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Optimization of quantitative proteomic analysis of clots

ABSTRACT: 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. We used a multiple enzyme digestion filter aided sample preparation (MED FASP) method combined with LC-MS/MS analysis performed on a Proxeon Easy-nLC System coupled to 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. Our proteomic approach revealed >500 proteins repeatedly identified in the plasma fibrin clots from patients with venous thromboembolism. The multienzyme digestion (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 and double step digestion. Peptide fractionation with SAX protocol slightly increased the depth of proteomic analyses, but also extended the time needed for sample analysis with LC-MS/MS.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood Plasma

SUBMITTER: Jacek Wisniewski

LAB HEAD: Jacek R Wisniewski

PROVIDER: PXD006814 | Pride | 2017-12-08

REPOSITORIES: Pride

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

Stachowicz Aneta A Siudut Jakub J Suski Maciej M Olszanecki Rafał R Korbut Ryszard R Undas Anetta A Wiśniewski Jacek R JR

Clinical proteomics 20171128

<h4>Background</h4>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 ...[more]

PMID: 29209155

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Project description:Antiphospholipid syndrome (APS) is associated with arterial and venous thrombosis. The unfavorable fibrin clot phenotype, including formation of dense and poorly lysable clots, has been reported both in thrombotic APS and venous thromboembolism (VTE). The presence and amount of different proteins within a plasma clot, not only associated with the coagulation system, may influence clot properties. To our knowledge, there is a lack of data on plasma fibrin-clot bound proteins in patients with thrombotic APS or VTE. The aim of our study was to perform a quantitative proteomic analysis of fibrin clots prepared from citrated plasma from subjects with thrombotic APS and prior VTE, along with fibrin clot permeability (Ks) and clot lysis time (CLT) assessed ex vivo. We investigated 23 consecutive patients with APS, 18 with a history of first-ever VTE, and 20 age and sex matched healthy subjects. A multiple enzyme digestion filter aided sample preparation and 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 were used. The proteomic analysis revealed that clot composition regarding 117 proteins in APS patients and 48 proteins in VTE patients was changed as compared to healthy controls, while 72 clot-bounded proteins differed between APS and VTE subjects. In healthy controls, Ks was associated with fibrinogen alpha and gamma chains (r=0.46 and r=0.46, both p<0.05, respectively) or apolipoprotein B-100 (r=-0.53, p<0.05), while CLT correlated with annexin A2 (r=-0.58, p<0.05), apolipoportein(a) (r=0.47, p<0.05), or platelet glycoprotein 4 (r=0.59, p<0.05). In VTE patients correlations of Ks with complement C1q and histone H2B, as factors closely linked with thrombosis, were observed (r=-0.52 and r=-0.47, both p<0.05, respectively). In patients with thrombotic APS all above-mentioned associations were not found. This study is the first to show that different proteins are able to influence the clot formation, structure, and properties. Since, prothrombotic conditions abolished associations observed in healthy subjects fibrin clots, differences in protein clot components might explain the links between prothrombotic fibrin clot phenotype and thromboembolic events.

Dataset Information

Optimization of quantitative proteomic analysis of clots

Publications

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

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