Proteome-wide Manner Research Articles

PathMHC: a workflow to selectively target pathogen-derived MHC peptides in discovery immunopeptidomics experiments for vaccine target identification.

Vaccine-elicited T cell responses can contribute to immune protection against emerging infectious disease risks such as antimicrobials-resistant (AMR) microbial pathogens and viruses with pandemic potential, but rapidly identifying appropriate targets for T cell priming vaccines remains challenging. Mass spectrometry (MS) analysis of peptides presented on major histocompatibility complexes (MHCs) can identify potential targets for protective T cell responses in a proteome-wide manner. However, pathogen-derived peptides are outnumbered by self peptides in the MHC repertoire and may be missed in untargeted MS analyses. Here we present a novel approach, termed PathMHC, that uses computational analysis of untargeted MS data followed by targeted MS to discover novel pathogen-derived MHC peptides more efficiently than untargeted methods alone. We applied this workflow to identify MHC peptides derived from multiple microbes, including potential vaccine targets presented on MHC-I by human dendritic cells infected with Mycobacterium tuberculosis . PathMHC will facilitate antigen discovery campaigns for vaccine development.

PSSMSearch: a server for modeling, visualization, proteome-wide discovery and annotation of protein motif specificity determinants.

There is a pressing need for in silico tools that can aid in the identification of the complete repertoire of protein binding (SLiMs, MoRFs, miniMotifs) and modification (moiety attachment/removal, isomerization, cleavage) motifs. We have created PSSMSearch, an interactive web-based tool for rapid statistical modeling, visualization, discovery and annotation of protein motif specificity determinants to discover novel motifs in a proteome-wide manner. PSSMSearch analyses proteomes for regions with significant similarity to a motif specificity determinant model built from a set of aligned motif-containing peptides. Multiple scoring methods are available to build a position-specific scoring matrix (PSSM) describing the motif specificity determinant model. This model can then be modified by a user to add prior knowledge of specificity determinants through an interactive PSSM heatmap. PSSMSearch includes a statistical framework to calculate the significance of specificity determinant model matches against a proteome of interest. PSSMSearch also includes the SLiMSearch framework’s annotation, motif functional analysis and filtering tools to highlight relevant discriminatory information. Additional tools to annotate statistically significant shared keywords and GO terms, or experimental evidence of interaction with a motif-recognizing protein have been added. Finally, PSSM-based conservation metrics have been created for taxonomic range analyses. The PSSMSearch web server is available at http://slim.ucd.ie/pssmsearch/.

Tandem Affinity Purification of Protein Complexes from Arabidopsis Cell Cultures.

Tandem affinity purification (TAP) coupled to mass spectrometry has become a powerful approach to identify protein-protein interactions from different biological systems, including plants, in a proteome-wide manner. By using two sequential affinity purification steps, TAP allows for isolation of high-purity TAP-tagged proteins of interest and their associated proteins. Here we describe optimized procedures to use the GSRhino TAP technology for protein complex isolation from Arabidopsis cell suspension cultures.

Strategy Based on Deglycosylation, Multiprotease, and Hydrophilic Interaction Chromatography for Large-Scale Profiling of Protein Methylation.

Reversible methylation of proteins regulates the majority of cellular processes, including signal transduction, mRNA splicing, transcriptional control, DNA repair, and protein translocation. A fundamental understanding of these biological processes at the molecular level requires comprehensive characterization of the methylated proteins. Methylation is often substoichiometric, and only a very limited number of methylated proteins and sites have been confidently identified to date. Although the intrinsically basic/hydrophilic methylated peptides can be enriched by the hydrophilic interaction liquid chromatography (HILIC), other hydrophilic peptides can coelute during the enrichment process and suppress the detection of methylated peptides. In addition, the modified Arg and Lys residues cannot be efficiently cleaved by trypsin, the most commonly used enzyme in shotgun proteomics. To overcome these caveats, we develop a novel de-glyco-assisted methylation site identification (DOMAIN) strategy which enables straightforward, fast, and reproducible analysis of protein methylation in a proteome-wide manner. Combining multidimensional fractionation and multiprotease digestion, our method enabled the identification of 573 methylated forms in 270 proteins, including 311 new methylation forms, in A549 cells. Combining this technique with stable isotope labeling quantitative proteomics and RNA interference, we determined the differential regulation of several putative methylated sites that are related to the protein arginine N-methyltransferase 3 (PRMT3). Collectively, our integrated proteomics workflow for comprehensive mapping of methylation sites enables a better understanding of protein methylation, while providing a rapid and effective approach for global protein methylation analysis in biomedical research.

Identification of RNA-binding domains of RNA-binding proteins in cultured cells on a system-wide scale with RBDmap.

