Panoramic Perspective on Human Phosphosites.

Protein phosphorylation is the most common reversible post-translational modification of proteins and is key in the regulation of many cellular processes. Due to this importance, phosphorylation is extensively studied, resulting in the availability of a large amount of mass spectrometry-based phospho-proteomics data. Here, we leverage the information in these large-scale phospho-proteomics data sets, as contained in Scop3P, to analyze and characterize proteome-wide protein phosphorylation sites (P-sites). First, we set out to differentiate correctly observed P-sites from false-positive sites using five complementary site properties. We then describe the context of these P-sites in terms of the protein structure, solvent accessibility, structural transitions and disorder, and biophysical properties. We also investigate the relative prevalence of disease-linked mutations on and around P-sites. Moreover, we assess the structural dynamics of P-sites in their phosphorylated and unphosphorylated states. As a result, we show how large-scale reprocessing of available proteomics experiments can enable a more reliable view on proteome-wide P-sites. Furthermore, adding the structural context of proteins around P-sites helps uncover possible conformational switches upon phosphorylation. Moreover, by placing sites in different biophysical contexts, we show the differential preference in protein dynamics at phosphorylated sites when compared to the nonphosphorylated counterparts.

Sensitive and Specific Spectral Library Searching with CompOmics Spectral Library Searching Tool and Percolator.

Maintaining high sensitivity while limiting false positives is a key challenge in peptide identification from mass spectrometry data. Here, we investigate the effects of integrating the machine learning-based postprocessor Percolator into our spectral library searching tool COSS (CompOmics Spectral library Searching tool). To evaluate the effects of this postprocessing, we have used 40 data sets from 2 different projects and have searched these against the NIST and MassIVE spectral libraries. The searching is carried out using 2 spectral library search tools, COSS and MSPepSearch with and without Percolator postprocessing, and using sequence database search engine MS-GF+ as a baseline comparator. The addition of the Percolator rescoring step to COSS is effective and results in a substantial improvement in sensitivity and specificity of the identifications. COSS is freely available as open source under the permissive Apache2 license, and binaries and source code are found at https://github.com/compomics/COSS.

The CEP5 Peptide Promotes Abiotic Stress Tolerance, As Revealed by Quantitative Proteomics, and Attenuates the AUX/IAA Equilibrium in Arabidopsis.

Peptides derived from non-functional precursors play important roles in various developmental processes, but also in (a)biotic stress signaling. Our (phospho)proteome-wide analyses of C-TERMINALLY ENCODED PEPTIDE 5 (CEP5)-mediated changes revealed an impact on abiotic stress-related processes. Drought has a dramatic impact on plant growth, development and reproduction, and the plant hormone auxin plays a role in drought responses. Our genetic, physiological, biochemical, and pharmacological results demonstrated that CEP5-mediated signaling is relevant for osmotic and drought stress tolerance in Arabidopsis, and that CEP5 specifically counteracts auxin effects. Specifically, we found that CEP5 signaling stabilizes AUX/IAA transcriptional repressors, suggesting the existence of a novel peptide-dependent control mechanism that tunes auxin signaling. These observations align with the recently described role of AUX/IAAs in stress tolerance and provide a novel role for CEP5 in osmotic and drought stress tolerance.

COSS: A Fast and User-Friendly Tool for Spectral Library Searching.

Spectral similarity searching to identify peptide-derived MS/MS spectra is a promising technique, and different spectrum similarity search tools have therefore been developed. Each of these tools, however, comes with some limitations, mainly because of low processing speed and issues with handling large databases. Furthermore, the number of spectral data formats supported is typically limited, which also creates a threshold to adoption. We have therefore developed COSS (CompOmics Spectral Searching), a new and user-friendly spectral library search tool supporting two scoring functions. COSS also includes decoy spectra generation for result validation. We have benchmarked COSS on three different spectral libraries and compared the results with established spectral searching tools and a sequence database search tool. Our comparison showed that COSS more reliably identifies spectra, is capable of handling large data sets and libraries, and is an easy to use tool that can run on low computer specifications. COSS binaries and source code can be freely downloaded from https://github.com/compomics/COSS.

Scop3P: A Comprehensive Resource of Human Phosphosites within Their Full Context.

