A search for ceramide binding proteins using bifunctional lipid analogs yields CERT-related protein StarD7.

Ceramides are central intermediates of sphingolipid metabolism with dual roles as mediators of cellular stress signaling and mitochondrial apoptosis. How ceramides exert their cytotoxic effects is unclear and their poor solubility in water hampers a search for specific protein interaction partners. Here, we report the application of a photoactivatable and clickable ceramide analog, pacCer, to identify ceramide binding proteins and unravel the structural basis by which these proteins recognize ceramide. Besides capturing ceramide transfer protein (CERT) from a complex proteome, our approach yielded CERT-related steroidogenic acute regulatory protein D7 (StarD7) as novel ceramide binding protein. Previous work revealed that StarD7 is required for efficient mitochondrial import of phosphatidylcholine (PC) and serves a critical role in mitochondrial function and morphology. Combining site-directed mutagenesis and photoaffinity labeling experiments, we demonstrate that the steroidogenic acute regulatory transfer domain of StarD7 harbors a common binding site for PC and ceramide. While StarD7 lacks robust ceramide transfer activity in vitro, we find that its ability to shuttle PC between model membranes is specifically affected by ceramides. Besides demonstrating the suitability of pacCer as a tool to hunt for ceramide binding proteins, our data point at StarD7 as a candidate effector protein by which ceramides may exert part of their mitochondria-mediated cytotoxic effects.

PRIME-XS, a European infrastructure for proteomics.

The PRIME-XS consortium is a pan-European infrastructure for proteomics. As a prologue to this special issue of Molecular & Cellular Proteomics on the research activities of the PRIME-XS consortium, we, as the guest editors of this issue, provide an overview of the structure and activities of this consortium, which is funded by the European Union's 7th Framework Programme for Research and Technological Development.

The human peptidylarginine deiminases type 2 and type 4 have distinct substrate specificities.

Human peptidylarginine deiminases (hPADs) have been implicated in several diseases, particularly in rheumatoid arthritis. Since hPAD2 and hPAD4 are the isotypes expressed in the inflamed joints of RA patients and protein citrullination by PADs has been proposed to play a pathophysiological role, they represent unique therapeutic targets. To facilitate the development of substrate-based PAD inhibitors the substrate specificity of hPAD2 and hPAD4 was determined. Recombinant hPADs were expressed in bacteria or mammalian cell lines and allowed to citrullinate proteins in cell lysates, as well as a series of synthetic peptides. The citrullinated residues in proteins and the efficiency of peptide citrullination were determined by mass spectrometry. In total 320 hPAD2 and 178 hPAD4 citrullination sites were characterized. Amino acid residues most commonly found in citrullination sites for both isotypes are Gly at +1 and Tyr at +3 relative to the target arginine. For hPAD4 several additional amino acids were observed to be preferred at various positions from -4 to +4. The substrate motifs determined by amino acid substitution analysis partially confirmed these preferences, although peptide context dependent differences were also observed. Taken together, our data show that the enzyme specificity for cellular substrates and synthetic peptides differs for hPAD2 and hPAD4. hPAD4 shows more restrictive substrate specificity compared to hPAD2. Consensus sequences, which can be used as the basis for the development of PAD inhibitors, were derived for the citrullination sites of both hPAD2 and hPAD4.

Dis3-like 1: a novel exoribonuclease associated with the human exosome.

The exosome is an exoribonuclease complex involved in the degradation and maturation of a wide variety of RNAs. The nine-subunit core of the eukaryotic exosome is catalytically inactive and may have an architectural function and mediate substrate binding. In Saccharomyces cerevisiae, the associated Dis3 and Rrp6 provide the exoribonucleolytic activity. The human exosome-associated Rrp6 counterpart contributes to its activity, whereas the human Dis3 protein is not detectably associated with the exosome. Here, a proteomic analysis of immunoaffinity-purified human exosome complexes identified a novel exosome-associated exoribonuclease, human Dis3-like exonuclease 1 (hDis3L1), which was confirmed to associate with the exosome core by co-immunoprecipitation. In contrast to the nuclear localization of Dis3, hDis3L1 exclusively localized to the cytoplasm. The hDis3L1 isolated from transfected cells degraded RNA in an exoribonucleolytic manner, and its RNB domain seemed to mediate this activity. The siRNA-mediated knockdown of hDis3L1 in HeLa cells resulted in elevated levels of poly(A)-tailed 28S rRNA degradation intermediates, indicating the involvement of hDis3L1 in cytoplasmic RNA decay. Taken together, these data indicate that hDis3L1 is a novel exosome-associated exoribonuclease in the cytoplasm of human cells.

