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Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification
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Quantifying protein measurands by peptide measurements: where do errors arise?
Irene van den Broek1, Fred P H T M Romijn, Nico P M Smit
1Department of Clinical Chemistry and Laboratory Medicine, Leiden University Medical Center (LUMC) , Albinusdreef 2, 2333 ZA Leiden, The Netherlands.
Journal of Proteome Research
|December 16, 2014
Summary
This study validates a mass spectrometry (MS) method for quantifying apolipoproteins A-I and B, identifying sample preparation and proteolysis as key error sources. External calibration with stable-isotope labeled apolipoproteins significantly improved precision by normalizing matrix effects.
Area of Science:
- Clinical Chemistry
- Analytical Chemistry
- Mass Spectrometry
Background:
- Accurate protein biomarker quantification via mass spectrometry (MS) is crucial for clinical diagnostics.
- Laboratory-developed tests require validation per EN ISO 15189:2012 for competence and traceability.
- Understanding error sources in the MS workflow, including pre-analytical and proteolysis phases, is essential.
Purpose of the Study:
- To thoroughly validate the bias and imprecision of an MS method for quantifying serum apolipoproteins A-I and B.
- To identify and quantify major sources of error in the MS-based protein quantification workflow.
- To provide recommendations for assessing and controlling analytical quality for clinical use.
Main Methods:
- Validation of bias and imprecision using Clinical and Laboratory Standards Institute (CLSI) guidelines EP15-A2 and EP09-A3.
- Analysis of 100 patient sera using stable-isotope labeled (SIL) peptides or SIL-Apolipoprotein A-I (Apo A-I) as internal standards.
- Systematic overview of error components across the entire workflow, including sample preparation and proteolysis.
Main Results:
- Sample preparation before proteolysis was identified as the primary source (∼85%) of within-sample imprecision without external calibration.
- Using SIL-Apo A-I as an internal standard did not improve imprecision compared to SIL-peptides alone.
- External calibration combined with SIL-Apo A-I significantly improved imprecision (∼9% to ∼6%) by normalizing matrix effects.
- Specimen-specific biases were observed, linked to modified peptide sequences or altered protease activity, indicating matrix effects on digestion.
Conclusions:
- Sample preparation and proteolysis are critical steps influencing the imprecision of MS-based protein quantification.
- External calibration is vital for mitigating matrix effects and improving the accuracy of MS-based protein measurements.
- The study provides a framework for understanding and controlling errors in MS assays, ensuring analytical quality for clinical applications.
