Quantification of seven apolipoproteins in human plasma by proteotypic peptides using fast LC‐MS/MS

Abstract

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 A po A‐I and A po B. Results: Tryptic digestion times ranged between 5 min (A po A‐I, A po E, A po A‐IV) and 16 h (A po 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 A po A‐I, A po A‐IV, A po B‐100, A po C‐I, A po C‐III, A po E, and <1.4 μmol/L for A po A‐II. CV <13% were determined. Comparison with immunoassays showed a good agreement for A po A‐I and A po B. Conclusion and clinical relevance: The validated preanalytical protocol enables a reliable simultaneous analysis of seven A pos in human serum without depletion. The method can now be applied in clinical studies to investigate the A po distributions in cardiovascular diseases.