2D-LC–MS/MS to Measure Cleaved High-Molecular-Weight Kininogen in Human Plasma as A Biomarker for C1-Inh-Hae

• DOI: 10.4155/bio-2017-0105
• Publication Date: 2017-10-21T12:48:34Z
• Authors: Guodong Zhang; Daniel J Sexton; Ryan R Faucette; Yongchang Qiu; Jiang Wu
• Keywords: Kininogen, High-Molecular-Weight; Hereditary Angioedema Types I and II; Solid Phase Extraction; 01 natural sciences; 0104 chemical sciences; Tandem Mass Spectrometry; Proteolysis; Humans; Amino Acid Sequence; Complement C1 Inhibitor Protein; Biomarkers; Blood Chemical Analysis; Chromatography, Liquid

C1-INH-HAE is caused by activation of plasma kallikrein which subsequently cleaves high-molecular-weight kininogen (HMWK) to generate bradykinin and cHMWK.A novel ion-pair 2D LC-MS/MS assay was developed to measure the 46 kDa cHMWK in plasma as a biomarker for C1-INH-HAE. The sample preparation included sodium dodecyl sulfate denaturation, methanol crash, chymotryptic digestion and peptide enrichment by solid phase extraction.The LLOQ was 200 ng/ml. The overall cHMWK recovery combining crash and digestion was 57.5%. The precision of the method was ≤12.7% and accuracy ≤-13.8%.A reagent-free LC-MS assay has been developed for the quantitation of 46 kDa cHMWK, which was shown to be elevated in plasma of C1-INH-HAE patients due to C1-INH deficiency relative to that of healthy subjects.