Optimization of quantitative proteomic analysis of clots generated from plasma of patients with venous thromboembolism

Clinical Proteomics

Maciej Suski¹  Rafał Olszanecki¹  Aneta Stachowicz¹  Ryszard Korbut¹  Jacek R. Wiśniewski²  Jakub Siudut³  Anetta Undas³ 

[1] Chair of Pharmacology, Jagiellonian University Medical College;Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry;Institute of Cardiology, Jagiellonian University Medical College;

关键词: Proteomics;    Clot;    Fibrin;    Venous thromboembolism;    MED FASP;   

DOI  :  10.1186/s12014-017-9173-x

来源: DOAJ

【 摘 要 】

Abstract Background It is well known that fibrin network binds a large variety of proteins, including inhibitors and activators of fibrinolysis, which may affect clot properties, such as stability and susceptibility to fibrinolysis. Specific plasma clot composition differs between individuals and may change in disease states. However, the plasma clot proteome has not yet been in-depth analyzed, mainly due to technical difficulty related to the presence of a highly abundant protein—fibrinogen and fibrin that forms a plasma clot. Methods The aim of our study was to optimize quantitative proteomic analysis of fibrin clots prepared ex vivo from citrated plasma of the peripheral blood drawn from patients with prior venous thromboembolism (VTE). We used a multiple enzyme digestion filter aided sample preparation, a multienzyme digestion (MED) FASP method combined with LC–MS/MS analysis performed on a Proxeon Easy-nLC System coupled to the Q Exactive HF mass spectrometer. We also evaluated the impact of peptide fractionation with pipet-tip strong anion exchange (SAX) method on the obtained results. Results Our proteomic approach revealed 476 proteins repeatedly identified in the plasma fibrin clots from patients with VTE including extracellular vesicle-derived proteins, lipoproteins, fibrinolysis inhibitors, and proteins involved in immune responses. The MED FASP method using three different enzymes: LysC, trypsin and chymotrypsin increased the number of identified peptides and proteins and their sequence coverage as compared to a single step digestion. Peptide fractionation with a pipet-tip strong anion exchange (SAX) protocol increased the depth of proteomic analyses, but also extended the time needed for sample analysis with LC–MS/MS. Conclusions The MED FASP method combined with a label-free quantification is an excellent proteomic approach for the analysis of fibrin clots prepared ex vivo from citrated plasma of patients with prior VTE.

【 授权许可】

Unknown