Foresight in clinical proteomics: current status, ethical considerations, and future perspectives.

With the advent of robust and high-throughput mass spectrometric technologies and bioinformatics tools to analyze large data sets, proteomics has penetrated broadly into basic and translational life sciences research. More than 95% of FDA-approved drugs currently target proteins, and most diagnostic tests are protein-based. The introduction of proteomics to the clinic, for instance to guide patient stratification and treatment, is already ongoing. Importantly, ethical challenges come with this success, which must also be adequately addressed by the proteomics and medical communities. Consortium members of the H2020 European Union-funded proteomics initiative: European Proteomics Infrastructure Consortium-providing access (EPIC-XS) met at the Core Technologies for Life Sciences (CTLS) conference to discuss the emerging role and implementation of proteomics in the clinic. The discussion, involving leaders in the field, focused on the current status, related challenges, and future efforts required to make proteomics a more mainstream technology for translational and clinical research. Here we report on that discussion and provide an expert update concerning the feasibility of clinical proteomics, the ethical implications of generating and analyzing large-scale proteomics clinical data, and recommendations to ensure both ethical and effective implementation in real-world applications.

Deep proteome profiling of circulating granulocytes reveals bactericidal/permeability-increasing protein as a biomarker for severe atherosclerotic coronary stenosis.

Coronary atherosclerosis represents the major cause of death in Western societies. As atherosclerosis typically progresses over years without giving rise to clinical symptoms, biomarkers are urgently needed to identify patients at risk. Over the past decade, evidence has accumulated suggesting cross-talk between the diseased vasculature and cells of the innate immune system. We therefore employed proteomics to search for biomarkers associated with severe atherosclerotic coronary lumen stenosis in circulating leukocytes. In a two-phase approach, we first performed in-depth quantitative profiling of the granulocyte proteome on a small pooled cohort of patients suffering from chronic (sub)total coronary occlusion and matched control patients using stable isotope peptide labeling, two-dimensional LC-MS/MS and data-dependent decision tree fragmentation. Over 3000 proteins were quantified, among which 57 candidate biomarker proteins remained after stringent filtering. The most promising biomarker candidates were subsequently verified in the individual samples of the discovery cohort using label-free, single-run LC-MS/MS analysis, as well as in an independent verification cohort of 25 patients with total coronary occlusion (CTO) and 19 matched controls. Our data reveal bactericidal/permeability-increasing protein (BPI) as a promising biomarker for severe atherosclerotic coronary stenosis, being down-regulated in circulating granulocytes of CTO patients.

Applications of stable isotope dimethyl labeling in quantitative proteomics.

Mass spectrometry has proven to be an indispensable tool for protein identification, characterization, and quantification. Among the possible methods in quantitative proteomics, stable isotope labeling by using reductive dimethylation has emerged as a cost-effective, simple, but powerful method able to compete at any level with the present alternatives. In this review, we briefly introduce experimental and software methods for proteome analysis using dimethyl labeling and provide a comprehensive overview of reported applications in the analysis of (1) differential protein expression, (2) posttranslational modifications, and (3) protein interactions.

Fully automated isotopic dimethyl labeling and phosphopeptide enrichment using a microfluidic HPLC phosphochip.

Quantitative detection of phosphorylation levels is challenging and requires an expertise in both stable isotope labeling as well as enrichment of phosphorylated peptides. Recently, a microfluidic device incorporating a nanoliter flow rate reversed phase column as well as a titania (TiO(2)) enrichment column was released. This HPLC phosphochip allows excellent recovery and separation of phosphorylated peptides in a robust and reproducible manner with little user intervention. In this work, we have extended the abilities of this chip by defining the conditions required for on-chip stable isotope dimethyl labeling allowing for automated quantitation. The resulting approach will make quantitative phosphoproteomics more accessible.

Profiling of N-acetylated protein termini provides in-depth insights into the N-terminal nature of the proteome.

