Translation regulation is a critical means by which cells control growth, division, and apoptosis. To gain further insight into translation and related processes, we performed multifaceted mass spectrometry-based proteomic screens of yeast ribosomal complexes and discovered an association of 77 uncharacterized yeast proteins with ribosomes. Immunoblotting revealed an EDTA-dependent cosedimentation with ribosomes in sucrose gradients for 11 candidate translation-machinery-associated (TMA) proteins. Tandem affinity purification linked one candidate, LSM12, to the RNA processing proteins PBP1 and PBP4. A second candidate, TMA46, interacted with RBG1, a GTPase that interacts with ribosomes. By adapting translation assays to high-throughput screening methods, we showed that null yeast strains harboring deletions for several of the TMA genes had alterations in protein synthesis rates (TMA7 and TMA19), susceptibility to drugs that inhibit translation (TMA7), translation fidelity (TMA20), and polyribosome profiles (TMA7, TMA19, and TMA20). TMA20 has significant sequence homology with the oncogene MCT-1. Expression of human MCT-1 in the ⌬tma20 yeast mutant complemented translation-related defects, strongly implying that MCT-1 functions in translation-related processes. Together these findings implicate the TMA proteins and, potentially, their human homologs, in translation related processes.[Keywords: Mass spectrometry; proteomics; ribosome; Saccharomyces cerevisiae; translation] Supplemental material is available at http://www.genesdev.org.

In conclusion, we demonstrate a microfiltration isolation method that preserves the exosome structure, reduces contamination from higher abundant urinary proteins, and can be easily implemented into mass spectrometry analysis for biomarker discovery efforts or incorporation into routine clinical laboratory applications to yield higher sample throughput.

CD8 + T cells (TCD8) confer protective immunity against many infectious diseases, suggesting that microbial TCD8 determinants are promising vaccine targets. Nevertheless, current T cell antigen identification approaches do not discern which epitopes drive protective immunity during active infection -information that is critical for the rational design of TCD8-targeted vaccines. We employed a proteomics-based approach for large-scale discovery of naturally processed determinants derived from a complex pathogen, vaccinia virus (VACV), that are presented by the most frequent representatives of four major HLA class I supertypes. Immunologic characterization revealed that many previously unidentified VACV determinants were recognized by smallpox-vaccinated human peripheral blood cells in a variegated manner. Many such determinants were recognized by HLA class I-transgenic mouse immune TCD8 too and elicited protective TCD8 immunity against lethal intranasal VACV infection. Notably, efficient processing and stable presentation of immune determinants as well as the availability of naive TCD8 precursors were sufficient to drive a multifunctional, protective TCD8 response. Our approach uses fundamental insights into T cell epitope processing and presentation to define targets of protective TCD8 immunity within human pathogens that have complex proteomes, suggesting that this approach has general applicability in vaccine sciences.

The SAGA histone acetyltransferase and TFIID complexes play key roles in eukaryotic transcription. Using hierarchical cluster analysis of mass spectrometry data to identify proteins that copurify with components of the budding yeast TFIID transcription complex, we discovered that an uncharacterized protein corresponding to the YPL047W open reading frame significantly associated with shared components of the TFIID and SAGA complexes. Using mass spectrometry and biochemical assays, we show that YPL047W (SGF11, 11-kDa SAGAassociated factor) is an integral subunit of SAGA. However, SGF11 does not appear to play a role in SAGA-mediated histone acetylation. DNA microarray analysis showed that SGF11 mediates transcription of a subset of SAGA-dependent genes, as well as SAGA-independent genes. SAGA purified from a sgf11⌬ deletion strain has reduced amounts of Ubp8p, and a ubp8⌬ deletion strain shows changes in transcription similar to those seen with the sgf11⌬ deletion strain. Together, these data show that Sgf11p is a novel component of the yeast SAGA complex and that SGF11 regulates transcription of a subset of SAGA-regulated genes. Our data suggest that the role of SGF11 in transcription is independent of SAGA's histone acetyltransferase activity but may involve Ubp8p recruitment to or stabilization in SAGA.A major component in the regulation of eukaryotic transcription initiation is the recruitment of transcriptional machinery to promoter elements that are arranged in tightly packed chromatin structures (21,23,30,34). A number of multiprotein complexes that mediate chromatin remodeling and promote transcriptional activity have been characterized (22,32). There are two general mechanisms by which these complexes alter chromatin structure. One class of complexes alters chromatin structure in an ATP-dependent manner (19,20). A second class regulates the chromatin structure by covalently modifying core histone proteins (H2A, H2B, H3, and H4) (31,45). Although several different histone modifications have been observed including phosphorylation, ubiquitination, and methylation (7,16,28,47), lysine acetylation catalyzed by histone acetyltransferase (HAT) enzymes is the best-characterized. Several multiprotein HAT complexes have been characterized in yeast. These include SAGA, ADA, NuA4, and NuA3 (reviewed in reference 43).Of the four known HAT complexes in yeast, SAGA is the best characterized. The name SAGA refers to its composition of Spt proteins (Spt3p, Spt7p, Spt8p, and Spt20p), Ada proteins (Ada1p, Ada2p, and Ada3p), and Gcn5p acetyltransferase (15). In addition to these components, SAGA also contains Tra1p and a subset of TATA-binding protein associated factors (TAFs), proteins originally identified as members of the TFIID transcription complex (13, 32). These include Taf5p, Taf6p, Taf9p, Taf10p, and Taf12p. A variant of the SAGA complex, named SALSA (SAGA altered, Spt8p absent) or SLIK (SAGA-like), has also been described (35,42). This version of SAGA lacks Spt8p and has a truncated form of Spt7p. It has been show...

