= Emerging. More than 5 years before clinical availability. (16.60%, 2024)
= Expected to be clinically available in 1 to 4 years. (37.02%, 2024)
= Clinically available now. (46.38%, 2024)
MSACL 2024 : Kruijt

MSACL 2024 Abstract

Self-Classified Topic Area(s): Proteomics > Precision Medicine > Emerging Technologies

Podium Presentation in Steinbeck 2 on Thursday at 11:10 (Chair: Carrie Adler / Kwasi Mawuenyega)

Molecular Characterization of Antithrombin by Mass Spectrometry: Lessons Learned From Antithrombin Deficiency and CDG Cohorts

Mirjam Kruijt (1), Maria Eugenia de la Morena-Barrio (2), Javier Corral (2), Christa M. Cobbaert (1), L. Renee Ruhaak (1)
(1) Department of Clinical Chemistry and Laboratory Medicine, Leiden University Medical Center, Leiden, The Netherlands (2) Servicio de Hematología, Hospital Universitario Morales Meseguer, Centro Regional de Hemodonación, Universidad de Murcia, IMIB-Pascual Parrilla, CIBERER-ISCIII, Murcia, Spain

Mirjam Kruijt, MSc (Presenter)

Presenter Bio: Mirjam is currently working as a 4th year PhD-student in the lab of prof.dr. Christa Cobbaert under the supervision of dr. Renee Ruhaak. Her focus is on the development of a new mass spectrometry test for quantitating antithrombin while simultaneously identifying antithrombin proteoforms by molecular characterization. The goal of this project is to improve patient diagnostics and advance the clinical care pathway for patients with hereditary antithrombin deficiency. Furthermore, the molecular characterization of antithrombin serves as a proof-of-concept for the simultaneous quantitative and qualitative characterization of any protein existing in multiple proteoforms using mass spectrometry.


Antithrombin (AT) is an N-glycosylated serpin that plays key roles in the coagulation cascade. Thus, AT deficiency sharply increases risk of (recurrent) thrombosis. A cause of hereditary AT deficiency lies in over 350 reported mutations of SERPINC1, the coding gene, which have a varying effect on the concentration and functionality of AT. Likewise, altered glycosylation of the four N-glycosylation sites may also cause AT deficiency. Current diagnostic tests for AT are based on functional assays using chromogenic substrates, a strategy developed over 30 years ago. Underdiagnosis of certain types of AT deficiency and limited information on the impact of specific proteoforms hamper the use of AT activity tests for diagnosing thrombophilia. To address this unmet clinical need, a mass spectrometry test for quantitative and qualitative characterization of AT proteoforms was developed. The test was analytically validated for quantitative precision, and we here present its qualitative potential.

This study examined whether molecular characterization of AT using mass spectrometry could identify patients with aberrant AT proteoforms due to hereditary AT deficiency or altered glycosylation caused by congenital disorders of glycosylation (CDGs).

Two cohorts of anonymized patients with clinically relevant AT proteoforms were analyzed (with local METC approval): a cohort of 91 patients with hereditary AT deficiency and a cohort of 41 patients with AT deficiency caused by CDG type 1. Both cohorts were previously screened for AT activity and genetic mutations in SERPINC1 or CDG-related genes. Citrate plasma samples were measured using our in-house developed MS test for AT, which monitors 23 proteotypic (wildtype) peptides. To confirm alterations in wildtype peptides, mutation-specific transitions were developed.

The study generated an extensive amount of information per patient. To facilitate robust and efficient analysis, an R-script was developed that verifies the overall AT concentration of each sample and identifies peptides that deviate from this concentration and are thus suspected of containing multiple proteoforms. Quantitative defects were observed in 95.6 % of AT deficiency patients and 87.8 % of CDG patients and (median concentration 0.73 and 0.86 µmol/L respectively, ref.int. 1.07 – 1.49 µmol/L). To confirm mutations in patients with AT deficiency, variant specific transitions were established for various mutations, including p.Val30Glu (AT Dublin), p.Arg45Trp, p.Pro73Leu (AT Basel), p.Leu131Phe (AT Budapest3), p.Arg177Cys, p.Arg425Cys. Interestingly, variant peptides were also identified in patients with type-1 AT deficiency, a subtype which is believed to cause a quantitative defect without expression and secretion of the variant proteoform. In CDG-patients the amount of alpha-AT proteoform (containing a glycan at site N167) was significantly decreased compared to healthy controls (71.1 % versus 94.6 %, respectively). Moreover, altered N-glycosylation was observed for a second N-glycosylation site (N224).

The LC-MRM-MS test for AT proves to identify patients with AT deficiency: congenital defects and CDG. By combining quantitative and qualitative proteoform information, a molecular diagnosis can be generated, identifying pathogenic variants in the sample at the amino acid and/or PTM level. We anticipate that quantitation and molecular characterization of AT has potential in precision diagnostics of thrombophilia and will refine the intended uses of AT testing in clinical care pathways. To summarize, by using our next-generation LC-MRM-MS test for AT, we expose the molecular puzzle underlying the clinical heterogeneity of AT deficiency, which cannot be captured by traditional activity tests.

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