Proteomics investigation of human platelets by shotgun nUPLC-MSE and 2DE experimental strategies: a comparative study

Platelets, the smallest human blood cells component, have a key role in the control of haemostasis and thrombosis but they have also been shown to be implicated in a number of different pathological states because of their involvement also in the process of inflammation end its resolution. Their peculiar anucleated morphology render the proteomics an intriguing approach to understand their biology. Given the high impact of platelet in different diseases we have started a systematic investigation of protein repertoire in controlled platelet preparation.

Platelets have been extracted from blood of healthy donors (n=6) collected by venipuncture in Vacutainer. The quality of the preparation was assessed by observation and enumeration in a Bürker chamber with a conventional tissue culture microscope. To characterize human platelets proteome we analysed the pool of purified platelets combining two proteomic approaches: 2-DE separation combined with Mass Spectrometry and nanoscale ultra performances LC-MS(E) shotgun proteomics experiments.

The 2D gel analysis leads an average of 1900 protein spots, after the filtering of "noise" and "false positive" spots, over 500 were selected to be eligible for further analysis given their optimal spot quality value. To perform the analysis by ion accounting shotgun proteomic approach, based on nano ultra performance liquid chromatography (nUPLC) coupled to MS(E) processing of continuum LC-MS data, the same pool of samples was subject to liquid phase tryptic digestion and the peptide obtained used for the experiments. All the data obtained were analysed using ProteinLynx GlobalServer v2.3 (PLGS, Waters). Three analytical replicates run were acquire in high/low energy modes and associated to a human protein database returning the identification of 100 distinct genes. Comparative analysis of the Gene Ontology has been performed to evaluate the differential functional representation of the molecular repertoire investigated with these two orthogonal approaches.

The overall molecular function classification revealed differences between the two proteomic approaches. In particular, we found significant differences in cytoskeletal proteins (19.65% 2-DE versus 45.60 Shotgun) and receptors (0,92% 2-DE versus 6.90% Shotgun).