N-terminal processing of proteins is a process affecting a large part of the eukaryotic proteome. Although N-terminal processing is an essential process, not many large inventories are available, in particular not for human proteins. Here we show that by using dedicated mass spectrometry-based proteomics techniques it is possible to unravel N-terminal processing in a semicomprehensive way. Our multiprotease approach led to the identification of 1391 acetylated human protein N termini in HEK293 cells and revealed that the role of the penultimate position on the cleavage efficiency by the methionine aminopeptidases is essentially conserved from Escherichia coli to human. Sequence analysis and comparisons of amino acid frequencies in the data sets of experimentally derived N-acetylated peptides from Drosophila melanogaster, Saccharomyces cerevisiae, and Halobacterium salinarum showed an exceptionally higher frequency of alanine residues at the penultimate position of human proteins, whereas the penultimate position in S. cerevisiae and H. salinarum is predominantly a serine. Genome-wide comparisons revealed that this effect is not related to protein N-terminal processing but can be traced back to characteristics of the genome.

RockerBox: analysis and filtering of massive proteomics search results.

A major problem in the analysis of mass spectrometry-based proteomics data is the vast growth of data volume, caused by improvements in sequencing speed of mass spectrometers. This growth affects analysis times and storage requirements so severely that many analysis tools are no longer able to cope with the increased file sizes. We present a tool, RockerBox, to address size problems for search results obtained from the widely used Mascot search engine. RockerBox allows for a fast evaluation of large result files by means of a number of commonly accepted metrics that can often be viewed through charts. Moreover, result files can be filtered without altering their informative content, based on a number of FDR calculation methods. File sizes can be reduced dramatically, often to a tenth of their original size, thus relaxing the need for storage and computation power, and boosting analysis of current and future proteomics experiments.

Site-specific methionine oxidation in calmodulin affects structural integrity and interaction with Ca2+/calmodulin-dependent protein kinase II.

Methionine oxidation in the ubiquitous calcium signaling protein calmodulin (CaM) is known to disrupt downstream signaling and target CaM for proteasomal degradation. The susceptibility of CaM to oxidation in the different conformations that are sampled during calcium signaling is currently not well defined. Using an integrative mass spectrometry (MS) approach, applying both native MS and LC/MS/MS, we unravel molecular details of CaM methionine oxidation in the context of its interaction with the Ca(2+)/CaM-dependent protein kinase II (CaMKII). Sensitivity to methionine oxidation in CaM was found to vary according to the conformational state. Three methionine residues (Met71, 72, 145) show increased reactivity in calcium-saturated CaM (holo-CaM) compared to calcium-free CaM (apo-CaM), which has important consequences for oxidation-targeted proteasomal degradation. In addition, all four methionines in the C-terminal lobe (Met109, 124, 144 and 145) are found to be protected from oxidation in a peptide-based model of the CaMKII-bound conformation (cbp-CaM). We furthermore demonstrate that the oxidation of Met144 and 145 inhibits the interaction of CaM with CaMKII. cbp-CaM, in contrast to apo- and holo-CaM, maintains its ability to bind CaMKII under simulated conditions of oxidative stress and is also protected from oxidation-induced unfolding. Thus, we show that the susceptibility towards oxidation of specific residues in CaM is tightly linked to its signaling state and conformation, which has direct implications for calcium/CaM-CaMKII related signaling.

Oxidative stress-induced modifications of histidyl-tRNA synthetase affect its tRNA aminoacylation activity but not its immunoreactivity.

The aminoacyl-tRNA synthetases are ubiquitously expressed enzymes that catalyze the esterification of amino acids to their cognate tRNAs. Autoantibodies against several aminoacyl-tRNA synthetases are found in autoimmune polymyositis and dermatomyositis patients. Because necrosis is often found in skeletal muscle biopsies of these patients, we hypothesized that cell-death-induced protein modifications may help in breaking immunological tolerance. Since cell death is associated with oxidative stress, the effect of oxidative stress on the main myositis-specific autoantibody target Jo-1 (histidyl-tRNA synthetase; HisRS) was studied in detail. The exposure of Jurkat cells to hydrogen peroxide resulted in the detection of several oxidized methionines and one oxidized tryptophan residue in the HisRS protein, as demonstrated by mass spectrometry. Unexpectedly, the tRNA aminoacylation activity of HisRS appeared to be increased upon oxidative modification. The analysis of myositis patient sera did not lead to the detection of autoantibodies that are specifically reactive with the modified HisRS protein. The results of this study demonstrate that the Jo-1/HisRS autoantigen is modified under oxidative stress conditions. The consequences of these modifications for the function of HisRS and its autoantigenicity are discussed.

Evaluation of the deuterium isotope effect in zwitterionic hydrophilic interaction liquid chromatography separations for implementation in a quantitative proteomic approach.

