title: Automated Multiplex LC-MS/MS Assay for Quantifying Serum Apolipoproteins A-I, B, C-I, C-II, C-III, and E with Qualitative Apolipoprotein E Phenotyping.
abstract: BACKGROUND
  Direct and calculated measures of lipoprotein fractions for cardiovascular risk assessment suffer from analytical inaccuracy in certain dyslipidemic and pathological states, most commonly hypertriglyceridemia. LC-MS/MS has proven suitable for multiplexed quantification and phenotyping of apolipoproteins. We developed and provisionally validated an automated assay for quantification of apolipoprotein (apo) A-I, B, C-I, C-II, C-III, and E and simultaneous qualitative assessment of apoE phenotypes.
  
  
  METHODS
  We used 5 value-assigned human serum pools for external calibration. Serum proteins were denatured, reduced, and alkylated according to standard mass spectrometry-based proteomics procedures. After trypsin digestion, peptides were analyzed by LC-MS/MS. For each peptide, we measured 2 transitions. We compared LC-MS/MS results to those obtained by an immunoturbidimetric assay or ELISA.
  
  
  RESULTS
  Intraassay CVs were 2.3%-5.5%, and total CVs were 2.5%-5.9%. The LC-MS/MS assay correlated (R = 0.975-0.995) with immunoturbidimetric assays with Conformité Européenne marking for apoA-I, apoB, apoC-II, apoC-III, and apoE in normotriglyceridemic (n = 54) and hypertriglyceridemic (n = 46) sera. Results were interchangeable for apoA-I ≤3.0 g/L (Deming slope 1.014) and for apoB-100 ≤1.8 g/L (Deming slope 1.016) and were traceable to higher-order standards.
  
  
  CONCLUSIONS
  The multiplex format provides an opportunity for new diagnostic and pathophysiologic insights into types of dyslipidemia and allows a more personalized approach for diagnosis and treatment of lipid abnormalities.
authors.affiliation: Department of Clinical Chemistry and Laboratory Medicine.
authors.forename: Irene
authors.initials: I
authors.lastname: van den Broek
authors.affiliation: Department of Clinical Chemistry and Laboratory Medicine.
authors.forename: Fred P H T M
authors.initials: FP
authors.lastname: Romijn
authors.affiliation: Department of Clinical Chemistry and Laboratory Medicine.
authors.forename: Jan
authors.initials: J
authors.lastname: Nouta
authors.affiliation: Department of Clinical Chemistry and Laboratory Medicine, Department of Cardiology.
authors.forename: Arnoud
authors.initials: A
authors.lastname: van der Laarse
authors.affiliation: Department of Immunohematology and Blood Transfusion, and.
authors.forename: Jan W
authors.initials: JW
authors.lastname: Drijfhout
authors.affiliation: Department of Clinical Chemistry and Laboratory Medicine.
authors.forename: Nico P M
authors.initials: NP
authors.lastname: Smit
authors.affiliation: Department of Clinical Chemistry and Laboratory Medicine, Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, the Netherlands.
authors.forename: Yuri E M
authors.initials: YE
authors.lastname: van der Burgt
authors.affiliation: Department of Clinical Chemistry and Laboratory Medicine, c.m.cobbaert@lumc.nl.
authors.forename: Christa M
authors.initials: CM
authors.lastname: Cobbaert
mesh_terms: D001053:Apolipoproteins / Q000097:blood*; D001331:Automation; D002851:Chromatography, High Pressure Liquid; D050171:Dyslipidemias / Q000097:blood* / Q000175:diagnosis*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D009391:Nephelometry and Turbidimetry; D010641:Phenotype; D040901:Proteomics; D053719:Tandem Mass Spectrometry
chemical_list: D001053:Apolipoproteins