Protein phosphorylation is a key post-translational modification in many biological processes and is associated to human diseases such as cancer and metabolic disorders. The accurate identification, annotation, and functional analysis of phosphosites are therefore crucial to understand their various roles. Phosphosites are mainly analyzed through phosphoproteomics, which has led to increasing amounts of publicly available phosphoproteomics data. Several resources have been built around the resulting phosphosite information, but these are usually restricted to the protein sequence and basic site metadata. What is often missing from these resources, however, is context, including protein structure mapping, experimental provenance information, and biophysical predictions. We therefore developed Scop3P: a comprehensive database of human phosphosites within their full context. Scop3P integrates sequences (UniProtKB/Swiss-Prot), structures (PDB), and uniformly reprocessed phosphoproteomics data (PRIDE) to annotate all known human phosphosites. Furthermore, these sites are put into biophysical context by annotating each phosphoprotein with per-residue structural propensity, solvent accessibility, disordered probability, and early folding information. Scop3P, available at https://iomics.ugent.be/scop3p, presents a unique resource for visualization and analysis of phosphosites and for understanding of phosphosite structure-function relationships.

Bcl-2 and IP3 compete for the ligand-binding domain of IP3Rs modulating Ca2+ signaling output.

Bcl-2 proteins have emerged as critical regulators of intracellular Ca2+ dynamics by directly targeting and inhibiting the IP3 receptor (IP3R), a major intracellular Ca2+-release channel. Here, we demonstrate that such inhibition occurs under conditions of basal, but not high IP3R activity, since overexpressed and purified Bcl-2 (or its BH4 domain) can inhibit IP3R function provoked by low concentration of agonist or IP3, while fails to attenuate against high concentration of agonist or IP3. Surprisingly, Bcl-2 remained capable of inhibiting IP3R1 channels lacking the residues encompassing the previously identified Bcl-2-binding site (a.a. 1380-1408) located in the ARM2 domain, part of the modulatory region. Using a plethora of computational, biochemical and biophysical methods, we demonstrate that Bcl-2 and more particularly its BH4 domain bind to the ligand-binding domain (LBD) of IP3R1. In line with this finding, the interaction between the LBD and Bcl-2 (or its BH4 domain) was sensitive to IP3 and adenophostin A, ligands of the IP3R. Vice versa, the BH4 domain of Bcl-2 counteracted the binding of IP3 to the LBD. Collectively, our work reveals a novel mechanism by which Bcl-2 influences IP3R activity at the level of the LBD. This allows for exquisite modulation of Bcl-2's inhibitory properties on IP3Rs that is tunable to the level of IP3 signaling in cells.

Scop3D: Online Visualization of Mutation Rates on Protein Structure.

Scop3D is a tool that automatically annotates protein structure with sequence conservation starting from a set of protein sequence variants. We present a complete upgrade and rewrite of Scop3D. We have included a DNA module that allows the analysis of single nucleotide polymorphisms in relation to the structural context of the protein. Scop3D therefore forms a bridge between genomics and protein structure. Moreover, Scop3D is now also available through an intuitive web-interface that makes the tool highly user-friendly.

Cross-linked peptide identification: A computational forest of algorithms.

Chemical cross-linking analyzed by mass spectrometry (XL-MS) has become an important tool in unravelling protein structure, dynamics, and complex formation. Because the analysis of cross-linked proteins with mass spectrometry results in specific computational challenges, many computational tools have been developed to identify cross-linked peptides from mass spectra and subsequently interpret the identified cross-links within their structural context. In this review, we will provide an overview of the different tools that are currently available to tackle the computational part of an XL-MS experiment. First, we give an introduction on the computational challenges encountered when processing data from a cross-linking experiment. We then discuss available tools to identify peptides that are linked by intact or MS-cleavable cross-linkers, and we provide an overview of tools to interpret cross-linked peptides in the context of protein structure. Finally, we give an outlook on data management and dissemination challenges and opportunities for cross-linking experiments.

Resolution of protein structure by mass spectrometry.