Quantitative methodologies for the global in-depth comparison of proteomes are frequently based on chemical derivatization of peptides with isotopically distinguishable labeling agents. In the present work, we set out to study the feasibility of the dimethyl labeling method in combination with ZIC-cHILIC (zwitterionic hydrophilic interaction liquid chromatography) technology for quantitative proteomics. We first addressed the potential issue of isotope effects perturbing the essential coelution of differently labeled peptides under ZIC-cHILIC separation. The deuterium incorporation-induced effect can be largely eliminated by favoring the mixed-mode ZIC-cHILIC separation based on combined hydrophilic and ionic interactions. Then, we evaluated the performance and applicability of this strategy using a sample consisting of human cell lysate. We demonstrate that our approach is suitable to perform unbiased quantitative proteome analysis, still quantifying more than 2500 proteins when analyzing only a few micrograms of sample.

Exploring the human leukocyte phosphoproteome using a microfluidic reversed-phase-TiO2-reversed-phase high-performance liquid chromatography phosphochip coupled to a quadrupole time-of-flight mass spectrometer.

The study of protein phosphorylation events is one of the most important challenges in proteome analysis. Despite the importance of phosphorylation for many regulatory processes in cells and many years of phosphoprotein and phosphopeptide research, the identification and characterization of phosphorylation by mass spectrometry is still a challenging task. Recently, we introduced an approach that facilitates the analysis of phosphopeptides by performing automated, online, TiO(2) enrichment of phosphopeptides prior to mass spectrometry (MS) analysis. The implementation of that method on a "plug-and-play" microfluidic high-performance liquid chromatography (HPLC) chip design will potentially open up efficient phosphopeptide enrichment methods enabling phosphoproteomics analyses by a broader research community. Following our initial proof of principle, whereby the device was coupled to an ion trap, we now show that this so-called phosphochip is capable of the enrichment of large numbers of phosphopeptides from complex cellular lysates, which can be more readily identified when coupled to a higher resolution quadrupole time-of-flight (Q-TOF) mass spectrometer. We use the phosphochip-Q-TOF setup to explore the phosphoproteome of nonstimulated primary human leukocytes where we identify 1012 unique phosphopeptides corresponding to 960 different phosphorylation sites providing for the first time an overview of the phosphoproteome of these important circulating white blood cells.

Automated online sequential isotope labeling for protein quantitation applied to proteasome tissue-specific diversity.

Quantitation of protein abundance is a vital component in the proteomic analysis of biological systems, which can be achieved by differential stable isotopic labeling. To analyze tissue-derived samples, the isotopic labeling can be performed using chemical labeling of the peptides post-digestion. Standard chemical labeling procedures often require many manual sample handling steps, reducing the accuracy of measurements. Here, we describe a fully automated, online (in nanoLC columns), labeling procedure, which allows protein quantitation using differential isotopic dimethyl labeling of peptide N termini and lysine residues. We show that the method allows reliable quantitation over a wide dynamic range and can be used to quantify differential protein abundances in lysates and, more targeted, differences in composition between purified protein complexes. We apply the method to determine the differences in composition between bovine liver and spleen 20 S core proteasome complexes. We find that although all catalytically active immunoproteasome subunits were up-regulated in spleen (compared with liver), only one of the normal catalytic subunits was down-regulated, suggesting that the tissue-specific immunoproteasome assembly is more diverse than previously assumed.

Phosphatidylethanolamine-binding proteins, including RKIP, exhibit affinity for phosphodiesterase-5 inhibitors.

Identifying protein "interactors" of drugs is of great importance to understand their mode of action and possible cross-reactivity to off-target protein binders. In this study, we profile proteins that bind to PF-3717842, a high-affinity phosphodiesterase-5 (PDE5) inhibitor, by using a refined affinity pulldown approach with PF-3717842 immobilized beads. By performing these pulldowns in rat testis tissue lysate, we strongly and specifically enriched for PDE5 and a few other PDEs. In addition to these expected affinity-enriched proteins we also detect rodent-specific phosphatidylethanolamine-binding protein 2 (PEBP2), as a putative binder to the PDE5 inhibitor. By using recombinant forms of the related murine mPEBP2, mPEBP1 and human hPEBP1 (also known as Raf kinase inhibitor protein or RKIP) we confirm that they all can bind strongly to immobilized as well as soluble PF-3717842. As the phosphatidylethanolamine-binding proteins are involved in various important signal transduction pathways, the synthetic PDE5 inhibitor used here might form a platform to synthesize enhanced binders/inhibitors of the family of PEBP proteins. Our approach shows how chemical proteomics might be used to profile the biochemical space (interactome) of small molecule inhibitors.

