Detecting molecular forms of antithrombin by LC-MRM-MS: defining the measurands

Author:

Ruhaak L. Renee1ORCID,Romijn Fred P.H.T.M.2,Smit Nico P.M.2,van der Laarse Arnoud2,Pieterse Mervin M.2,de Maat Moniek P.M.3,Haas Fred J.L.M.3,Kluft Cornelis3,Amiral Jean4,Meijer Piet3,Cobbaert Christa M.2

Affiliation:

1. Department of Clinical Chemistry and Laboratory Medicine , Leiden University Medical Center , Postzone E2-P, Albinusdreef 2 , 2333 ZA Leiden , The Netherlands , Phone: +31-71526-6397

2. Department of Clinical Chemistry and Laboratory Medicine , Leiden University Medical Center , Leiden , The Netherlands

3. ECAT Foundation , Voorschoten , The Netherlands

4. HYPHEN BioMed , Neuville sur Oise , France

Abstract

Abstract Background: Antithrombin (AT) is a critical regulator of coagulation, and its overall activity is typically measured using functional tests. A large number of molecular forms of AT have been identified and each individual carries multiple molecular proteoforms representing variable activities. Conventional functional tests are completely blind for these proteoforms. A method that ensures properly defined measurands for AT is therefore needed. We here assess whether mass spectrometry technology, in particular multiple reaction monitoring (MRM), is suitable for the quantification of AT and the qualitative detection of its molecular proteoforms. Methods: Plasma proteins were denatured, reduced and alkylated prior to enzymatic digestion. MRM transitions were developed towards tryptic peptides and glycopeptides using AT purified from human plasma. For each peptide, three transitions were measured, and stable isotope-labeled peptides were used for quantitation. Completeness of digestion was assessed using digestion time curves. Results: MRM transitions were developed for 19 tryptic peptides and 4 glycopeptides. Two peptides, FDTISEK and FATTFYQHLADSK, were used for quantitation, and using a calibration curve of isolated AT in 40 g/L human serum albumin, CVs below 3.5% were obtained for FDTISEK, whereas CVs below 8% were obtained for FATTFYQHLADSK. Of the 26 important AT mutations, 20 can be identified using this method, while altered glycosylation profiles can also be detected. Conclusions: We here show the feasibility of the liquid chromatography multiple reaction monitoring mass spectrometry (LC-MRM-MS) technique for the quantitation of AT and the qualitative analysis of most of its molecular proteoforms. Knowing the measurands will enable standardization of AT tests by providing in-depth information on the molecular proteoforms of AT.

Publisher

Walter de Gruyter GmbH

Subject

Biochemistry, medical,Clinical Biochemistry,General Medicine

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