Related Experiment Video
Updated: May 8, 2026
A High Throughput, Multiplexed and Targeted Proteomic CSF Assay to Quantify Neurodegenerative Biomarkers and Apolipoprotein E Isoforms Status
Published on: October 20, 2016
Quantification of seven apolipoproteins in human plasma by proteotypic peptides using fast LC-MS/MS
Uta Ceglarek1, Julia Dittrich, Susen Becker
1Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University Hospital Leipzig, Leipzig, Germany; LIFE-Leipzig Research Center for Civilization Diseases, University of Leipzig, Leipzig, Germany.
Purpose:
We investigated different sample pretreatment strategies and developed a standardized sample pretreatment protocol for absolute quantification of seven apolipoproteins (Apos) in human serum by LC-MS/MS using proteotypic peptides and corresponding stable isotope-labeled peptides as internal standards.
Experimental Design:
Micro-LC was coupled with quadrupole-linear ion trap MS for quantification and peptide confirmation. Denaturation, reduction, alkylation, and tryptic digestion including ultrasound and microwave assistance were investigated. Method comparison of 50 plasma samples with an immunoassay was performed for Apo A-I and Apo B.
Results:
Tryptic digestion times ranged between 5 min (Apo A-I, Apo E, Apo A-IV) and 16 h (Apo A-II). Ultrasound and microwave assistance did not improve the digestion yield. Linearity was found between 0.1 nmol/L and 100 mmol/L. The lower limits of quantification were ≤ 0.4 μmol/L for Apo A-I, Apo A-IV, Apo B-100, Apo C-I, Apo C-III, Apo E, and <1.4 μmol/L for Apo A-II. CV <13% were determined. Comparison with immunoassays showed a good agreement for Apo A-I and Apo B.
Conclusion And Clinical Relevance:
The validated preanalytical protocol enables a reliable simultaneous analysis of seven Apos in human serum without depletion. The method can now be applied in clinical studies to investigate the Apo distributions in cardiovascular diseases.
