Application of 2-dimensional difference gel electrophoresis (2D-DIGE) to the study of thrombin-activated human platelet secretome.
Thrombin is an agonist inducing platelet activation. We combined two-dimensional difference gel electrophoresis (2D-DIGE) and mass spectrometry (MALDI-TOF MS) to analyse differentially expressed proteins secreted from thrombin-stimulated platelets. Human washed platelets, from healthy volunteers, were stimulated with thrombin 0.5 U/ml at 37 degrees C without stirring and the secreted proteins were resolved by 2D-DIGE. By image analysis, 1094 spots were detected in the 2D gel. The spots whose mean intensity showed at least a five-fold change intensity increase or decrease in the thrombin-activated platelet gel in comparison with unstimulated control were digested by trypsin and subjected to MALDI-TOF MS analysis. Peptides from mass spectra of in-gel digest samples were matched against available databases, using the Mascot search engine (Matrix Science) for peptide mass fingerprint. In the activated platelet secretome, transferrin, glutathione-transferase, WD repeat protein, ER-60, thrombospondin-1 precursor and thrombospondin were the most abundant. Also lamin A, a nuclear protein, not previously identified in platelets, appeared to be released. The novel strategy to combine 2D-DIGE with MALDI-TOF MS is a useful approach for a quantitative analysis of the effect of thrombin on the secretome profile of human platelets.
Della Corte
Anna
A
Research Laboratories, John Paul II Centre for High Technology Research and Education in Biomedical Sciences, Catholic University, Largo, 86100 Campobasso, Italy. anna.dellacorte@rm.unicatt.it
Maugeri
Norma
N
Pampuch
Agnieszka
A
Cerletti
Chiara
C
de Gaetano
Giovanni
G
Rotilio
Domenico
D
D000328:Adult; D001792:Blood Platelets / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D005260:Female; D006801:Humans; D008297:Male; D015539:Platelet Activation / Q000187:drug effects / Q000502:physiology*; D020543:Proteome / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D013917:Thrombin / Q000494:pharmacology
D020543:Proteome; D013917:Thrombin