The human platelet proteome mapped by peptide‐centric proteomics: A functional protein profile

Abstract

Several studies have been published in which holistic approaches were used to characterise the proteome and transcriptome of human platelets. The key intent being that a deeper understanding of the normal and aberrant physiological functions of platelets can only be achieved if most biomolecular building blocks are mapped. Here we present the application of recently developed novel technologies that overcome some of the shortcomings of gel-based proteomics. Central in our approach is the so-called combined fractional diagonal chromatography (COFRADIC)-technology in which sets of representative peptides are sorted in a diagonal RP chromatographic system through a specific modification of their side chain. In this study we combined three different COFRADIC sorting techniques to analyse the proteome of human platelets. Methionyl, cysteinyl and amino terminal peptides were isolated and analysed by MS/MS. Merging the peptide identifications obtained after database searching resulted in a core set of 641 platelet proteins, which comprises the largest set identified today. In comparison to previously published platelet proteomes, we identified 404 novel platelet proteins containing a high number of hydrophobic membrane proteins and hypothetical proteins. Furthermore we discuss the observed characteristics and potential benefits of each of the different COFRADIC technologies for proteome analysis and highlight important issues that need to be considered when searching sequence databases using data obtained in peptide-centric, non-gel proteomics studies.

HUPO Brain Proteome Project: Summary of the pilot phase and introduction of a comprehensive data reprocessing strategy

“…This will become mandatory as the journals already recognised the need for reliable data sets [6,7,23].…”

“…This phenomenon is specified by Martens et al (this Issue) describing, e.g. that the overlap between the HUPO BPP and the HUPO PPP [22] human pilot study is slightly above 15, but nearly 50% between the HUPO BPP and the platelet proteome data set [23] after normalisation to the latest IPI version. The proteins concerned mostly belong to housekeeping genes.…”

What Can Proteomics Tell Us About Platelets?

“…115 Owing to the enrichment provided by specific probes, activity-based protein profiling 116-121 B, These studies are mainly (40%) based on 2-dimensional gel electrophoresis (2-DE)/ difference gel electrophoresis (DIGE), 46,52,53,80,82,83,85,87,88,90,91,[93][94][95][96]98,102,106,110,112,113 whereas gelfree methods (23%) are still under-represented. 21,42,89,97,[114][115][116][117][118]121 Combination refers to studies using 2-DE/DIGE in combination with other approaches. COFRADIC indicates combined fractional diagonal chromatography; and MudPIT, multidimensional protein identification technology.…”

Targeted Analysis of Protein Termini

“…11,12 Several "terminal" proteomics approaches have been described. 8,[13][14][15][16][17] Many of these approaches are labor-intensive since they require extensive chemical modification and/or repeated separation of the sample. Gevaert et al used combined fractional diagonal chromatography (COFRADIC) in which free amino groups are chemically acetylated on the protein level, and N-termini of tryptic cleavage products are blocked with trinitrobenzene (TNBS) on the peptide level.…”