Characterization of Gla proteoforms and non-Gla peptides of gamma carboxylated proteins: Application to quantification of prothrombin proteoforms in human plasma

Gamma (gamma) carboxylation is an essential post-translational modification in vitamin K-dependent proteins (VKDPs), involved in maintaining critical biological homeostasis. Alterations in the abundance or activity of these proteins have pharmacological and pathological consequences. Importantly, low levels of fully gamma-carboxylated clotting factors increase plasma des-gamma-carboxy precursors resulting in little or no biological activity. Therefore, it is important to characterize the levels of gamma-carboxylation that reflect the active state of these proteins. The conventional enzyme-linked immunosorbent assay for protein induced by vitamin K absence or antagonist II (PIVKA-II) quantification uses an antibody that is not applicable to distinguish different gamma-carboxylation states. Liquid chromatography-mass spectrometry (LC-MS) approaches have been utilized to distinguish different gamma-carboxylated proteoforms, however, these attempts were impeded by poor sensitivity due to spontaneous neutral loss of CO2 and simultaneous cleavage of the backbone bond in the collision cell. In this study, we utilized an alkaline mobile phase in combination with polarity switching (positive and negative ionization modes) to simultaneously identify and quantify gamma-carboxylated VKDPs. The method was applied to compare Gla proteomics of prothrombin (FII) in 10 mu L plasma samples of healthy control and warfarin-treated adults. We also identified surrogate non-Gla peptides for seven other VKDPs to quantify total (active plus inactive) protein levels. The total protein approach (TPA) was used to quantify absolute levels of the VKDPs in human plasma.