finding a balance between both parameters produced the greatest identification and quantification rates. Finally, we demonstrate that S/N can be a useful surrogate for relative abundance ratios when only a single species is detected.

    View details for DOI 10.1021/pr800333e

    View details for Web of Science ID 000260792000015

    View details for PubMedID 18798661

  • Proteomic Profiling of gamma-Secretase Substrates and Mapping of Substrate Requirements PLOS BIOLOGY Hemming, M. L., Elias, J. E., Gygi, S. P., Selkoe, D. J. 2008; 6 (10): 2314-2328


    The presenilin/gamma-secretase complex, an unusual intramembrane aspartyl protease, plays an essential role in cellular signaling and membrane protein turnover. Its ability to liberate numerous intracellular signaling proteins from the membrane and also mediate the secretion of amyloid-beta protein (Abeta) has made modulation of gamma-secretase activity a therapeutic goal for cancer and Alzheimer disease. Although the proteolysis of the prototypical substrates Notch and beta-amyloid precursor protein (APP) has been intensely studied, the full spectrum of substrates and the determinants that make a transmembrane protein a substrate remain unclear. Using an unbiased approach to substrate identification, we surveyed the proteome of a human cell line for targets of gamma-secretase and found a relatively small population of new substrates, all of which are type I transmembrane proteins but have diverse biological roles. By comparing these substrates to type I proteins not regulated by gamma-secretase, we determined that besides a short ectodomain, gamma-secretase requires permissive transmembrane and cytoplasmic domains to bind and cleave its substrates. In addition, we provide evidence for at least two mechanisms that can target a substrate for gamma cleavage: one in which a substrate with a short ectodomain is directly cleaved independent of sheddase association, and a second where a substrate requires ectodomain shedding to instruct subsequent gamma-secretase processing. These findings expand our understanding of the mechanisms of substrate selection as well as the diverse cellular processes to which gamma-secretase contributes.

    View details for DOI 10.1371/journal.pbio.0060257

    View details for Web of Science ID 000260423900022

    View details for PubMedID 18942891

  • Assessing enzyme activities using stable isotope Labeling and mass spectrometry MOLECULAR & CELLULAR PROTEOMICS Everley, P. A., Gartner, C. A., Haas, W., Saghatelian, A., Elias, J. E., Cravatt, B. F., Zetter, B. R., Gygi, S. P. 2007; 6 (10): 1771-1777


    Activity-based protein profiling has emerged as a valuable technology for labeling, enriching, and assessing protein activities from complex mixtures. This is primarily accomplished via a two-step identification and quantification process. Here we show a highly quantitative and streamlined method, termed catch-and-release activity profiling of enzymes (CAPE), which reduces this procedure to a single step. Furthermore the CAPE approach has the ability to detect small quantitative changes that may have been missed by alternative mass spectrometry-based techniques.

    View details for DOI 10.1074/mcp.M700057-MCP200

    View details for Web of Science ID 000250092600010

    View details for PubMedID 17627935

  • Large-scale phosphorylation analysis of alpha-factor-arrested Saccharomyces cerevisiae JOURNAL OF PROTEOME RESEARCH Li, X., Gerber, S. A., Rudner, A. D., Beausoleil, S. A., Haas, W., Villen, J., Elias, J. E., Gygi, S. P. 2007; 6 (3): 1190-1197


    Protein phosphorylation is essential for numerous cellular processes. Large-scale profiling of phosphoproteins continues to enhance the depth and speed at which we understand these processes. The development of effective phosphoprotein and peptide enrichment techniques and improvements to mass spectrometric instrumentation have intensified phosphoproteomic research in recent years, leading to unprecedented achievements. Here, we describe a large-scale phosphorylation analysis of alpha-factor-arrested yeast. Using a multidimensional separation strategy involving preparative SDS-PAGE for prefractionation, in-gel digestion with trypsin, and immobilized metal affinity chromatography (IMAC) enrichment of phosphopeptides, followed by LC-MS/MS analysis employing a hybrid LTQ-Orbitrap mass spectrometer, we were able to catalog a substantial portion of the phosphoproteins present in yeast whole-cell lysate. This analysis yielded the confident identification of 2288 nonredundant phosphorylation sites from 985 proteins. The ambiguity score (Ascore) algorithm was utilized to determine the certainty of site localization for the entire data set. In addition, the size of the data set permitted extraction of known and novel kinase motifs using the Motif-X algorithm. Finally, a large number of members of the pheromone signaling pathway were found as phosphoproteins and are discussed.