This protocol is an extension to: Nat. Protoc. 8, 491-500 (2013); doi:10.1038/nprot.2013.020; published online 14 February 2013RBDmap is a method for identifying, in a proteome-wide manner, the regions of RNA-binding proteins (RBPs) engaged in native interactions with RNA. In brief, cells are irradiated with UV light to induce protein-RNA cross-links. Following stringent denaturing washes, the resulting covalently linked protein-RNA complexes are purified with oligo(dT) magnetic beads. After elution, RBPs are subjected to partial proteolysis, in which the protein regions still bound to the RNA and those released to the supernatant are separated by a second oligo(dT) selection. After sample preparation and mass-spectrometric analysis, peptide intensity ratios between the RNA-bound and released fractions are used to determine the RNA-binding regions. As a Protocol Extension, this article describes an adaptation of an existing Protocol and offers additional applications. The earlier protocol (for the RNA interactome capture method) describes how to identify the active RBPs in cultured cells, whereas this Protocol Extension also enables the identification of the RNA-binding domains of RBPs. The experimental workflow takes 1 week plus 2 additional weeks for proteomics and data analysis. Notably, RBDmap presents numerous advantages over classic methods for determining RNA-binding domains: it produces proteome-wide, high-resolution maps of the protein regions contacting the RNA in a physiological context and can be adapted to different biological systems and conditions. Because RBDmap relies on the isolation of polyadenylated RNA via oligo(dT), it will not provide RNA-binding information on proteins interacting exclusively with nonpolyadenylated transcripts. Applied to HeLa cells, RBDmap uncovered 1,174 RNA-binding sites in 529 proteins, many of which were previously unknown.

A Quantitative Proteomic Analysis of In Vitro Assembled Chromatin

The structure of chromatin is critical for many aspects of cellular physiology and is considered to be the primary medium to store epigenetic information. It is defined by the histone molecules that constitute the nucleosome, the positioning of the nucleosomes along the DNA and the non-histone proteins that associate with it. These factors help to establish and maintain a largely DNA sequence-independent but surprisingly stable structure. Chromatin is extensively disassembled and reassembled during DNA replication, repair, recombination or transcription in order to allow the necessary factors to gain access to their substrate. Despite such constant interference with chromatin structure, the epigenetic information is generally well maintained. Surprisingly, the mechanisms that coordinate chromatin assembly and ensure proper assembly are not particularly well understood. Here, we use label free quantitative mass spectrometry to describe the kinetics of in vitro assembled chromatin supported by an embryo extract prepared from preblastoderm Drosophila melanogaster embryos. The use of a data independent acquisition method for proteome wide quantitation allows a time resolved comparison of in vitro chromatin assembly. A comparison of our in vitro data with proteomic studies of replicative chromatin assembly in vivo reveals an extensive overlap showing that the in vitro system can be used for investigating the kinetics of chromatin assembly in a proteome-wide manner.

Carboxyterminal protein processing in health and disease: key actors and emerging technologies.

Carboxypeptidases are important mediators of cellular behavior. Through C-terminal truncations, they alter protein functionality and participate in proteome turnover. Similarly, carboxypeptidases shape the human peptidome by targeting neuroendocrine and vasoactive peptides, thereby regulating signaling pathways in the nervous and cardiovascular systems as well as in embryonic development. Carboxypeptidases are widely connected to various pathological processes such as carcinogenesis and neurodegenerative and cardiovascular diseases. The repertoire of carboxypeptidase in vivo substrates still remains poorly defined, largely due to the lack of suitable experimental approaches. Understanding the precise role of carboxypeptidases is pivotal in the future development of diagnostic/prognostic markers in such diseases. To date, very little attention has been paid to the implication of carboxypeptidases in shaping the proteome as well as the peptidome. This review focuses on the patho-physiological function of carboxypeptidases and highlights the approaches by which proteomics-based technologies can be applied to characterize carboxypeptidases and to quantify the differential regulation of proteins by carboxypeptidases in a proteome-wide manner.

HLA Supertypes Contribute in HIV Type 1 Cytotoxic T Lymphocyte Epitope Clustering in Nef and Gag Proteins

Induction of HIV-1-specific cytotoxic T lymphocyte (CTL) responses largely depends upon the presentation of CTL epitopes to the CD8(+) T cells aided by a large number of different HLA class I alleles. Although several studies showed the clustering pattern of HIV-1 CTL epitopes, the underlying reason for this tendency remains unresolved. Moreover, the hypothesis that the CTL epitope clusters tend to coincide with the conserved and hydrophobic regions of HIV-1 proteins has been challenged in recent times. The present study aims to characterize and compare the HIV-1 CTL epitope clusters in terms of restricting HLA alleles, hydrophobicity, and sequence conservation in a proteome-wide manner by including a large number of experimentally validated CTL epitopes from the HIV Molecular Immunology Database. CTL epitope cluster distribution analysis in a proteome-wide manner revealed that only two HIV-1 proteins, namely Nef and Gag, have significant cluster-forming capacity where their epitope localization coincides with the hydrophobic and conserved regions. Furthermore, analyses of proteasomal cleavage sites and HLA anchoring motif frequencies in the epitope-dense regions highlighted the role of specific HLA supertypes such as HLA B*07, HLA B*58, HLA A*02, and HLA A*03 in selecting the hydrophobic and conserved amino acid positions within Nef and Gag proteins to be presented as epitopes. Based on our results, we hypothesize that the cluster-forming tendency of HIV-1 CTL epitopes is not a proteome-wide feature confined to Nef and Gag proteins. Their cluster-forming tendency largely depends on the host HLA alleles that contribute significantly in selecting functionally constrained hydrophobic regions within the HIV-1 proteome.