Although generating large amounts of proteomic data using tandem mass spectrometry has become routine, there is currently no single set of comprehensive tools for the rigorous analysis of tandem mass spectrometry results given the large variety of possible experimental aims. Currently available applications are typically designed for displaying proteins and posttranslational modifications from the point of view of the mass spectrometrist and are not versatile enough to allow investigators to develop biological models of protein function, protein structure, or cell state. In addition, storage and dissemination of mass spectrometry-based proteomic data are problems facing the scientific community. To address these issues, we have developed a relational database model that efficiently stores and manages large amounts of tandem mass spectrometry results. We have developed an integrated suite of multifunctional analysis software for interpreting, comparing, and displaying these results. The rapid advance of proteomics is driving the development of new methods and applications for dissecting networks of protein interactions, identifying posttranslational modifications that modulate biological processes, and monitoring changes in whole proteomes. Over the past several years, LC-MS/MS has emerged as a powerful method to identify and quantify large numbers of proteins. Using LC-ESI-MS/MS or LC-MALDI-MS/MS, tens to hundreds of thousands of fragmentation spectra can be collected in a single tandem mass spectrometry experiment from purified protein complexes, subcellular fractions, or whole cell lysates (1-5). Analyzing this wealth of data to construct models of protein function, protein structure, or cell states is a major challenge. Current software applications to identify proteins from tandem mass spectrometry experiments typically deliver a simple list of accession numbers and gene names with minimal biological annotation. Most output reports focus on the experimental mass spectrometry information rather than the biological content. Presenting proteins in a more biologically intuitive format based on functional similarity and expression levels via graphical summaries would significantly simplify the interpretation of large lists of proteins.With the many spectra being collected, primary interpretation of the data to identify peptides and proteins has become dependent on computer analysis. Numerous computer algorithms have been developed to compare the measured values of the precursor ion and its fragmentation ions to the theoretical masses of peptides and fragmentation products derived from protein sequences in a database. Three of the most widely used search algorithms of this type, SEQUEST, MAS-COT, and X!Tandem, return for each spectrum the peptide sequences in a protein sequence database that best match the spectral data (6 -11). However, determining whether the peptide sequence truly represents the data is more difficult. MASCOT and X!Tandem calculate the probability that the identified peptide is not a stochastic...

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To identify protein–protein interactions and phosphorylated amino acid sites in eukaryotic mRNA translation, replicate TAP‐MudPIT and control experiments are performed targeting Saccharomyces cerevisiae genes previously implicated in eukaryotic mRNA translation by their genetic and/or functional roles in translation initiation, elongation, termination, or interactions with ribosomal complexes. Replicate tandem affinity purifications of each targeted yeast TAP‐tagged mRNA translation protein coupled with multidimensional liquid chromatography and tandem mass spectrometry analysis are used to identify and quantify copurifying proteins. To improve sensitivity and minimize spurious, nonspecific interactions, a novel cross‐validation approach is employed to identify the most statistically significant protein–protein interactions. Using experimental and computational strategies discussed herein, the previously described protein composition of the canonical eukaryotic mRNA translation initiation, elongation, and termination complexes is calculated. In addition, statistically significant unpublished protein interactions and phosphorylation sites for S. cerevisiae’s mRNA translation proteins and complexes are identified.

Rational design of CD8+ T cell (TCD8)-based vaccines requires knowledge of the immunogenic and protective epitopes presented during infection, information which is currently lacking. Using the clinically successful smallpox vaccine as a model, ~200 novel naturally processed vaccinia viral peptides presented by HLA-A*0201 and -B*0702 molecules were identified and characterized. Humans showed a variegated response to these determinants, reminiscent of the hierarchic response seen in HLA class I transgenic mice. Importantly, multiple TCD8 epitopes were commonly recognized by humans and mice and identified potential targets for protective TCD8-mediated immunity. After acute infection, both dominant and subdominant TCD8 specificities exhibited all of the immunologic features necessary for protection. However, early and efficient presentation of immune determinants during infection ensured protective responses, regardless of dominance, such that subunit vaccination targeting subdominant or recessive TCD8 specificities conferred protection against lethal poxvirus challenge. Hence, an in-depth knowledge of naturally processed T cell epitopes coupled with the identification of TCD8-based targets that protect from lethal poxviral infection are essential for rational vaccine design.