Myelin localization of peptidylarginine deiminases 2 and 4: comparison of PAD2 and PAD4 activities.

An understanding of the structure and composition of the myelin sheath is essential to understand the pathogenesis of demyelinating diseases such as multiple sclerosis (MS). The presence of citrulline in myelin proteins in particular myelin basic protein (MBP) causes an important change in myelin structure, which destabilizes myelin. The peptidylarginine deiminases (PADs) are responsible for converting arginine in proteins to citrulline. Two of these, PAD2 and PAD4, were localized to the myelin sheath by immunogold electron microscopy. Deimination of MBP by the recombinant forms of these enzymes showed that it was extensive, that is, PAD2 deiminated 18 of 19 arginyl residues in MBP, whereas PAD4 deiminated 14 of 19 residues. In the absence of PAD2 (the PAD2-knockout mouse) PAD4 remained active with limited deimination of arginyl residues. In myelin isolated from patients with MS, the amounts of both PAD2 and PAD4 enzymes were increased compared with that in normals, and the citrullinated proteins were also increased. These data support the view that an increase in citrullinated proteins resulting from increased PAD2 and 4 is an important change in the pathogenesis of MS.

Methylation of arginine residues interferes with citrullination by peptidylarginine deiminases in vitro.

Peptidylarginine deiminase (PAD) enzymes catalyze the conversion of arginine residues in proteins to citrulline residues. Citrulline is a non-standard amino acid that is not incorporated in proteins during translation, but can be generated post-translationally by the PAD enzymes. Although the existence of citrulline residues in proteins has been known for a long time, only a few proteins have been reported to contain this amino acid under normal conditions. These include the nuclear histones, which also contain a wide variety of other post-translational modifications, as for instance methylation of arginine residues. It has been suggested that citrullination and methylation of arginine residues are competing processes and that PAD enzymes might "reverse" the methylation of arginine residues by converting monomethylated arginine into citrulline. However, conflicting data have been reported on the capacity of PADs to citrullinate monomethylated peptidylarginine. Using synthetic peptides that contain either arginine or methylated arginine residues, we show that the human PAD2, PAD3 and PAD4 enzymes and PAD enzyme present in several mouse tissues in vitro can only convert non-methylated peptidylarginine into peptidylcitrulline and that hPAD6 does not show any deiminating activity at all. A comparison of bovine histones either treated or untreated with PAD by amino acid analysis also supported the interference of deimination by arginine methylation. Taken together, these data indicate that it is unlikely that methyl groups at the guanidino position of peptidylarginine can be removed by peptidylarginine deiminases, which has important implications for the recently reported role of these enzymes in gene regulation.

Increased citrullination of histone H3 in multiple sclerosis brain and animal models of demyelination: a role for tumor necrosis factor-induced peptidylarginine deiminase 4 translocation.

Modification of arginine residues by citrullination is catalyzed by peptidylarginine deiminases (PADs), of which five are known, generating irreversible protein structural modifications. We have shown previously that enhanced citrullination of myelin basic protein contributed to destabilization of the myelin membrane in the CNS of multiple sclerosis (MS) patients. We now report increased citrullination of nucleosomal histones by PAD4 in normal-appearing white matter (NAWM) of MS patients and in animal models of demyelination. Histone citrullination was attributable to increased levels and activity of nuclear PAD4. PAD4 translocation into the nucleus was attributable to elevated tumor necrosis factor-alpha (TNF-alpha) protein. The elevated TNF-alpha in MS NAWM was not associated with CD3+ or CD8+ lymphocytes, nor was it associated with CD68+ microglia/macrophages. GFAP, a measure of astrocytosis, was the only cytological marker that was consistently elevated in the MS NAWM, suggesting that TNF-alpha may have been derived from astrocytes. In cell cultures of mouse and human oligodendroglial cell lines, PAD4 was predominantly cytosolic but TNF-alpha treatment induced its nuclear translocation. To address the involvement of TNF-alpha in targeting PAD4 to the nucleus, we found that transgenic mice overexpressing TNF-alpha also had increased levels of citrullinated histones and elevated nuclear PAD4 before demyelination. In conclusion, high citrullination of histones consequent to PAD4 nuclear translocation is part of the process that leads to irreversible changes in oligodendrocytes and may contribute to apoptosis of oligodendrocytes in MS.