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)
Ph.D., Harvard University, Cell Biology (2006)
B.S., Cornell University, Biology, Genetics (1998)
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..
Independent Studies (8)
- Biomedical Informatics Teaching Methods
BIOMEDIN 290 (Aut, Win, Spr, Sum)
- Directed Reading and Research
BIOMEDIN 299 (Aut, Win, Spr, Sum)
- Directed Reading in Chemical and Systems Biology
CSB 299 (Aut, Win, Spr, Sum)
- Graduate Research
CSB 399 (Aut, Win, Spr, Sum)
- Medical Scholars Research
BIOMEDIN 370 (Aut, Win, Spr, Sum)
- Medical Scholars Research
CSB 370 (Aut, Win, Spr, Sum)
- Out-of-Department Advanced Research Laboratory in Bioengineering
BIOE 191X (Aut, Win, Sum)
- Undergraduate Research
CSB 199 (Aut, Win, Spr, Sum)
- Biomedical Informatics Teaching Methods
Prior Year Courses
- Research Seminar
CSB 270 (Win)
- Mass Spectrometry and Proteomics: Opening the Black Box
BIOS 227 (Win)
- Research Seminar
CSB 270 (Win)
- Research Seminar
CSB 270 (Win)
- Research Seminar
Application of de Novo Sequencing to Large-Scale Complex Proteomics Data Sets
JOURNAL OF PROTEOME RESEARCH
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 Web of Science ID 000371754100007
View details for PubMedID 26743026
Host-Microbiota Interactions in the Pathogenesis of Antibiotic-Associated Diseases
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 2015; 9 (9): 1908-1915
Host-centric Proteomics of Stool: A Novel Strategy Focused on intestinal Responses to the Gut Microbiota
MOLECULAR & CELLULAR PROTEOMICS
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 Web of Science ID 000328816000022
View details for PubMedID 23982161
The Phosphoproteomes of Plasmodium falciparum and Toxoplasma gondii Reveal Unusual Adaptations Within and Beyond the Parasites' Boundaries
CELL HOST & MICROBE
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
A function for cyclin D1 in DNA repair uncovered by protein interactome analyses in human cancers
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
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
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
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
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
- Reproductive Aging Drives Protein Accumulation in the Uterus and Limits Lifespan in C. elegans PLOS GENETICS 2015; 11 (12)
Quantitative Imaging of Gut Microbiota Spatial Organization
CELL HOST & MICROBE
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
A Proteomic Strategy Identifies Lysine Methylation of Splicing Factor snRNP70 by the SETMAR Enzyme
JOURNAL OF BIOLOGICAL CHEMISTRY
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
Calcineurin regulates the yeast synaptojanin Inp53/Sjl3 during membrane stress
MOLECULAR BIOLOGY OF THE CELL
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 Web of Science ID 000351945000016
- Role of disease-associated tolerance in infectious superspreaders PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 2014; 111 (44): 15780-15785
Role of disease-associated tolerance in infectious superspreaders.
Proceedings of the National Academy of Sciences of the United States of America
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
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
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 Web of Science ID 000340517500030
- The Calcium-Dependent Protein Kinase 3 of Toxoplasma Influences Basal Calcium Levels and Functions beyond Egress as Revealed by Quantitative Phosphoproteome Analysis PLOS PATHOGENS 2014; 10 (6)
The calcium-dependent protein kinase 3 of toxoplasma influences basal calcium levels and functions beyond egress as revealed by quantitative phosphoproteome analysis.
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
A Role for the MRN Complex in ATR Activation via TOPBP1 Recruitment
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
Transcriptional role of cyclin D1 in development revealed by a genetic-proteomic screen
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.)
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
Identification of beta-Secretase (BACE1) Substrates Using Quantitative Proteomics
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
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
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
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
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
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
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
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
2005; 4 (10): 1459-1470
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
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
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
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
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 http://llama.med.harvard.edu/Software.html.
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
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
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
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