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

  • L. Renee Ruhaak ORCID logo EMAIL logo , Fred P.H.T.M. Romijn , Nico P.M. Smit , Arnoud van der Laarse , Mervin M. Pieterse , Moniek P.M. de Maat , Fred J.L.M. Haas , Cornelis Kluft , Jean Amiral , Piet Meijer und Christa M. Cobbaert
Veröffentlicht/Copyright: 1. Mai 2018
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Clinical Chemistry and Laboratory Medicine (CCLM)
Aus der Zeitschrift Clinical Chemistry and Laboratory Medicine (CCLM) Band 56 Heft 10

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.

Keywords: antithrombin; defined measurands; molecular proteoforms; quantitative protein analysis; triple quadrupole mass spectrometry

  1. Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.

  2. Research funding: None declared.

  3. Employment or leadership: None declared.

  4. Honorarium: None declared.

  5. Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.

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Supplementary Material:

The online version of this article offers supplementary material (https://doi.org/10.1515/cclm-2017-1111).

Received: 2017-11-28
Accepted: 2018-03-16
Published Online: 2018-05-01
Published in Print: 2018-09-25

©2018 Walter de Gruyter GmbH, Berlin/Boston

Artikel in diesem Heft

  1. Frontmatter
  2. Editorials
  3. Harmonization in laboratory medicine: Blowin’ in the wind
  4. Standardization and harmonization of autoimmune diagnostics
  5. On the complexity of hemostasis and the need for harmonization of test practice
  6. Harmonization of laboratory hematology: a long and winding journey
  7. Section 1: Current Harmonization Activities at Global Level
  8. Harmonization in laboratory medicine: more than clinical chemistry?
  9. Harmonization of External Quality Assessment Schemes and their role – clinical chemistry and beyond
  10. An overview of EFLM harmonization activities in Europe
  11. Metrological traceability and harmonization of medical tests: a quantum leap forward is needed to keep pace with globalization and stringent IVD-regulations in the 21st century!
  12. Assessment of bone turnover in osteoporosis: harmonization of the total testing process
  13. Recent initiatives in harmonization of hemostasis practice
  14. EASI – European Autoimmunity Standardisation Initiative: facing the challenges of diagnostics in autoimmunity
  15. Harmonization of microbiology processes and standards: work in progress
  16. Harmonization initiatives in the generation, reporting and application of biological variation data
  17. Harmonization of accreditation to ISO15189
  18. External quality assessment programs in the context of ISO 15189 accreditation
  19. Section 2: Pre-Pre and Pre-Analytical Phase
  20. Laboratory testing in the emergency department: an Italian Society of Clinical Biochemistry and Clinical Molecular Biology (SIBioC) and Academy of Emergency Medicine and Care (AcEMC) consensus report
  21. The EFLM strategy for harmonization of the preanalytical phase
  22. Section 3: The Analytical Phase
  23. The roadmap for harmonization: status of the International Consortium for Harmonization of Clinical Laboratory Results
  24. The quest for equivalence of test results: the pilgrimage of the Dutch Calibration 2.000 program for metrological traceability
  25. Current state and recommendations for harmonization of serum/plasma 17-hydroxyprogesterone mass spectrometry methods
  26. International normalized ratio (INR) testing in Europe: between-laboratory comparability of test results obtained by Quick and Owren reagents
  27. Detecting molecular forms of antithrombin by LC-MRM-MS: defining the measurands
  28. A design for external quality assessment for the analysis of thiopurine drugs: pitfalls and opportunities
  29. Harmonization of PCR-based detection of intestinal pathogens: experiences from the Dutch external quality assessment scheme on molecular diagnosis of protozoa in stool samples
  30. Harmonization of urine albumin/creatinine ratio (ACR) results: a study based on an external quality assessment program in Polish laboratories
  31. Standardization of autoimmune testing – is it feasible?
  32. Diagnostic laboratory tests for systemic autoimmune rheumatic diseases: unmet needs towards harmonization
  33. Clinically relevant discrepancies between different rheumatoid factor assays
  34. An international survey on anti-neutrophil cytoplasmic antibodies (ANCA) testing in daily clinical practice
  35. Predictive autoimmunity using autoantibodies: screening for anti-nuclear antibodies
  36. Harmonization in autoimmune thyroid disease diagnostics
  37. International consensus on antinuclear antibody patterns: definition of the AC-29 pattern associated with antibodies to DNA topoisomerase I
  38. Reference standards for the detection of anti-mitochondrial and anti-rods/rings autoantibodies
  39. International Consensus on Antinuclear Antibody Patterns: defining negative results and reporting unidentified patterns
https://doi.org/10.1515/cclm-2017-1111
Schlagwörter für diesen Artikel
antithrombin; defined measurands; molecular proteoforms; quantitative protein analysis; triple quadrupole mass spectrometry