    View details for DOI 10.1021/pr060559j

    View details for Web of Science ID 000244638400028

    View details for PubMedID 17330950

  • Catch-and-release reagents for broadscale quantitative proteomics analyses JOURNAL OF PROTEOME RESEARCH Gartner, C. A., Elias, J. E., Bakalarski, C. E., Gygi, S. P. 2007; 6 (4): 1482-1491


    The relative quantification of protein expression levels in different cell samples through the utilization of stable isotope dilution has become a standard method in the field of proteomics. We describe here the development of a new reductively cleavable reagent which facilitates the relative quantification of thousands of proteins from only tens of micrograms of starting protein. The ligand features a novel disulfide moiety that links biotin and a thiol-reactive entity. The disulfide is stable to reductive conditions employed during sample labeling but is readily cleaved under mild conditions using tris-(2-carboxyethyl) phosphine (TCEP). This unique chemical property allows for the facile use of immobilized avidin in a manner equivalent to the use of conventional reversible-binding affinity resins. Target peptides are bound to avidin resin, washed rigorously, then cleaved directly from the resin, resulting in simplified sample handling procedures and reduced nonspecific interactions. Here we demonstrate the stability of the linker under two different reducing conditions and show how this "catch-and-release (CAR)" reagent can be used to quantitatively compare protein abundances from two distinct cellular lysates. Starting with only 40 microg protein from each sample, 1840 individual proteins were identified in a single experiment. Using in-house software for automated peak integration, 1620 of these proteins were quantified for differential expression.

    View details for DOI 10.1021/pr060605f

    View details for Web of Science ID 000245510900026

    View details for PubMedID 17311443

  • Optimization and use of peptide mass measurement accuracy in shotgun proteomics MOLECULAR & CELLULAR PROTEOMICS Haas, W., Faherty, B. K., Gerber, S. A., Elias, J. E., Beausoleil, S. A., Bakalarski, C. E., Li, X., Villen, J., Gygi, S. P. 2006; 5 (7): 1326-1337


    Mass spectrometers that provide high mass accuracy such as FT-ICR instruments are increasingly used in proteomic studies. Although the importance of accurately determined molecular masses for the identification of biomolecules is generally accepted, its role in the analysis of shotgun proteomic data has not been thoroughly studied. To gain insight into this role, we used a hybrid linear quadrupole ion trap/FT-ICR (LTQ FT) mass spectrometer for LC-MS/MS analysis of a highly complex peptide mixture derived from a fraction of the yeast proteome. We applied three data-dependent MS/MS acquisition methods. The FT-ICR part of the hybrid mass spectrometer was either not exploited, used only for survey MS scans, or also used for acquiring selected ion monitoring scans to optimize mass accuracy. MS/MS data were assigned with the SEQUEST algorithm, and peptide identifications were validated by estimating the number of incorrect assignments using the composite target/decoy database search strategy. We developed a simple mass calibration strategy exploiting polydimethylcyclosiloxane background ions as calibrant ions. This strategy allowed us to substantially improve mass accuracy without reducing the number of MS/MS spectra acquired in an LC-MS/MS run. The benefits of high mass accuracy were greatest for assigning MS/MS spectra with low signal-to-noise ratios and for assigning phosphopeptides. Confident peptide identification rates from these data sets could be doubled by the use of mass accuracy information. It was also shown that improving mass accuracy at a cost to the MS/MS acquisition rate substantially lowered the sensitivity of LC-MS/MS analyses. The use of FT-ICR selected ion monitoring scans to maximize mass accuracy reduced the number of protein identifications by 40%.

