Home Optimizing charge state distribution is a prerequisite for accurate protein biomarker quantification with LC-MS/MS, as illustrated by hepcidin measurement
Article
Licensed
Unlicensed Requires Authentication

Optimizing charge state distribution is a prerequisite for accurate protein biomarker quantification with LC-MS/MS, as illustrated by hepcidin measurement

  • Ellen M.H. Schmitz , Niels M. Leijten , Joost L.J. van Dongen , Maarten A.C. Broeren , Lech G. Milroy , Luc Brunsveld , Volkher Scharnhorst and Daan van de Kerkhof EMAIL logo
Published/Copyright: May 19, 2018
Become an author with De Gruyter Brill
Submit Manuscript Author Information
Clinical Chemistry and Laboratory Medicine (CCLM)
From the journal Clinical Chemistry and Laboratory Medicine (CCLM) Volume 56 Issue 9

Abstract

Background:

Targeted quantification of protein biomarkers with liquid chromatography-tandem mass spectrometry (LC-MS/MS) has great potential, but is still in its infancy. Therefore, we elucidated the influence of charge state distribution and matrix effects on accurate quantification, illustrated by the peptide hormone hepcidin.

Methods:

An LC-MS/MS assay for hepcidin, developed based on existing literature, was improved by using 5 mM ammonium formate buffer as mobile phase A and as an elution solution for solid phase extraction (SPE) to optimize the charge state distribution. After extensive analytical validation, focusing on interference and matrix effects, the clinical consequence of this method adjustment was studied by performing receiving operating characteristic (ROC)-curve analysis in patients with iron deficiency anemia (IDA, n=44), anemia of chronic disease (ACD, n=42) and non-anemic patients (n=93).

Results:

By using a buffered solution during sample preparation and chromatography, the most abundant charge state was shifted from 4+ to 3+ and the charge state distribution was strongly stabilized. The matrix effects which occurred in the 4+ state were therefore avoided, eliminating bias in the low concentration range of hepcidin. Consequently, sensitivity, specificity and positive predictive value (PPV) for detection of IDA patients with the optimized assay (96%, 97%, 91%, respectively) were much better than for the original assay (73%, 70%, 44%, respectively).

Conclusions:

Fundamental improvements in LC-MS/MS assays greatly impact the accuracy of protein quantification. This is urgently required for improved diagnostic accuracy and clinical value, as illustrated by the validation of our hepcidin assay.

Keywords: charge state distribution; hepcidin; LC-MS/MS; mass spectrometry; protein quantification
Corresponding author: Dr. Daan van de Kerkhof, Algemeen Klinisch Laboratorium Catharina Ziekenhuis, Michelangelolaan 2, 5623 EJ Eindhoven, The Netherlands, Phone: 040 – 239 8639, Expert Center Clinical Chemistry Eindhoven, Eindhoven, The Netherlands; and Catharina Hospital Eindhoven, Clinical Laboratory, Eindhoven, The Netherlands

  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.

References

1. Jain KK. Biomarkers – technologies, markets and companies. Jain PharmaBiotech Report No.: ID 328357 2017.Search in Google Scholar

2. Ezan E, Bitsch F. Critical comparison of MS and immunoassays for the bioanalysis of therapeutic antibodies. Bioanalysis 2009;1:1375–88.10.4155/bio.09.121Search in Google Scholar PubMed

3. Ketha SS, Singh RJ, Ketha H. Role of mass spectrometry in clinical endocrinology. Endocrinol Metab Clin North Am 2017;46:593–613.10.1016/j.ecl.2017.04.001Search in Google Scholar PubMed

4. Keevil BG. LC-MS/MS analysis of steroids in the clinical laboratory. Clin Biochem 2016;49:989–97.10.1016/j.clinbiochem.2016.04.009Search in Google Scholar PubMed

5. Chappell DL, Lassman ME, McAvoy T, Lin M, Spellman DS, Laterza OF. Quantitation of human peptides and proteins via MS: review of analytically validated assays. Bioanalysis 2014;6:1843–57.10.4155/bio.14.145Search in Google Scholar PubMed