Typically, mass spectrometry is used to identify the peptides present in a complex peptide mixture and subsequently the precursor proteins. As such, mass spectrometry focuses mainly on the primary structure, the (modified) amino acid sequence of peptides and proteins. In contrast, the three-dimensional structure of a protein is typically determined with protein X-ray crystallography or NMR. Despite the close relationship between these two aspects of protein studies (sequence and structure), mass spectrometry and structure determination are not frequently combined. Nevertheless, this combination of approaches, dubbed conformational proteomics, can offer insight into the function, working mechanism, and conformational status of a protein. In this review, we will discuss the developments at the intersection of mass spectrometry-based proteomics and protein structure determination and start from a brief overview of the classic approaches to identify protein structure along with their advantages and disadvantages. We will subsequently discuss the ability of mass spectrometry to overcome some of the hurdles of these classic methods. Finally, we will provide an outlook on the interplay of mass spectrometry and protein structure determination, and highlight several recent experiments in which mass spectrometry was successfully used to either aid or complement structure elucidation. © 2014 Wiley Periodicals, Inc. Mass Spec Rev 35:653-665, 2016.

A Pipeline for Differential Proteomics in Unsequenced Species.

Shotgun proteomics experiments often take the form of a differential analysis, where two or more samples are compared against each other. The objective is to identify proteins that are either unique to a specific sample or a set of samples (qualitative differential proteomics), or that are significantly differentially expressed in one or more samples (quantitative differential proteomics). However, the success depends on the availability of a reliable protein sequence database for each sample. To perform such an analysis in the absence of a database, we here propose a novel, generic pipeline comprising an adapted spectral similarity score derived from database search algorithms that compares samples at the spectrum level to detect unique spectra. We applied our pipeline to compare two parasitic tapeworms: Taenia solium and Taenia hydatigena, of which only the former poses a threat to humans. Furthermore, because the genome of T. solium recently became available, we were able to prove the effectiveness and reliability of our pipeline a posteriori.

Xilmass: A New Approach toward the Identification of Cross-Linked Peptides.

Chemical cross-linking coupled with mass spectrometry plays an important role in unravelling protein interactions, especially weak and transient ones. Moreover, cross-linking complements several structural determination approaches such as cryo-EM. Although several computational approaches are available for the annotation of spectra obtained from cross-linked peptides, there remains room for improvement. Here, we present Xilmass, a novel algorithm to identify cross-linked peptides that introduces two new concepts: (i) the cross-linked peptides are represented in the search database such that the cross-linking sites are explicitly encoded, and (ii) the scoring function derived from the Andromeda algorithm was adapted to score against a theoretical tandem mass spectrometry (MS/MS) spectrum that contains the peaks from all possible fragment ions of a cross-linked peptide pair. The performance of Xilmass was evaluated against the recently published Kojak and the popular pLink algorithms on a calmodulin-plectin complex data set, as well as three additional, published data sets. The results show that Xilmass typically had the highest number of identified distinct cross-linked sites and also the highest number of predicted cross-linked sites.

Scop3D: three-dimensional visualization of sequence conservation.

The integration of a protein's structure with its known sequence variation provides insight on how that protein evolves, for instance in terms of (changing) function or immunogenicity. Yet, collating the corresponding sequence variants into a multiple sequence alignment, calculating each position's conservation, and mapping this information back onto a relevant structure is not straightforward. We therefore built the Sequence Conservation on Protein 3D structure (scop3D) tool to perform these tasks automatically. The output consists of two modified PDB files in which the B-values for each position are replaced by the percentage sequence conservation, or the information entropy for each position, respectively. Furthermore, text files with absolute and relative amino acid occurrences for each position are also provided, along with snapshots of the protein from six distinct directions in space. The visualization provided by scop3D can for instance be used as an aid in vaccine development or to identify antigenic hotspots, which we here demonstrate based on an analysis of the fusion proteins of human respiratory syncytial virus and mumps virus.

PepShell: visualization of conformational proteomics data.

Proteins are dynamic molecules; they undergo crucial conformational changes induced by post-translational modifications and by binding of cofactors or other molecules. The characterization of these conformational changes and their relation to protein function is a central goal of structural biology. Unfortunately, most conventional methods to obtain structural information do not provide information on protein dynamics. Therefore, mass spectrometry-based approaches, such as limited proteolysis, hydrogen-deuterium exchange, and stable-isotope labeling, are frequently used to characterize protein conformation and dynamics, yet the interpretation of these data can be cumbersome and time consuming. Here, we present PepShell, a tool that allows interactive data analysis of mass spectrometry-based conformational proteomics studies by visualization of the identified peptides both at the sequence and structure levels. Moreover, PepShell allows the comparison of experiments under different conditions, including different proteolysis times or binding of the protein to different substrates or inhibitors.

Limited Proteolysis Combined with Stable Isotope Labeling Reveals Conformational Changes in Protein (Pseudo)kinases upon Binding Small Molecules.