    View details for DOI 10.1074/mcp.M500339-MCP200

    View details for Web of Science ID 000238674800013

    View details for PubMedID 16635985

  • Enhanced analysis of metastatic prostate cancer using stable isotopes and high mass accuracy instrumentation JOURNAL OF PROTEOME RESEARCH Everley, P. A., Bakalarski, C. E., ELIAS, J. E., Waghorne, C. G., Beausoleil, S. A., Gerber, S. A., Faherty, B. K., ZETTER, B. R., Gygi, S. P. 2006; 5 (5): 1224-1231


    The primary goal of proteomics is to gain a better understanding of biological function at the protein expression level. As the field matures, numerous technologies are being developed to aid in the identification, quantification and characterization of protein expression and post-translational modifications on a near-global scale. Stable isotope labeling by amino acids in cell culture is one such technique that has shown broad biological applications. While we have recently shown the application of this technology to a model of metastatic prostate cancer, we now report a substantial improvement in quantitative analysis using a linear ion-trap Fourier transform ion cyclotron resonance mass spectrometer (LTQ FT) and novel quantification software. This resulted in the quantification of nearly 1400 proteins, a greater than 3-fold increase in comparison to our earlier study. This dramatic increase in proteome coverage can be attributed to (1) use of a double-labeling strategy, (2) greater sensitivity, speed and mass accuracy provided by the LTQ FT mass spectrometer, and (3) more robust quantification software. Finally, by using a concatenated target/decoy protein database for our peptide searches, we now report these data in the context of an estimated false-positive rate of one percent.

    View details for DOI 10.1021/pr0504891

    View details for Web of Science ID 000237390200021

    View details for PubMedID 16674112

  • Characterization of mouse spleen cells by subtractive proteomics MOLECULAR & CELLULAR PROTEOMICS Dieguez-Acuna, F. J., Gerber, S. A., Kodama, S., ELIAS, J. E., Beausoleil, S. A., Faustman, D., Gygi, S. P. 2005; 4 (10): 1459-1470


    Major analytical challenges encountered by shotgun proteome analysis include both the diversity and dynamic range of protein expression. Often new instrumentation can provide breakthroughs in areas where other analytical improvements have not been successful. In the current study, we utilized new instrumentation (LTQ FT) to characterize complex protein samples by shotgun proteomics. Proteomic analyses were performed on murine spleen tissue separated by magnetic beads into distinct CD45- and CD45+ cell populations. Using shotgun protein analysis we identified approximately 2,000 proteins per cell group by over 12,000 peptides with mass deviations of less than 4.5 ppm. Datasets obtained by LTQ FT analysis provided a significant increase in the number of proteins identified and greater confidence in those identifications and improved reproducibility in replicate analyses. Because CD45- and not CD45+ cells are able to regenerate functional pancreatic islet cells in a mouse model of type I diabetes, protein expression was further compared by a subtractive proteomic approach in search of an exclusive protein expression profile in CD45- cells. Characterization of the proteins exclusively identified in CD45- cells was performed using gene ontology terms via the Javascript GoMiner. The CD45- cell subset readily revealed proteins involved in development, suggesting the persistence of a fetal stem cell in an adult animal.

    View details for DOI 10.1074/mcp.M500137-MCP200

    View details for Web of Science ID 000232207900004

    View details for PubMedID 16037072

  • The Parkinson's disease-associated DJ-1 protein is a transcriptional co-activator that protects against neuronal apoptosis HUMAN MOLECULAR GENETICS Xu, J., Zhong, N., Wang, H. Y., ELIAS, J. E., Kim, C. Y., Woldman, I., Pifl, C., Gygi, S. P., Geula, C., Yankner, B. A. 2005; 14 (9): 1231-1241


    Mutations in the DJ-1 gene cause early-onset autosomal recessive Parkinson's disease (PD), although the role of DJ-1 in the degeneration of dopaminergic neurons is unresolved. Here we show that the major interacting-proteins with DJ-1 in dopaminergic neuronal cells are the nuclear proteins p54nrb and pyrimidine tract-binding protein-associated splicing factor (PSF), two multifunctional regulators of transcription and RNA metabolism. PD-associated DJ-1 mutants exhibit decreased nuclear distribution and increased mitochondrial localization, resulting in diminished co-localization with co-activator p54nrb and repressor PSF. Unlike pathogenic DJ-1 mutants, wild-type DJ-1 acts to inhibit the transcriptional silencing activity of the PSF. In addition, the transcriptional silencer PSF induces neuronal apoptosis, which can be reversed by wild-type DJ-1 but to a lesser extent by PD-associated DJ-1 mutants. DJ-1-specific small interfering RNA sensitizes cells to PSF-induced apoptosis. Both DJ-1 and p54nrb block oxidative stress and mutant alpha-synuclein-induced cell death. Thus, DJ-1 is a neuroprotective transcriptional co-activator that may act in concert with p54nrb and PSF to regulate the expression of a neuroprotective genetic program. Mutations that impair the transcriptional co-activator function of DJ-1 render dopaminergic neurons vulnerable to apoptosis and may contribute to the pathogenesis of PD.

