Application of 2-dimensional difference gel electrophoresis (2D-DIGE) to the study of thrombin-activated human platelet secretome

Anna Della Corte, Norma Maugeri, Agnieszka Pampuch, Chiara Cerletti, Giovanni De Gaetano, Domenico Rotilio

Research output: Contribution to journalArticle

38 Citations (Scopus)

Abstract

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°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.

Original languageEnglish
Pages (from-to)43-50
Number of pages8
JournalPlatelets
Volume19
Issue number1
DOIs
Publication statusPublished - Feb 2008

Fingerprint

Two-Dimensional Difference Gel Electrophoresis
Thrombin
Blood Platelets
Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry
Gels
Lamin Type A
Thrombospondin 1
Thrombospondins
Search Engine
Proteins
Peptide Mapping
Platelet Activation
Transferrin
Nuclear Proteins
Glutathione Transferase
Trypsin
Mass Spectrometry
Healthy Volunteers
Databases
Peptides

Keywords

  • DIGE
  • Lamin A thrombin
  • Platelet secretome
  • Proteomics

ASJC Scopus subject areas

  • Hematology
  • Cell Biology

Cite this

Application of 2-dimensional difference gel electrophoresis (2D-DIGE) to the study of thrombin-activated human platelet secretome. / Della Corte, Anna; Maugeri, Norma; Pampuch, Agnieszka; Cerletti, Chiara; De Gaetano, Giovanni; Rotilio, Domenico.

In: Platelets, Vol. 19, No. 1, 02.2008, p. 43-50.

Research output: Contribution to journalArticle

Della Corte, Anna ; Maugeri, Norma ; Pampuch, Agnieszka ; Cerletti, Chiara ; De Gaetano, Giovanni ; Rotilio, Domenico. / Application of 2-dimensional difference gel electrophoresis (2D-DIGE) to the study of thrombin-activated human platelet secretome. In: Platelets. 2008 ; Vol. 19, No. 1. pp. 43-50.
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