Likely due to conformational rearrangements, small molecule inhibitors may stabilize the active conformation of protein kinases and paradoxically promote tumorigenesis. We combined limited proteolysis with stable isotope labeling MS to monitor protein conformational changes upon binding of small molecules. Applying this method to the human serine/threonine kinase B-Raf, frequently mutated in cancer, we found that binding of ATP or its nonhydrolyzable analogue AMP-PNP, but not ADP, stabilized the structure of both B-Raf(WT) and B-Raf(V600E). The ATP-competitive type I B-Raf inhibitor vemurafenib and the type II inhibitor sorafenib stabilized the kinase domain (KD) but had distinct effects on the Ras-binding domain. Stabilization of the B-Raf(WT) KD was confirmed by hydrogen/deuterium exchange MS and molecular dynamics simulations. Our results are further supported by cellular assays in which we assessed cell viability and phosphorylation profiles in cells expressing B-Raf(WT) or B-Raf(V600E) in response to vemurafenib or sorafenib. Our data indicate that an overall stabilization of the B-Raf structure by specific inhibitors activates MAPK signaling and increases cell survival, helping to explain clinical treatment failure. We also applied our method to monitor conformational changes upon nucleotide binding of the pseudokinase KSR1, which holds high potential for inhibition in human diseases.

Unraveling the specificities of the different human methionine sulfoxide reductases.

The oxidation of free and protein-bound methionine into methionine sulfoxide is a frequently occurring modification caused by ROS. Most organisms express methionine sulfoxide reductases (MSR enzymes) to repair this potentially damaging modification. Humans express three different MSRB enzymes which reside in different cellular compartments. In this study, we have explored the specificity of the human MSRB enzymes both by in silico modeling and by experiments on oxidized peptides. We found that MSRB1 is the least specific MSRB enzyme, which is in agreement with the observation that MSRB1 is the only MSRB enzyme found in the cytosol and the nucleus, and therefore requires a broad specificity to reduce all possible substrates. MSRB2 and MSRB3, which are both found in mitochondria, are more specific but because of their co-occurrence they can likely repair all possible substrates.

Getting intimate with trypsin, the leading protease in proteomics.

Nowadays, mass spectrometry-based proteomics is carried out primarily in a bottom-up fashion, with peptides obtained after proteolytic digest of a whole proteome lysate as the primary analytes instead of the proteins themselves. This experimental setup crucially relies on a protease to digest an abundant and complex protein mixture into a far more complex peptide mixture. Full knowledge of the working mechanism and specificity of the used proteases is therefore crucial, both for the digestion step itself as well as for the downstream identification and quantification of the (fragmentation) mass spectra acquired for the peptides in the mixture. Targeted protein analysis through selected reaction monitoring, a relative newcomer in the specific field of mass spectrometry-based proteomics, even requires a priori understanding of protease behavior for the proteins of interest. Because of the rapidly increasing popularity of proteomics as an analytical tool in the life sciences, there is now a renewed demand for detailed knowledge on trypsin, the workhorse protease in proteomics. This review addresses this need and provides an overview on the structure and working mechanism of trypsin, followed by a critical analysis of its cleavage behavior, typically simply accepted to occur exclusively yet consistently after Arg and Lys, unless they are followed by a Pro. In this context, shortcomings in our ability to understand and predict the behavior of trypsin will be highlighted, along with the downstream implications. Furthermore, an analysis is carried out on the inherent shortcomings of trypsin with regard to whole proteome analysis, and alternative approaches will be presented that can alleviate these issues. Finally, some reflections on the future of trypsin as the workhorse protease in mass spectrometry-based proteomics will be provided.

Protein structure as a means to triage proposed PTM sites.

PTMs such as phosphorylation are often important actors in protein regulation and recognition. These functions require both visibility and accessibility to other proteins; that the modification is located at the surface of the protein. Currently, many repositories provide information on PTMs but structural information is often lacking. This study, which focuses on phosphorylation sites available in UniProtKB/Swiss-Prot, illustrates that most phosphorylation sites are indeed found at the surface of the protein, but that some sites are found buried in the core of the protein. Several of these identified buried phosphorylation sites can easily become accessible upon small conformational changes while others would require the whole protein to unfold and are hence most unlikely modification sites. Subsequent analysis of phosphorylation sites available in PRIDE demonstrates that taking the structure of the protein into account would be a good guide in the identification of the actual phosphorylated positions in phophoproteomics experiments. This analysis illustrates that care must be taken when simply accepting the position of a PTM without first analyzing its position within the protein structure if the latter is available.