    View details for DOI 10.1093/hmg/ddi134

    View details for Web of Science ID 000228835500012

    View details for PubMedID 15790595

  • BRCA1-dependent ubiquitination of gamma-tubulin regulates centrosome number MOLECULAR AND CELLULAR BIOLOGY Starita, L. M., Machida, Y., Sankaran, S., ELIAS, J. E., Griffin, K., Schlegel, B. P., Gygi, S. P., Parvin, J. D. 2004; 24 (19): 8457-8466


    Proper centrosome duplication and spindle formation are crucial for prevention of chromosomal instability, and BRCA1 plays a role in this process. In this study, transient inhibition of BRCA1 function in cell lines derived from mammary tissue caused rapid amplification and fragmentation of centrosomes. Cell lines tested that were derived from nonmammary tissues did not amplify the centrosome number in this transient assay. We tested whether BRCA1 and its binding partner, BARD1, ubiquitinate centrosome proteins. Results showed that centrosome components, including gamma-tubulin, are ubiquitinated by BRCA1/BARD1 in vitro. The in vitro ubiquitination of gamma-tubulin was specific, and function of the carboxy terminus was necessary for this reaction; truncated BRCA1 did not ubiquitinate gamma-tubulin. BRCA1/BARD1 ubiquitinated lysines 48 and 344 of gamma-tubulin in vitro, and expression in cells of gamma-tubulin K48R caused a marked amplification of centrosomes. This result supports the notion that the modification of these lysines in living cells is critical in the maintenance of centrosome number. One of the key problems in understanding the biology of BRCA1 has been the identification of a specific target of BRCA1/BARD1 ubiquitination and its effect on mammary cell biology. The results of this study identify a ubiquitination target and suggest a biological impact important in the etiology of breast cancer.

    View details for DOI 10.1128/MCB.24.19.8457-8466.2004

    View details for Web of Science ID 000223990100014

    View details for PubMedID 15367667

  • Large-scale characterization of HeLa cell nuclear phosphoproteins PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Beausoleil, S. A., Jedrychowski, M., Schwartz, D., ELIAS, J. E., Villen, J., Li, J. X., Cohn, M. A., Cantley, L. C., Gygi, S. P. 2004; 101 (33): 12130-12135


    Determining the site of a regulatory phosphorylation event is often essential for elucidating specific kinase-substrate relationships, providing a handle for understanding essential signaling pathways and ultimately allowing insights into numerous disease pathologies. Despite intense research efforts to elucidate mechanisms of protein phosphorylation regulation, efficient, large-scale identification and characterization of phosphorylation sites remains an unsolved problem. In this report we describe an application of existing technology for the isolation and identification of phosphorylation sites. By using a strategy based on strong cation exchange chromatography, phosphopeptides were enriched from the nuclear fraction of HeLa cell lysate. From 967 proteins, 2,002 phosphorylation sites were determined by tandem MS. This unprecedented large collection of sites permitted a detailed accounting of known and unknown kinase motifs and substrates.

    View details for DOI 10.1073/pnas.0404720101

    View details for Web of Science ID 000223410100041

    View details for PubMedID 15302935

  • SILVER helps assign peptides to tandem mass spectra using intensity-based scoring JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY Gibbons, F. D., ELIAS, J. E., Gygi, S. P., Roth, F. P. 2004; 15 (6): 910-912


    Tandem mass spectrometry is commonly used to identify peptides (and thereby proteins) that are present in complex mixtures. Peptide identification from tandem mass spectra is partially automated, but still requires human curation to resolve "borderline" peptide-spectrum matches (PSMs). SILVER is web-based software that assists manual curation of tandem mass spectra, using a recently developed intensity-based machine-learning approach to scoring PSMs, Elias et al. In this method, a large training set of peptide, fragment, and peak-intensity properties for both matched and mismatched PSMs was used to develop a score measuring consistency between each predicted fragment ion of a candidate peptide and its corresponding observed spectral peak intensity. The SILVER interface provides a visual representation of match quality between each candidate fragment ion and the observed spectrum, thereby expediting manual curation of tandem mass spectra. SILVER is available online at

