A Novel LC‐MS/MS Assay for Quantification of Des‐carboxy Prothrombin and Characterization of Warfarin‐Induced Changes

Figures

• 

  Figure 1. Schematic representation of sample preparation and analysis protocols. Proteins from plasma samples (10 µL) were precipitated with 0.1% trifluoroacetic acid (TFA) in isopropanol (IPA). Extracted proteins were reduced with dithiothreitol (DTT), alkylated with Iodoacetamide and digested with trypsin. Finally, digested samples were analyzed by liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). Prothrombin surrogate peptides from both glutamic acid (Gla)‐region and non‐Gla region (highlighted by blue color) were selected for the analysis.

• 

  Figure 2. Tandem mass spectrometry (MS/MS) spectra of carboxy and des‐carboxy forms of a representative peptide, ANTFLEEVRK. (a) MS/MS spectrum of parent ion, m/z 607.8386 (ANTFLEEVRK – des‐carboxy peptide) showing y‐ions as dominant fragment, (b) MS/MS spectrum of parent ion, m/z 829.8301 (ANTFLE[E]VRK – mono–carboxy peptide) showing ions with one CO2 loss (y‐CO2) as dominant fragment and (c) MS/MS spectrum of parent ion, 651.7717 (ANTFL[E][E]VRK – di‐carboxy peptide) showing ions with two CO2 losses (y‐2CO2) as dominant fragments.

• 

  Figure 3. Assay performance. The linearity of the liquid chromatography‐tandem mass spectrometry assay was determined by spiking heavy peptide into human serum albumin (a‐ 2.5–100 µg/mL) and human serum matrix (b‐ 10, 40, and 80 µg/mL). Recovery was calculated by spiking the standard prothrombin at 40 and 80 µg/mL into pooled control serum samples (c).

• 

  Figure 4. Comparison of abundance of total prothrombin, carboxy peptide (ANTFL[E][E]VRK) and des‐carboxy peptides (ANTFLEEVRK and GLNER) in serum samples from control and warfarin treated individuals. Each dot and square represent the individual subjects from control, and warfarin treated groups, respectively. Error bars indicate the standard deviation. Significant reduction in the levels of total prothrombin (P = 0.02) and carboxylated prothrombin (P < 0.0001) in warfarin treated subjects compared with control subjects was observed. Des‐carboxylated prothrombin was significantly (P < 0.0001) elevated in warfarin treated subjects. Des‐carboxy peptide ANTFLEEVRK is a low response peptide, which was quantifiable in first analysis only (n = 1). Mann‐Whitney test. *P value < 0.05, **P value < 0.01 and ***P value < 0.001.

• 

  Figure 5. Correlation between international normalized ratio (INR) value with abundance of total prothrombin, carboxy peptide, and des‐carboxy peptides in serum samples from warfarin‐treated subjects. (a–d) represent correlations with INR in warfarin‐treated samples (group 1). (e,f) Represent the correlation between carboxy peptide ANTFL[E][E]VRK with des‐carboxy peptides GNLER and ANTFLEEVRK in the same sample set. INR values negatively correlated with carboxy peptides while positively correlated with des‐carboxy peptides. In addition, there was a strong negative correlation between carboxy peptide and des‐carboxy peptides.