Proteomic characterization of human platelet-derived microparticles

• Shotgun proteomic study of platelet-derived microparticles.
• Low molecular weight protein enrichment by hydrogel nanoparticles.
• NanoHPLC-MS/MS analysis of in-solution tryptic digests of microparticle proteins.

Microparticles (MPs) are small fragments of apoptotic or activated cells that may contribute to pathological processes in many diseases. Platelet-derived MPs (PMPs) are the most abundant type of MPs in human blood. To characterize the proteins in PMPs we used a shotgun proteomics approach by nanoHPLC separation followed by MS analysis on an LTQ Orbitrap XL. PMPs were produced from isolated platelets stimulated with adenosine diphosphate (ADP). We developed an analytical platform constituted by two different steps: in the first one we used a standard shotgun strategy; in the second one, to improve low-molecular weight, low-abundance-proteins identification, the samples were fractionated using hydrogel nanoparticles, an enrichment system based on a mixed mechanism of dimensional exclusion and colorant affinity. This was chosen to tackle a common issue with shotgun approaches, in which the low-abundance proteins are not detected when surveys are on a broad scale. By means of the entire analytical platform, we identified 603 proteins, 243 of which were not previously identified. A simple and straightforward procedure for the study of PMPs was provided, producing a tool for further understanding their biological and pathological roles, and a baseline for future studies aimed at discovering biomarkers involved in several diseases.

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