6. Nemeth E, Tuttle MS, Powelson J, Vaughn MB, Donovan A, Ward DM, et al. Hepcidin regulates cellular iron efflux by binding to ferroportin and inducing its internalization. Science 2004;306:2090–3.10.1126/science.1104742Search in Google Scholar PubMed

7. Nicolas G, Chauvet C, Viatte L, Danan JL, Bigard X, Devaux I, et al. The gene encoding the iron regulatory peptide hepcidin is regulated by anemia, hypoxia, and inflammation. J Clin Invest 2002;110:1037–44.10.1172/JCI0215686Search in Google Scholar

8. Nemeth E, Valore EV, Territo M, Schiller G, Lichtenstein A, Ganz T. Hepcidin, a putative mediator of anemia of inflammation, is a type II acute-phase protein. Blood 2003;101:2461–3.10.1182/blood-2002-10-3235Search in Google Scholar PubMed

9. Wagner M, Ashby DR, Kurtz C, Alam A, Busbridge M, Raff U, et al. Hepcidin-25 in diabetic chronic kidney disease is predictive for mortality and progression to end stage renal disease. PLoS One 2015;10:e0123072.10.1371/journal.pone.0123072Search in Google Scholar PubMed PubMed Central

10. Bregman DB, Morris D, Koch TA, He A, Goodnough LT. Hepcidin levels predict nonresponsiveness to oral iron therapy in patients with iron deficiency anemia. Am J Hematol 2013;88:97–101.10.1002/ajh.23354Search in Google Scholar PubMed

11. Ganz T, Olbina G, Girelli D, Nemeth E, Westerman M. Immunoassay for human serum hepcidin. Blood 2008;112:4292–7.10.1182/blood-2008-02-139915Search in Google Scholar PubMed

12. Koliaraki V, Marinou M, Vassilakopoulos TP, Vavourakis E, Tsochatzis E, Pangalis GA, et al. A novel immunological assay for hepcidin quantification in human serum. PLoS One 2009;4:e4581.10.1371/journal.pone.0004581Search in Google Scholar PubMed PubMed Central

13. Butterfield AM, Luan P, Witcher DR, Manetta J, Murphy AT, Wroblewski VJ, et al. A dual-monoclonal sandwich ELISA specific for hepcidin-25. Clin Chem 2010;56:1725–32.10.1373/clinchem.2010.151522Search in Google Scholar PubMed

14. Rochat B, Peduzzi D, McMullen J, Favre A, Kottelat E, Favrat B, et al. Validation of hepcidin quantification in plasma using LC-HRMS and discovery of a new hepcidin isoform. Bioanalysis 2013;5:2509–20.10.4155/bio.13.225Search in Google Scholar PubMed

15. Murphy AT, Witcher DR, Luan P, Wroblewski VJ. Quantitation of hepcidin from human and mouse serum using liquid chromatography tandem mass spectrometry. Blood 2007;110:1048–54.10.1182/blood-2006-11-057471Search in Google Scholar PubMed

16. Li H, Rose MJ, Tran L, Zhang J, Miranda LP, James CA, et al. Development of a method for the sensitive and quantitative determination of hepcidin in human serum using LC-MS/MS. J Pharmacol Toxicol Methods 2009;59:171–80.10.1016/j.vascn.2009.02.004Search in Google Scholar PubMed

17. Bansal SS, Abbate V, Bomford A, Halket JM, Macdougall IC, Thein SL, et al. Quantitation of hepcidin in serum using ultra-high-pressure liquid chromatography and a linear ion trap mass spectrometer. Rapid Commun Mass Spectrom 2010;24:1251–9.10.1002/rcm.4512Search in Google Scholar PubMed

18. Itkonen O, Parkkinen J, Stenman U-H, Hämäläinen E. Preanalytical factors and reference intervals for serum hepcidin LC–MS/MS method. Clin Chim Acta 2012;413:696–701.10.1016/j.cca.2011.12.015Search in Google Scholar PubMed

19. Wolff F, Deleers M, Melot C, Gulbis B, Cotton F. Hepcidin-25: measurement by LC-MS/MS in serum and urine, reference ranges and urinary fractional excretion. Clin Chim Acta Int J Clin Chem 2013;423:99–104.10.1016/j.cca.2013.04.021Search in Google Scholar PubMed

