Quantitative protein mass spectrometry for evaluation of the coagulation protein profile
Plasma proteins involved in coagulation and fibrinolysis are essential in maintaining hemostatic balance, and their circulating levels are critical determinants of bleeding and thrombosis risk. However, current laboratory tests measure only one or a few proteins at a time and therefore fail to reflect the overall balance between pro- and anticoagulant pathways. Quantitative protein mass spectrometry (QPMS) enables multiplexed and precise quantification of plasma proteins, providing an opportunity for comprehensive profiling of coagulation proteins in both research and clinical settings.
We developed two complementary multiplexed QPMS assays for plasma protein quantification. The first is an explorative research assay that allows multiplexed measurement of 159 plasma proteins, including 23 involved in coagulation, enabling large-scale profiling in research settings across conditions associated with venous thromboembolism risk. The second, a coagulation-focused QPMS assay, was developed according to clinical chemistry standards with the aim to provide precise and standardized quantification of coagulation proteins suitable for both research and diagnostic use. This assay was initially established for five key proteins: fibrinogen (FBG), von Willebrand factor (VWF), FVIII, FIX and FXI, and is currently being expanded to include all liver-produced coagulation proteins to comprehensively evaluate the coagulation protein profile. For both assays, proteins are enzymatically digested with trypsin to generate representative peptides which are quantified with liquid chromatography-mass spectrometry relative to stable isotope-labeled (SIL) internal standards. Analytical validation included assessments of precision, linearity, stability, and comparability with routine laboratory assays.
The explorative plasma protein profile assay showed excellent analytical performance (median CV < 7.5%, mean R² = 0.92), and was successfully applied in clinical cohorts including antiphospholipid syndrome, weight loss, and Cushing’s disease, revealing condition-specific protein signatures. The coagulation-focused QPMS assay demonstrated high analytical precision and strong agreement with routine laboratory tests for FBG, VWF, FIX, and FXI (FBG: r=0.991, VWF: r=0.970, FIX: r=0.926, FXI: r=0.871). FVIII showed lower signal intensity due to its low plasma abundance, and enrichment strategies are under development to improve detection. Validation of the extended assay covering all liver-produced coagulation proteins is ongoing and preliminary data indicate good analytical validity.
These QPMS assays provide a powerful approach for multiplexed quantitative assessment of plasma proteins involved in coagulation and fibrinolysis, proving valuable in research settings and holding promise for future diagnostic applications.