title: Quantification of seven apolipoproteins in human plasma by proteotypic peptides using fast LC-MS/MS.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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.
  
  
  CONCLUSIONS
  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.
authors: Institute 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.
authors: Uta
authors: U
authors: Ceglarek
authors: Julia
authors: J
authors: Dittrich
authors: Susen
authors: S
authors: Becker
authors: Frank
authors: F
authors: Baumann
authors: Linda
authors: L
authors: Kortz
authors: Joachim
authors: J
authors: Thiery
mesh_terms: D001053:Apolipoproteins / Q000032:analysis* / Q000737:chemistry; D001774:Blood Chemical Analysis / Q000379:methods*; D002853:Chromatography, Liquid; D006801:Humans; D010455:Peptides / Q000737:chemistry*; D053719:Tandem Mass Spectrometry
chemical_list: D001053:Apolipoproteins; D010455:Peptides
keywords: Apolipoproteins; Liquid chromatography-linear ion trap mass spectrometry; Proteotypic peptide