20. Lefebvre T, Dessendier N, Houamel D, Ialy-Radio N, Kannengiesser C, Manceau H, et al. LC-MS/MS method for hepcidin-25 measurement in human and mouse serum: clinical and research implications in iron disorders. Clin Chem Lab Med 2015;53:1557–67.10.1515/cclm-2014-1093Search in Google Scholar PubMed

21. Swensen AC, Finnell JG, Matias C, Gross AJ, Prince JT, Watt RK, et al. Whole blood and urine bioactive Hepcidin-25 determination using liquid chromatography mass spectrometry. Anal Biochem 2017;517:23–30.10.1016/j.ab.2016.10.023Search in Google Scholar PubMed

22. Kroot JJ, Kemna EH, Bansal SS, Busbridge M, Campostrini N, Girelli D, et al. Results of the first international round robin for the quantification of urinary and plasma hepcidin assays: need for standardization. Haematologica 2009;94:1748–52.10.3324/haematol.2009.010322Search in Google Scholar PubMed PubMed Central

23. Kroot JJ, van Herwaarden AE, Tjalsma H, Jansen RT, Hendriks JC, Swinkels DW. Second round robin for plasma hepcidin methods: first steps toward harmonization. Am J Hematol 2012;87:977–83.10.1002/ajh.23289Search in Google Scholar PubMed

24. Chan W, White P, editors. Fmoc solid phase peptide synthesis: a practical approach. Oxford, New York: Oxford University Press, 1999.10.1093/oso/9780199637256.001.0001Search in Google Scholar

25. CLSI. EP06, Evaluation of the linearity of quantitative measurement procedures: a statistical approach: approved guideline. Clinical and Laboratory Standards Institute, 2003.Search in Google Scholar

26. CLSI. EP15-A3, User verification of precision and estimation of bias; approved guideline, 3rd Edition. Clinical and Laboratory Standards Institute, 2014.Search in Google Scholar

27. ICH. Q2(R1), Validation of analytical procedures: text and methodology. International council for harmonisation of technical requirements for pharmaceuticals for human use, 2005.Search in Google Scholar

28. CLSI. EP17-A2, Evaluation of detection capability for clinical laboratory measurement procedures; approved guideline, 2nd ed. Clinical and Laboratory Standards Institute, 2012.Search in Google Scholar

29. Hwang S-I, Lee Y-Y, Park J-O, Norton HJ, Clemens E, Schrum LW, et al. Effects of a single dose of oral iron on hepcidin concentrations in human urine and serum analyzed by a robust LC-MS/MS method. Clin Chim Acta Int J Clin Chem 2011;412:2241–7.10.1016/j.cca.2011.08.014Search in Google Scholar PubMed PubMed Central

30. Van den Broek I, van Dongen WD. LC-MS-based quantification of intact proteins: perspective for clinical and bioanalytical applications. Bioanalysis 2015;7:1943–58.10.4155/bio.15.113Search in Google Scholar PubMed

31. Hewavitharana AK, Herath HM, Shaw PN, Cabot PJ, Kebarle P. Effect of solvent and electrospray mass spectrometer parameters on the charge state distribution of peptides–a case study using liquid chromatography/mass spectrometry method development for beta-endorphin assay. Rapid Commun Mass Spectrom 2010;24:3510–4.10.1002/rcm.4806Search in Google Scholar PubMed

32. Taylor PJ. Matrix effects: the Achilles heel of quantitative high-performance liquid chromatography–electrospray–tandem mass spectrometry. Clin Biochem 2005;38:328–34.10.1016/j.clinbiochem.2004.11.007Search in Google Scholar PubMed

33. Anderson DS, Kirchner M, Kellogg M, Kalish LA, Jeong J-Y, Vanasse G, et al. Design and validation of a high-throughput matrix-assisted laser desorption ionization time-of-flight mass spectrometry method for quantification of hepcidin in human plasma. Anal Chem 2011;83:8357–62.10.1021/ac2020905Search in Google Scholar PubMed

Supplementary Material:

The online version of this article offers supplementary material (https://doi.org/10.1515/cclm-2018-0013).