Cathepsin S Mediates Gastric Cancer Cell Migration and Invasion via a Putative Network of Metastasis-Associated Proteins

Cancer progression is governed by multifaceted interactions of cancer cells with their microenvironment and one of these ways is through secreted compounds. Substances released by gastric cancer cells have not being profiled in a proteome-wide manner. ITRAQ-based tandem mass spectrometry was employed to quantify proteins secreted by HFE145 normal, MKN7 well-differentiated, and MKN45 poorly differentiated gastric cancer cell lines. The expression levels of 237 proteins were found to be significantly different between normal and cancer cells. Further examination of 16 gastric cell lines and 115 clinical samples validated the up-regulation of CTSS expression in gastric cancer. Silencing CTSS expression suppressed the migration and invasion of gastric cancer cells in vitro. Subsequent secretomics revealed that CTSS silencing resulted in changes in expression levels of 197 proteins, one-third of which are implicated in cellular movement. Proteome-wide comparative secretomes of normal and gastric cancer cells were produced that constitute a useful resource for gastric cancer research. CTSS was demonstrated to play novel roles in gastric cancer cell migration and invasion, putatively via a network of proteins associated with cell migration, invasion, or metastasis. Cathepsin S is member of a large group of extracellular proteases, which are attractive drug targets. The implicated role of CTSS in gastric cancer metastasis provides an opportunity to test existing compounds against CTSS for adjuvant therapy and/or treatment of metastatic gastric cancers.

Phosphoproteomics by mass spectrometry: insights, implications, applications and limitations

Phosphorylation of proteins is a predominant, reversible post-translational modification. It is central to a wide variety of physiological responses and signaling mechanisms. Recent advances have allowed the global scope of phosphorylation to be addressed by mass spectrometry using phosphoproteomic approaches. In this perspective, we discuss four aspects of phosphoproteomics: the insights and implications from recently published phosphoproteomic studies and the applications and limitations of current phosphoproteomic strategies. Since approximately 50,000 known phosphorylation sites do not yet have any ascribed function, we present our perspectives on a major function of protein phosphorylation that may be of predictive value in hypothesis-based investigations. Finally, we discuss strategies to measure the stoichiometry of phosphorylation in a proteome-wide manner that is not provided by current phosphoproteomic approaches.

Proteomic and Biochemical Studies of Calcium- and Phosphorylation-Dependent Calmodulin Complexes in Mammalian Cells

Protein conformational changes due to cofactor binding (e.g., metal ions, heme) and/or post-translational modifications (e.g., phosphorylation) modulate dynamic protein complexes. Calmodulin (CaM) plays an essential role in regulating calcium signaling and homeostasis. Herein, we report a straightforward and systematic approach to identify potential calcium- and phosphorylation-dependent CaM complexes in a proteome-wide manner. We have identified over 120 CaM-associated proteins encompassing four different classes of CaM binding in HeLa cells, namely, calcium-dependent and phosphorylation-dependent (e.g., EDD1), calcium-dependent and phosphorylation-independent (e.g., myosin IE), calcium-independent and phosphorylation-dependent (e.g., DDX3), and calcium-independent and phosphorylation-independent (e.g., DDX5). To demonstrate the utility of our method in understanding biological pathways, we showed that in vivo phosphorylation of inositol 1,4,5-triphosphate receptor type 1 (IP3R1) at Ser1598 significantly reduced the affinity of its Ca2+-dependent CaM binding. However, phosphorylation of IP3R1 did not substantially affect its Ca2+-independent CaM binding. These results shed new lights on the mechanism underlying the marked increase of Ca2+ release due to IP3R1 phosphorylation. We further showed that staurosporine-sensitive kinase(s) and phosphatase PP1 play a critical role in modulating the phosphorylation-dependent CaM binding of IP3R1. Our method may serve as a general strategy to identify and characterize phosphorylation-dependent protein complexes, to pinpoint the phosphorylation sites and associated kinase(s) and phosphatase(s) involved in the protein-protein interactions, and to functionally characterize these complexes in mammalian cells.