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OFFgel‐based multidimensional LC‐MS/MS approach to the cataloguing of the human platelet proteome for an interactomic profile
Author(s) -
Krishnan Shibu,
Gaspari Marco,
Corte Anna Della,
Bianchi Patrizia,
Crescente Marilena,
Cerletti Chiara,
Torella Daniele,
Indolfi Ciro,
de Gaetano Giovanni,
Donati Maria Benedetta,
Rotilio Domenico,
Cuda Giovanni
Publication year - 2011
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.201000592
Subject(s) - proteome , proteomics , chemistry , shotgun proteomics , platelet , chromatography , fractionation , isoelectric focusing , mass spectrometry , computational biology , biochemistry , biology , enzyme , gene , immunology
The proteome of quiescent human platelets was analyzed by a shotgun proteomics approach consisting of enzymatic digestion, peptide separation based on isoelectric point by the use of OFFgel fractionation and, finally, RP nanoscale chromatography coupled to MS/MS detection (nano‐LC‐MS/MS). OFFgel fractionation in the first dimension was effective in providing an additional dimension of separation, orthogonal to RP nano‐LC, thus generating an off‐line multidimensional separation platform that proved to be robust and easy to set up. The analysis identified 1373 proteins with high confidence (false discovery rate<0.25%). The core set of 1373 human platelet proteins was investigated by Ingenuity Pathway Analysis software from which ten canonical pathways and eight networks have been validated, to suggest that platelets behave either as inflammatory or immune cells, and plasma membrane and cytoskeleton proteins play a fundamental role in their function. Moreover, toxicity pathway in agreement with network analysis, supports the concept that platelet life span is governed by an apoptotic mechanism.