Received: 2018-01-05
Accepted: 2018-03-26
Published Online: 2018-05-19
Published in Print: 2018-08-28

©2018 Walter de Gruyter GmbH, Berlin/Boston

Articles in the same Issue

  1. Frontmatter
  2. Editorials
  3. Clinical Chemistry and Laboratory Medicine continues to shine brightly in the constellation of laboratory medicine
  4. The Theranos saga and the consequences
  5. Innovative approaches in diabetes diagnosis and monitoring: less invasive, less expensive… but less, equally or more efficient?
  6. Reviews
  7. Exploring the microbiota to better understand gastrointestinal cancers physiology
  8. Linking type 2 diabetes and gynecological cancer: an introductory overview
  9. Mini Reviews
  10. MicroRNAs as predictive biomarkers of response to tyrosine kinase inhibitor therapy in metastatic renal cell carcinoma
  11. Salivary biomarkers and cardiovascular disease: a systematic review
  12. Opinion Paper
  13. The meteoric rise and dramatic fall of Theranos: lessons learned for the diagnostic industry
  14. General Clinical Chemistry and Laboratory Medicine
  15. Uncertainty evaluation in clinical chemistry, immunoassay, hematology and coagulation analytes using only external quality assessment data
  16. Measurement uncertainty and metrological traceability of whole blood cyclosporin A mass concentration results obtained by UHPLC-MS/MS
  17. Computer-assisted interventions in the clinical laboratory process improve the diagnosis and treatment of severe vitamin B12 deficiency
  18. Trueness, precision and stability of the LIAISON 1-84 parathyroid hormone (PTH) third-generation assay: comparison to existing intact PTH assays
  19. Fibroblast growth factor 23 and renal function among young and healthy individuals
  20. Optimizing charge state distribution is a prerequisite for accurate protein biomarker quantification with LC-MS/MS, as illustrated by hepcidin measurement
  21. Quantification of human complement C2 protein using an automated turbidimetric immunoassay
  22. EE score: an index for simple differentiation of homozygous hemoglobin E and hemoglobin E-β0-thalassemia
  23. Reference Values and Biological Variations
  24. Algorithm on age partitioning for estimation of reference intervals using clinical laboratory database exemplified with plasma creatinine
  25. A simple transformation independent method for outlier definition
  26. Cancer Diagnostics
  27. Quantification of vanillylmandelic acid, homovanillic acid and 5-hydroxyindoleacetic acid in urine using a dilute-and-shoot and ultra-high pressure liquid chromatography tandem mass spectrometry method
  28. Cardiovascular Diseases
  29. Sialylated isoforms of apolipoprotein C-III and plasma lipids in subjects with coronary artery disease
  30. Diabetes
  31. Analysis of protein glycation in human fingernail clippings with near-infrared (NIR) spectroscopy as an alternative technique for the diagnosis of diabetes mellitus
  32. Letter to the Editor
  33. Preanalytical errors before and after implementation of an automatic blood tube labeling system in two outpatient phlebotomy centers
  34. Hemolysis interference studies: freeze method should be used in the preparation of hemolyzed samples
  35. The curious case of postprandial glucose less than fasting glucose: little things that matter much
  36. Finding best practice in internal quality control procedures using external quality assurance performance
  37. Evaluation of the analytical performance of a new ADVIA immunoassay using the Centaur XPT platform system for the measurement of cardiac troponin I
  38. Reference ranges of the Sebia free light chain ratio in patients with chronic kidney disease
  39. Antigen excess detection by automated assays for free light chains
  40. Multiple myeloma and macro creatine kinase type 1: the first case report
  41. Comparison of five cell-free DNA isolation methods to detect the EGFR T790M mutation in plasma samples of patients with lung cancer
  42. Can we use a point-of-care blood gas analyzer to measure the lactate concentration in cerebrospinal fluid of patients with suspected meningitis?
  43. Unstable haemoglobin variant Hb Leiden is detected on Sysmex XN-Series analysers
  44. Congress Abstracts
  45. 59th National Congress of the Hungarian Society of Laboratory Medicine
https://doi.org/10.1515/cclm-2018-0013
Keywords for this article
charge state distribution; hepcidin; LC-MS/MS; mass spectrometry; protein quantification

