Defining the Measurands for Antithrombin and its Proteoforms Using Mass Spectrometry Based Techniques Renee Ruhaak (Presenter) Leiden University Medical Center Presenter Bio: Renee Ruhaak is a passionate analytical scientist with a strong interest in the application of novel technology to better understand changes in physiology associated with states of health and disease. She obtained her PhD from Leiden University Medical Center, Leiden, The Netherlands and subsequently did a post-doc at the University of California, Davis, after which she became an assistant professor, first at MD Anderson Cancer Center in Houston, TX, but soon moved back to the Leiden University Medical center to join the department of Clinical Chemistry and Laboratory Medicine.

Authors: L. Renee Ruhaak[1], F.P.H.T.M Romijn[1], N.P.M. Smit[1] J. Nouta[2], M. Pieterse[1], Y.E.M. van der Burgt[1,2], M. Wuhrer[2] and C.M. Cobbaert[1]
1 Department of Clinical Chemistry and Laboratory Medicine, Leiden University Medical Center, Leiden, The Netherlands and 2 Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, The Netherlands

Current medical tests for antithrombin deficiency generally measure the overall activity, being blind for the actual proteoforms of AT that contribute to the test. Here, we present an MRM-based test that allows for the identification and quantification of clinically relevant proteoforms of AT according to predefined analytical performance specifications, as well as the characterization of AT candidate reference materials using high-end proteomics strategies. This now enables further unravelling of the individual contribution of the different AT-proteoforms to AT activity test results, which is a first and crucial step in understanding the value of the current AT tests offered in the clinical laboratory.

Introduction

Antithrombin (AT) is an anticoagulating protein, and reduced AT activity is associated with increased risk of thrombosis. Several genetic mutations that hamper AT activity have been reported [1] Moreover, protein glycosylation plays an important role in AT activity: β-AT, having only three of four glycosylation sites occupied, has 1000-fold increased activity compared to α-AT [2,3]. Currently, AT activity is often analysed using functional assays that measure overall activity. The molecular compounds that contribute to total activity assays are unknown because the specific contribution of α-AT, β-AT, other differentially glycosylated forms of AT and genetic variants goes unrecognized. Better assays, in which the measurands are well defined, are required to understand the value of the current tests. We here aim to assess the use of quantitative clinical chemistry proteomics by liquid chromatography coupled to mass spectrometry (LC-MS) to explore the molecular forms of AT.

Methods

For the development of an multiple reaction monitoring (MRM) LC-MS method, tryptic digests were prepared of citrated plasma samples, isolated AT and isolated AT at different concentrations in HSA (40 g/L) as well as other plasma samples. Four stable isotope labelled (SIL) peptides were added at the start of the sample preparation as internal standards. Transitions were developed for peptides and glycopeptides from AT to achieve four analytical aims: 1. The quantitation of the total AT concentration, 2. The detection of clinically relevant mutations in AT, 3. The differentiation of α-AT and β-AT and 4. The identification of other differentially glycosylated forms of AT. Separations were optimized using RP as well as HILIC stationary phases. Completeness of digestion was assessed using digestion time curves and accuracy of the developed test was evaluated using CLSI EP-15 protocol. Five different AT samples, either isolated from human origin or from recombinant sources, were identified as candidate reference materials. These samples were evaluated for their purity and proteoforms using three strategies: SDS-PAGE, CE-MS/MS of tryptic peptides, as well as CE-MS of intact AT protein.

Results

MRM transitions were developed for 19 tryptic peptides and 4 glycopeptides. On RP separation, two peptides, FDTISEK and FATTFYQHLADSK, were used for absolute quantitation of the overall AT concentration, 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 [4]. Out of 26 selected AT mutations, 20 can be identified using RP separation, whereas α-AT and β-AT cannot be separated due to lack of retention. However, this separation could be achieved using HILIC stationary phase, together with altered glycosylation profiles of AT. Three of the five candidate reference materials were shown to be of high purity and comprise proteoforms typically observed in human specimens.

Conclusions & Discussion

We developed an LC-MRM-MS method for detection and quantification of AT and its proteoforms. The in-depth characterization of the AT molecular forms will enable unravelling of the individual composition of human specimens, which is then foundational in defining criteria for the further assessment of candidate reference materials. Knowledge on the measurands of an analyte, which is enabled now for AT, is one of the most crucial steps for study design in test evaluation and for adequate implementation of the metrological traceability concept.

References

1. Patnaik, M. M.; Moll, S. Haemophilia 2008, 14, 1229-1239.

2. Orlando, C.; Heylen, O.; Lissens, W.; Jochmans, K. Thromb Res 2015, 135, 1179-1185.

3. Turk, B.; Brieditis, I.; Bock, S. C.; Olson, S. T.; Bjork, I. Biochemistry 1997, 36, 6682-6691.

4. Ruhaak, L. R.; Romijn, F.; Smit, N. P. M.; van der Laarse, A.; Pieterse, M. M.; de Maat, M. P. M.; Haas, F.; Kluft, C.; Amiral, J.; Meijer, P.; Cobbaert, C. M. Clin chem lab med 2018, in press.