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

Abstract

Aim: C1-INH-HAE is caused by activation of plasma kallikrein which subsequently cleaves high-molecular-weight kininogen (HMWK) to generate bradykinin and cHMWK. Materials & methods: 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. Results: 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%. Conclusion: 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.

Keywords::

• C1-INH-HAE biomarker
• cleaved high-molecular-weight kininogen
• ion-pair 2D-LC–MS/MS
• protein biomarker

Supplementary data

To view the supplementary data that accompany this paper, please visit the journal website at: www.tandfonline.com/doi/suppl/10.4155/bio-2017-0105

Financial & competing interests disclosure

All authors are full-time employees of Shire and own stock. Shire funded the study. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

Acknowledgements

HAE plasma was collected by the following biomarker study investigators: C Akin (Brigham and Womens Hospital), J Anderson (Clinical Research Center of Alabama, Alabama Allergy & Asthma Center), A Banerji (Massachusetts General Hospital), J Bernstein (University of Cincinnati), E Brooks (UT Health Science Center at San Antonio), P Busse (Mount Sinai School of Medicine), M Cicardi (University of Milan), H Li (Institute for Asthma and Allergy, MD, USA), D McNeil (Columbus, OH, USA), L Schwartz and J Wedner (Washington University School of Medicine).