Articles in the same Issue

  1. Frontmatter
  2. Editorials
  3. Clinical Chemistry and Laboratory Medicine continues to shine brightly in the constellation of laboratory medicine
  4. The Theranos saga and the consequences
  5. Innovative approaches in diabetes diagnosis and monitoring: less invasive, less expensive… but less, equally or more efficient?
  6. Reviews
  7. Exploring the microbiota to better understand gastrointestinal cancers physiology
  8. Linking type 2 diabetes and gynecological cancer: an introductory overview
  9. Mini Reviews
  10. MicroRNAs as predictive biomarkers of response to tyrosine kinase inhibitor therapy in metastatic renal cell carcinoma
  11. Salivary biomarkers and cardiovascular disease: a systematic review
  12. Opinion Paper
  13. The meteoric rise and dramatic fall of Theranos: lessons learned for the diagnostic industry
  14. General Clinical Chemistry and Laboratory Medicine
  15. Uncertainty evaluation in clinical chemistry, immunoassay, hematology and coagulation analytes using only external quality assessment data
  16. Measurement uncertainty and metrological traceability of whole blood cyclosporin A mass concentration results obtained by UHPLC-MS/MS
  17. Computer-assisted interventions in the clinical laboratory process improve the diagnosis and treatment of severe vitamin B12 deficiency
  18. Trueness, precision and stability of the LIAISON 1-84 parathyroid hormone (PTH) third-generation assay: comparison to existing intact PTH assays
  19. Fibroblast growth factor 23 and renal function among young and healthy individuals
  20. Optimizing charge state distribution is a prerequisite for accurate protein biomarker quantification with LC-MS/MS, as illustrated by hepcidin measurement
  21. Quantification of human complement C2 protein using an automated turbidimetric immunoassay
  22. EE score: an index for simple differentiation of homozygous hemoglobin E and hemoglobin E-β0-thalassemia
  23. Reference Values and Biological Variations
  24. Algorithm on age partitioning for estimation of reference intervals using clinical laboratory database exemplified with plasma creatinine
  25. A simple transformation independent method for outlier definition
  26. Cancer Diagnostics
  27. Quantification of vanillylmandelic acid, homovanillic acid and 5-hydroxyindoleacetic acid in urine using a dilute-and-shoot and ultra-high pressure liquid chromatography tandem mass spectrometry method
  28. Cardiovascular Diseases
  29. Sialylated isoforms of apolipoprotein C-III and plasma lipids in subjects with coronary artery disease
  30. Diabetes
  31. Analysis of protein glycation in human fingernail clippings with near-infrared (NIR) spectroscopy as an alternative technique for the diagnosis of diabetes mellitus
  32. Letter to the Editor
  33. Preanalytical errors before and after implementation of an automatic blood tube labeling system in two outpatient phlebotomy centers
  34. Hemolysis interference studies: freeze method should be used in the preparation of hemolyzed samples
  35. The curious case of postprandial glucose less than fasting glucose: little things that matter much
  36. Finding best practice in internal quality control procedures using external quality assurance performance
  37. Evaluation of the analytical performance of a new ADVIA immunoassay using the Centaur XPT platform system for the measurement of cardiac troponin I
  38. Reference ranges of the Sebia free light chain ratio in patients with chronic kidney disease
  39. Antigen excess detection by automated assays for free light chains
  40. Multiple myeloma and macro creatine kinase type 1: the first case report
  41. Comparison of five cell-free DNA isolation methods to detect the EGFR T790M mutation in plasma samples of patients with lung cancer
  42. Can we use a point-of-care blood gas analyzer to measure the lactate concentration in cerebrospinal fluid of patients with suspected meningitis?
  43. Unstable haemoglobin variant Hb Leiden is detected on Sysmex XN-Series analysers
  44. Congress Abstracts
  45. 59th National Congress of the Hungarian Society of Laboratory Medicine
Sign up now to receive a 20% welcome discount
Institutional Access
How does access work?
Have an idea on how to improve our website?
Please write us.
© 2025 De Gruyter Brill
Downloaded on 14.9.2025 from https://www.degruyterbrill.com/document/doi/10.1515/cclm-2018-0013/html
Scroll to top button