Artikel in diesem Heft

  1. Frontmatter
  2. Editorials
  3. Harmonization in laboratory medicine: Blowin’ in the wind
  4. Standardization and harmonization of autoimmune diagnostics
  5. On the complexity of hemostasis and the need for harmonization of test practice
  6. Harmonization of laboratory hematology: a long and winding journey
  7. Section 1: Current Harmonization Activities at Global Level
  8. Harmonization in laboratory medicine: more than clinical chemistry?
  9. Harmonization of External Quality Assessment Schemes and their role – clinical chemistry and beyond
  10. An overview of EFLM harmonization activities in Europe
  11. Metrological traceability and harmonization of medical tests: a quantum leap forward is needed to keep pace with globalization and stringent IVD-regulations in the 21st century!
  12. Assessment of bone turnover in osteoporosis: harmonization of the total testing process
  13. Recent initiatives in harmonization of hemostasis practice
  14. EASI – European Autoimmunity Standardisation Initiative: facing the challenges of diagnostics in autoimmunity
  15. Harmonization of microbiology processes and standards: work in progress
  16. Harmonization initiatives in the generation, reporting and application of biological variation data
  17. Harmonization of accreditation to ISO15189
  18. External quality assessment programs in the context of ISO 15189 accreditation
  19. Section 2: Pre-Pre and Pre-Analytical Phase
  20. Laboratory testing in the emergency department: an Italian Society of Clinical Biochemistry and Clinical Molecular Biology (SIBioC) and Academy of Emergency Medicine and Care (AcEMC) consensus report
  21. The EFLM strategy for harmonization of the preanalytical phase
  22. Section 3: The Analytical Phase
  23. The roadmap for harmonization: status of the International Consortium for Harmonization of Clinical Laboratory Results
  24. The quest for equivalence of test results: the pilgrimage of the Dutch Calibration 2.000 program for metrological traceability
  25. Current state and recommendations for harmonization of serum/plasma 17-hydroxyprogesterone mass spectrometry methods
  26. International normalized ratio (INR) testing in Europe: between-laboratory comparability of test results obtained by Quick and Owren reagents
  27. Detecting molecular forms of antithrombin by LC-MRM-MS: defining the measurands
  28. A design for external quality assessment for the analysis of thiopurine drugs: pitfalls and opportunities
  29. Harmonization of PCR-based detection of intestinal pathogens: experiences from the Dutch external quality assessment scheme on molecular diagnosis of protozoa in stool samples
  30. Harmonization of urine albumin/creatinine ratio (ACR) results: a study based on an external quality assessment program in Polish laboratories
  31. Standardization of autoimmune testing – is it feasible?
  32. Diagnostic laboratory tests for systemic autoimmune rheumatic diseases: unmet needs towards harmonization
  33. Clinically relevant discrepancies between different rheumatoid factor assays
  34. An international survey on anti-neutrophil cytoplasmic antibodies (ANCA) testing in daily clinical practice
  35. Predictive autoimmunity using autoantibodies: screening for anti-nuclear antibodies
  36. Harmonization in autoimmune thyroid disease diagnostics
  37. International consensus on antinuclear antibody patterns: definition of the AC-29 pattern associated with antibodies to DNA topoisomerase I
  38. Reference standards for the detection of anti-mitochondrial and anti-rods/rings autoantibodies
  39. International Consensus on Antinuclear Antibody Patterns: defining negative results and reporting unidentified patterns
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