Method for identifying marker proteins for the diagnosis and risk stratification of blood coagulation disorders

A method for identifying marker proteins for diagnosis and risk stratification of blood coagulation disorders, comprising the following steps:

• (i) providing a sample comprising whole blood, blood serum, or blood plasma containing thrombocytes,
• (ii) aliquoting the sample into at least two aliquots,
• (iii)(A) adding a thrombocyte activator to at least a first aliquot and
• (B) adding a thrombocyte inhibitor to at least a second aliquot, and repeating the addition once or more, and optionally
• (C) adding a thrombocyte activator and a thrombocyte inhibitor to at least a third aliquot, and optionally repeating the respective additions once or more,
• (iv) lysing cells in each of the first aliquot, the second aliquot, and optionally the third aliquot,
• (v) extracting proteins from each of the first aliquot, the second aliquot and optionally the third aliquot, and digesting the extracted proteins to form peptides by means of proteases,
• (vi) enriching and separating phosphorylated peptides from the peptides formed by digestion,
• (vii) performing an analysis of the phosphorylated peptides by means of quantitative mass spectrometry, and
• identifying phosphorylated peptides which at the same time demonstrate an increased or reduced phosphorylation from (iii)(A) over time and which demonstrate, oppositely, reduced or increased phosphorylation from (iii)(B) over time and which optionally demonstrate a reduced or increased phosphorylation in comparison with phosphorylated peptides from (iii)(C), wherein the identified phosphorylated peptides can serve as marker proteins for the diagnosis and the risk stratification of blood coagulation disorders.