    View details for Web of Science ID 000221707900016

    View details for PubMedID 15144981

  • Intensity-based protein identification by machine learning from a library of tandem mass spectra NATURE BIOTECHNOLOGY ELIAS, J. E., Gibbons, F. D., King, O. D., Roth, F. P., Gygi, S. P. 2004; 22 (2): 214-219


    Tandem mass spectrometry (MS/MS) has emerged as a cornerstone of proteomics owing in part to robust spectral interpretation algorithms. Widely used algorithms do not fully exploit the intensity patterns present in mass spectra. Here, we demonstrate that intensity pattern modeling improves peptide and protein identification from MS/MS spectra. We modeled fragment ion intensities using a machine-learning approach that estimates the likelihood of observed intensities given peptide and fragment attributes. From 1,000,000 spectra, we chose 27,000 with high-quality, nonredundant matches as training data. Using the same 27,000 spectra, intensity was similarly modeled with mismatched peptides. We used these two probabilistic models to compute the relative likelihood of an observed spectrum given that a candidate peptide is matched or mismatched. We used a 'decoy' proteome approach to estimate incorrect match frequency, and demonstrated that an intensity-based method reduces peptide identification error by 50-96% without any loss in sensitivity.

    View details for DOI 10.1038/nbt930

    View details for Web of Science ID 000188730500022

    View details for PubMedID 14730315

  • A proteomics approach to understanding protein ubiquitination NATURE BIOTECHNOLOGY Peng, J. M., Schwartz, D., ELIAS, J. E., Thoreen, C. C., Cheng, D. M., Marsischky, G., Roelofs, J., Finley, D., Gygi, S. P. 2003; 21 (8): 921-926


    There is a growing need for techniques that can identify and characterize protein modifications on a large or global scale. We report here a proteomics approach to enrich, recover, and identify ubiquitin conjugates from Saccharomyces cerevisiae lysate. Ubiquitin conjugates from a strain expressing 6xHis-tagged ubiquitin were isolated, proteolyzed with trypsin and analyzed by multidimensional liquid chromatography coupled with tandem mass spectrometry (LC/LC-MS/MS) for amino acid sequence determination. We identified 1,075 proteins from the sample. In addition, we detected 110 precise ubiquitination sites present in 72 ubiquitin-protein conjugates. Finally, ubiquitin itself was found to be modified at seven lysine residues providing evidence for unexpected diversity in polyubiquitin chain topology in vivo. The methodology described here provides a general tool for the large-scale analysis and characterization of protein ubiquitination.

    View details for DOI 10.1038/nbt849

    View details for Web of Science ID 000184484600036

    View details for PubMedID 12872131

  • Evaluation of multidimensional chromatography coupled with tandem mass spectrometry (LC/LC-MS/MS) for large-scale protein analysis: The yeast proteome JOURNAL OF PROTEOME RESEARCH Peng, J. M., ELIAS, J. E., Thoreen, C. C., Licklider, L. J., Gygi, S. P. 2003; 2 (1): 43-50


    Highly complex protein mixtures can be directly analyzed after proteolysis by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). In this paper, we have utilized the combination of strong cation exchange (SCX) and reversed-phase (RP) chromatography to achieve two-dimensional separation prior to MS/MS. One milligram of whole yeast protein was proteolyzed and separated by SCX chromatography (2.1 mm i.d.) with fraction collection every minute during an 80-min elution. Eighty fractions were reduced in volume and then re-injected via an autosampler in an automated fashion using a vented-column (100 microm i.d.) approach for RP-LC-MS/MS analysis. More than 162,000 MS/MS spectra were collected with 26,815 matched to yeast peptides (7,537 unique peptides). A total of 1,504 yeast proteins were unambiguously identified in this single analysis. We present a comparison of this experiment with a previously published yeast proteome analysis by Yates and colleagues (Washburn, M. P.; Wolters, D.; Yates, J. R., III. Nat. Biotechnol. 2001, 19, 242-7). In addition, we report an in-depth analysis of the false-positive rates associated with peptide identification using the Sequest algorithm and a reversed yeast protein database. New criteria are proposed to decrease false-positives to less than 1% and to greatly reduce the need for manual interpretation while permitting more proteins to be identified.

    View details for DOI 10.1021/pr025556v

    View details for Web of Science ID 000180874400005

    View details for PubMedID 12643542