Predicting tryptic cleavage from proteomics data using decision tree ensembles.

Trypsin is the workhorse protease in mass spectrometry-based proteomics experiments and is used to digest proteins into more readily analyzable peptides. To identify these peptides after mass spectrometric analysis, the actual digestion has to be mimicked as faithfully as possible in silico. In this paper we introduce CP-DT (Cleavage Prediction with Decision Trees), an algorithm based on a decision tree ensemble that was learned on publicly available peptide identification data from the PRIDE repository. We demonstrate that CP-DT is able to accurately predict tryptic cleavage: tests on three independent data sets show that CP-DT significantly outperforms the Keil rules that are currently used to predict tryptic cleavage. Moreover, the trees generated by CP-DT can make predictions efficiently and are interpretable by domain experts.

Bcl-xL acts as an inhibitor of IP3R channels, thereby antagonizing Ca2+-driven apoptosis.

Anti-apoptotic Bcl-2-family members not only act at mitochondria but also at the endoplasmic reticulum, where they impact Ca2+ dynamics by controlling IP3 receptor (IP3R) function. Current models propose distinct roles for Bcl-2 vs. Bcl-xL, with Bcl-2 inhibiting IP3Rs and preventing pro-apoptotic Ca2+ release and Bcl-xL sensitizing IP3Rs to low [IP3] and promoting pro-survival Ca2+ oscillations. We here demonstrate that Bcl-xL too inhibits IP3R-mediated Ca2+ release by interacting with the same IP3R regions as Bcl-2. Via in silico superposition, we previously found that the residue K87 of Bcl-xL spatially resembled K17 of Bcl-2, a residue critical for Bcl-2's IP3R-inhibitory properties. Mutagenesis of K87 in Bcl-xL impaired its binding to IP3R and abrogated Bcl-xL's inhibitory effect on IP3Rs. Single-channel recordings demonstrate that purified Bcl-xL, but not Bcl-xLK87D, suppressed IP3R single-channel openings stimulated by sub-maximal and threshold [IP3]. Moreover, we demonstrate that Bcl-xL-mediated inhibition of IP3Rs contributes to its anti-apoptotic properties against Ca2+-driven apoptosis. Staurosporine (STS) elicits long-lasting Ca2+ elevations in wild-type but not in IP3R-knockout HeLa cells, sensitizing the former to STS treatment. Overexpression of Bcl-xL in wild-type HeLa cells suppressed STS-induced Ca2+ signals and cell death, while Bcl-xLK87D was much less effective in doing so. In the absence of IP3Rs, Bcl-xL and Bcl-xLK87D were equally effective in suppressing STS-induced cell death. Finally, we demonstrate that endogenous Bcl-xL also suppress IP3R activity in MDA-MB-231 breast cancer cells, whereby Bcl-xL knockdown augmented IP3R-mediated Ca2+ release and increased the sensitivity towards STS, without altering the ER Ca2+ content. Hence, this study challenges the current paradigm of divergent functions for Bcl-2 and Bcl-xL in Ca2+-signaling modulation and reveals that, similarly to Bcl-2, Bcl-xL inhibits IP3R-mediated Ca2+ release and IP3R-driven cell death. Our work further underpins that IP3R inhibition is an integral part of Bcl-xL's anti-apoptotic function.

Massively parallel interrogation of protein fragment secretability using SECRiFY reveals features influencing secretory system transit.

While transcriptome- and proteome-wide technologies to assess processes in protein biogenesis are now widely available, we still lack global approaches to assay post-ribosomal biogenesis events, in particular those occurring in the eukaryotic secretory system. We here develop a method, SECRiFY, to simultaneously assess the secretability of >105 protein fragments by two yeast species, S. cerevisiae and P. pastoris, using custom fragment libraries, surface display and a sequencing-based readout. Screening human proteome fragments with a median size of 50-100 amino acids, we generate datasets that enable datamining into protein features underlying secretability, revealing a striking role for intrinsic disorder and chain flexibility. The SECRiFY methodology generates sufficient amounts of annotated data for advanced machine learning methods to deduce secretability patterns. The finding that secretability is indeed a learnable feature of protein sequences provides a solid base for application-focused studies.