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Clin Chem. 2016 Jan;62(1):188-97. doi: 10.1373/clinchem.2015.246702. Epub 2015 Nov 19.

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.

Author information

1
Department of Clinical Chemistry and Laboratory Medicine.
2
Department of Clinical Chemistry and Laboratory Medicine, Department of Cardiology.
3
Department of Immunohematology and Blood Transfusion, and.
4
Department of Clinical Chemistry and Laboratory Medicine, Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, the Netherlands.
5
Department of Clinical Chemistry and Laboratory Medicine, c.m.cobbaert@lumc.nl.

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.

PMID:
26585923
DOI:
10.1373/clinchem.2015.246702
[Indexed for MEDLINE]
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