Quantifying Protein Measurands by Peptide Measurements: Where Do Errors Arise?
Abstract
Clinically actionable quantification of protein biomarkers by mass spectrometry (MS) requires analytical performance in concordance with quality specifications for diagnostic tests. Laboratory-developed tests should, therefore, be validated in accordance with EN ISO 15189:2012 guidelines for medical laboratories to demonstrate competence and traceability along the entire workflow, including the selected standardization strategy and the phases before, during, and after proteolysis. In this study, bias and imprecision of a previously developed MS method for quantification of serum apolipoproteins A-I (Apo A-I) and B (Apo B) were thoroughly validated according to Clinical and Laboratory Standards Institute (CLSI) guidelines EP15-A2 and EP09-A3, using 100 patient sera and either stable-isotope labeled (SIL) peptides or SIL-Apo A-I as internal standard. The systematic overview of error components assigned sample preparation before the first 4 h of proteolysis as major source (∼85%) of within-sample imprecision without external calibration. No improvement in imprecision was observed with the use of SIL-Apo A-I instead of SIL-peptides. On the contrary, when the use of SIL-Apo A-I was combined with external calibration, imprecision improved significantly (from ∼9% to ∼6%) as a result of the normalization for matrix effects on linearity. A between-sample validation of bias in 100 patient sera further supported the presence of matrix effects on digestion completeness and additionally d…
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Manual sample preparation and matrix effects are major sources of error.
“We previously demonstrated that manual sample preparation and matrix effects are major sources of error (30) .”
From Automated multiplex LC-MS/MS assay for quantifying serum apolipoproteins A-I, B, C-I, C-II, C-III, and E with qualitative apolipoprotein E phenotyping (2016) · cites this paper as [30]
Clinical application of LC-MS/MS for protein biomarkers requires stringent criteria for bias and precision, which are particularly challenging in workflows involving enzymatic digestion of the protein measurand before LC-MS/MS peptide measurement.
“Clinical application of LC-MS/MS for protein biomarkers, furthermore, requires stringent criteria for bias and precision (28, 29) , which are particularly challenging in work flows involving enzymatic digestion of the protein measurand before LC-MS/MS peptide measurement (30) .”
From Automated multiplex LC-MS/MS assay for quantifying serum apolipoproteins A-I, B, C-I, C-II, C-III, and E with qualitative apolipoprotein E phenotyping (2016) · cites this paper as [30]
The previously reported manual sample preparation procedure for serum apoA-I and apoB was optimized to improve peptide recoveries and digestion rates for all apolipoproteins.
“Our previously reported manual sample preparation procedure for serum apoA-I and apoB (19, 30, 31) was optimized to improve peptide recoveries and digestion rates for all apolipoproteins (see online Supplemental Data and online Supplemental Fig. 4).”
From Automated multiplex LC-MS/MS assay for quantifying serum apolipoproteins A-I, B, C-I, C-II, C-III, and E with qualitative apolipoprotein E phenotyping (2016) · cites this paper as [30]
There was a consistent negative deviation for the MS response of apoA-I and apoB in sample NTG5, in concordance with previous observations as well as ITA measurements.
“In addition, there was a consistent negative deviation for the MS response of apoA-I and apoB in sample NTG5, in concordance with previous observations as well as ITA measurements (30) .”
From Automated multiplex LC-MS/MS assay for quantifying serum apolipoproteins A-I, B, C-I, C-II, C-III, and E with qualitative apolipoprotein E phenotyping (2016) · cites this paper as [30]
Cites
Lassman, M. E., McLaughlin, T. M., Somers, E. P., Stefanni, A. C., Chen, Z., Murphy, B. A., Bierilo, K. K., Flattery, A. M., Wong, K. K., Castro‐Perez, J. M., et al. (2012) · Rapid Communications in Mass Spectrometry · doi:10.1002/rcm.5296 · Cited by 51 · see citing works
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van den Broek, I., Romijn, F. P. H. T. M., Nouta, J., van der Laarse, A., Drijfhout, J. W., Smit, N. P. M., van der Burgt, Y. E. M., & Cobbaert, C. M. (2016) · Clinical Chemistry · doi:10.1373/clinchem.2015.246702 · Cited by 105 · see citing works
Self-citationvan den Broek, I., Fu, Q., Kushon, S., Kowalski, M. P., Millis, K., Percy, A., Holewinski, R. J., Venkatraman, V., & Van Eyk, J. E. (2017) · Clinical Mass Spectrometry · doi:10.1016/j.clinms.2017.08.004 · Cited by 62 · see citing works
- Authors
- van den Broek, I., Romijn, F. P. H. T. M., Smit, N. P. M., van der Laarse, A., Drijfhout, J. W., van der Burgt, Y. E. M., & Cobbaert, C. M.
- Affiliations
- Department of Clinical Chemistry and Laboratory Medicine, Leiden University Medical Center (LUMC), Albinusdreef 2, 2333 ZA Leiden, The Netherlands
- Leiden University Medical Center (NL)
- Center for Proteomics and Metabolomics, Leiden University Medical Center (LUMC), Albinusdreef 2, 2333 ZA Leiden, The Netherlands
- Venue
- Journal of Proteome Research
- Publisher
- American Chemical Society (ACS)
- Published
- 2015-01-06
- Volume
- 14
- Issue
- 2
- Pages
- 928-942
- ISSN
- 1535-3893 (ISSN-L)
- 1535-3907
- OpenAlex
- W2073637268
- Cited by
- 49
- References
- 1
- Citations by year
- 2025: 2 · 2024: 1 · 2023: 2 · 2022: 4 · 2021: 5 · 2020: 5 · 2018: 4 · 2017: 12 · 2016: 7 · 2015: 7
- FWCI
- 3.49
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Journal of Proteome Research (publishedVersion)
- PubMed (publishedVersion)
- Data Archiving and Networked Services (DANS) (submittedVersion)
- Leiden Repository (Leiden University) (submittedVersion)
- Topics
- Physical Sciences › Chemistry › Spectroscopy › Advanced Proteomics Techniques and Applications
- Health Sciences › Medicine › Physiology › Clinical Laboratory Practices and Quality Control
- Physical Sciences › Chemistry › Spectroscopy › Mass Spectrometry Techniques and Applications
- Keywords
- apolipoprotein A-I, apolipoprotein B, bias, isotope-labeled internal standards, external calibration, matrix effects, proteolysis, serum samples, laboratory-developed tests, CLSI guidelines
- MeSH terms
- Blood Proteins, Chromatography, Liquid, Humans, Isotope Labeling, Peptide Fragments, Trypsin, Reproducibility of Results, Proteomics, Tandem Mass Spectrometry
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