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Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification
Published on: August 17, 2015
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Quantitative protein mass-spectrometry requires a standardized pre-analytical phase
Nico P M Smit1, Fred P H T M Romijn1, Vanessa J J van Ham2
1Department of Clinical Chemistry and Laboratory Medicine, Leiden University Medical Center, Leiden, The Netherlands.
Clinical Chemistry and Laboratory Medicine
|September 7, 2022
Summary
Blood collection tubes significantly impact quantitative protein mass spectrometry (QPMS) results. Heparin plasma is comparable to serum, while citrate and EDTA plasma affect apolipoprotein quantification due to altered digestion.
Area of Science:
- Clinical Chemistry
- Proteomics
- Mass Spectrometry
Background:
- Quantitative protein mass-spectrometry (QPMS) relies on tryptic digestion and peptide measurement.
- The interchangeability of serum and plasma matrices and the impact of anticoagulants on QPMS are not well understood.
- Serum is often preferred for apolipoprotein QPMS, necessitating investigation into plasma matrix effects.
Purpose of the Study:
- To investigate the effect of different blood collection matrices (serum vs. plasma with various anticoagulants) on apolipoprotein quantification using mass spectrometry.
- To determine if serum and plasma are interchangeable for QPMS analyses.
- To assess the influence of in-vitro anticoagulants on peptide recovery and tryptic digestion kinetics.
Main Methods:
- Blood samples from 44 healthy donors were collected simultaneously into tubes for serum and plasma (heparin, citrate, EDTA).
- Nine apolipoproteins were quantified using standard operating procedures and serum calibrators.
- Tryptic digestion kinetics were monitored over 22 hours in each matrix to assess peptide formation.
Main Results:
- Citrate plasma showed a -14.6% bias in apolipoprotein recovery compared to serum.
- Heparin plasma yielded results comparable to serum ( -0.3% bias).
- EDTA plasma exhibited a more pronounced and variable reduction (-5.3% bias), particularly affecting apoA-I, A-II, and C-III peptides due to altered digestion.
Conclusions:
- Plasma anticoagulants significantly affect QPMS test outcomes.
- Heparin plasma is a suitable alternative to serum for apolipoprotein QPMS.
- EDTA and citrate plasma can lead to inaccurate results due to altered digestion or dilution, emphasizing the need for pre-analytical standardization in clinical chemistry.
