Antiplatelet treatment is of fundamental importance in combatting functions/dysfunction of platelets in the pathogenesis of cardiovascular and inflammatory diseases. Dysfunction of anucleate platelets is likely to be completely attributable to alterations in posttranslational modifications and protein expression. We therefore examined the proteome of platelets highly purified from fresh blood donations, using elaborate protocols to ensure negligible contamination by leukocytes, erythrocytes, and plasma. Using quantitative mass spectrometry, we created the first comprehensive and quantitative human platelet proteome, comprising almost 4000 unique proteins, estimated copy numbers for similar to 3700 of those, and assessed intersubject (4 donors) as well as intrasubject (3 different blood samples from 1 donor) variations of the proteome. For the first time, our data allow for a systematic and weighted appraisal of protein networks and pathways in human platelets, and indicate the feasibility of differential and comprehensive proteome analyses from small blood donations. Because 85% of the platelet proteome shows no variation between healthy donors, this study represents the starting point for disease-oriented platelet proteomics. In the near future, comprehensive and quantitative comparisons between normal and well-defined dysfunctional platelets, or between platelets obtained from donors at various stages of chronic cardiovascular and inflammatory diseases will be feasible

Tobacco mature pollen has extremely desiccated cytoplasm, and is metabolically quiescent. Upon re-hydration it becomes metabolically active and that results in later emergence of rapidly growing pollen tube. These changes in cytoplasm hydration and metabolic activity are accompanied by protein phosphorylation. In this study, we subjected mature pollen, 5-min-activated pollen, and 30-min-activated pollen to TCA/acetone protein extraction, trypsin digestion and phosphopeptide enrichment by titanium dioxide. The enriched fraction was subjected to nLC-MS/MS. We identified 471 phosphopeptides that carried 432 phosphorylation sites, position of which was exactly matched by mass spectrometry. These 471 phosphopeptides were assigned to 301 phosphoproteins, because some proteins carried more phosphorylation sites. Of the 13 functional groups, the majority of proteins were put into these categories: transcription, protein synthesis, protein destination and storage, and signal transduction. Many proteins were of unknown function, reflecting the fact that male gametophyte contains many specific proteins that have not been fully functionally annotated. The quantitative data highlighted the dynamics of protein phosphorylation during pollen activation; the identified phosphopeptides were divided into seven groups based on the regulatory trends. The major group comprised mature pollen-specific phosphopeptides that were dephosphorylated during pollen activation. Several phosphopeptides representing the same phosphoprotein had different regulation, which pinpointed the complexity of protein phosphorylation and its clear functional context. Collectively, we showed the first phosphoproteomics data on activated pollen where the position of phosphorylation sites was clearly demonstrated and regulatory kinetics was resolved. Molecular & Cellular

The transition between the quiescent mature and the metabolically active germinating pollen grain most probably involves changes in protein phosphorylation status, since phosphorylation has been implicated in the regulation of many cellular processes. Given that, only a minor proportion of cellular proteins are phosphorylated at any one time, and that phosphorylated and nonphosphorylated forms of many proteins can co-exist within a cell, the identification of phosphoproteins requires some prior enrichment from a crude protein extract. Here, we have used metal oxide/hydroxide affinity chromatography (MOAC) based on an aluminum hydroxide matrix for this purpose, and have generated a population of phosphoprotein candidates from both mature and in vitro activated tobacco pollen grains. Both electrophoretic and nonelectrophoretic methods, allied to MS, were applied to these extracts to identify a set of 139 phosphoprotein candidates. In vitro phosphorylation was also used to validate the spectrum of phosphoprotein candidates obtained by the MOAC phosphoprotein enrichment. Since only one phosphorylation site was detected by the above approach, titanium dioxide phosphopeptide enrichment of trypsinized mature pollen crude extract was performed as well. It resulted in a detection of additional 51 phosphorylation sites giving a total of 52 identified phosphosites in this set of 139 phosphoprotein candidates.

bThe structural polyprotein Gag of human immunodeficiency virus type 1 (HIV-1) is necessary and sufficient for formation of virus-like particles. Its C-terminal p6 domain harbors short peptide motifs that facilitate virus release from the plasma membrane and mediate incorporation of the viral Vpr protein. p6 has been shown to be the major viral phosphoprotein in HIV-1-infected cells and virions, but the sites and functional relevance of p6 phosphorylation are not clear. Here, we identified phosphorylation of several serine and threonine residues in p6 in purified virus preparations using mass spectrometry. Mutation of individual candidate phosphoacceptor residues had no detectable effect on virus assembly, release, and infectivity, however, suggesting that phosphorylation of single residues may not be functionally relevant. Therefore, a comprehensive mutational analysis was conducted changing all potentially phosphorylatable amino acids in p6, except for a threonine that is part of an essential peptide motif. To avoid confounding changes in the overlapping pol reading frame, mutagenesis was performed in a provirus with genetically uncoupled gag and pol reading frames. An HIV-1 derivative carrying 12 amino acid changes in its p6 region, abolishing all but one potential phosphoacceptor site, showed no impairment of Gag assembly and virus release and displayed only very subtle deficiencies in viral infectivity in T-cell lines and primary lymphocytes. All mutations were stable over 2 weeks of culture in primary cells. Based on these findings, we conclude that phosphorylation of p6 is dispensable for HIV-1 assembly, release, and infectivity in tissue culture.

With the increasing popularity of comparative studies of complex proteomes, reporter ion-based quantification methods such as iTRAQ and TMT have become commonplace in biological studies. Their appeal derives from simple multiplexing and quantification of several samples at reasonable cost. This advantage yet comes with a known shortcoming: precursors of different species can interfere, thus reducing the quantification accuracy. Recently, two methods were brought to the community alleviating the amount of interference via novel experimental design. Before considering setting up a new workflow, tuning the system, optimizing identification and quantification rates, etc. one legitimately asks: is it really worth the effort, time and money? The question is actually not easy to answer since the interference is heavily sample and system dependent. Moreover, there was to date no method allowing the inline estimation of error rates for reporter quantification. We therefore introduce a method called iQuARI to compute false discovery rates for reporter ion based quantification experiments as easily as Target/Decoy FDR for identification. With it, the scientist can accurately estimate the amount of interference in his sample on his system and eventually consider removing shadows subsequently, a task for which reporter ion quantification might not be the solution of choice.