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.

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.

[1]  J. Smith,et al.  Development of an MRM assay panel with application to biobank samples from patients with myocardial infarction. , 2013, Journal of proteomics.

[2]  Thomas Laurell,et al.  Moving towards high density clinical signature studies with a human proteome catalogue developing multiplexing mass spectrometry assay panels , 2011, Journal of Clinical Bioinformatics.

[3]  Susan E. Abbatiello,et al.  Targeted Peptide Measurements in Biology and Medicine: Best Practices for Mass Spectrometry-based Assay Development Using a Fit-for-Purpose Approach* , 2014, Molecular & Cellular Proteomics.

[4]  A. Van der Laarse,et al.  Metrological traceability in mass spectrometry-based targeted protein quantitation: a proof-of-principle study for serum apolipoproteins A-I and B100. , 2014, Journal of proteomics.

[5]  Christoph H Borchers,et al.  Multiple Reaction Monitoring-based, Multiplexed, Absolute Quantitation of 45 Proteins in Human Plasma* , 2009, Molecular & Cellular Proteomics.

[6]  A. Van der Laarse,et al.  Evaluation of interspecimen trypsin digestion efficiency prior to multiple reaction monitoring-based absolute protein quantification with native protein calibrators. , 2013, Journal of proteome research.

[7]  N. Shachter.  Apolipoproteins C-I and C-III as important modulators of lipoprotein metabolism , 2001, Current opinion in lipidology.

[8]  W. H. Hannon,et al.  International Federation of Clinical Chemistry standardization project for measurements of apolipoproteins A-I and B. IV. Comparability of apolipoprotein B values by use of International Reference Material. , 1994, Clinical chemistry.

[9]  L L Needham,et al.  Isotope dilution--mass spectrometric quantification of specific proteins: model application with apolipoprotein A-I. , 1996, Clinical chemistry.

[10]  D. Blom,et al.  Dysbetalipoproteinaemia: A mixed hyperlipidaemia of remnant lipoproteins due to mutations in apolipoprotein E , 2014, Critical reviews in clinical laboratory sciences.

[11]  W. H. Hannon,et al.  International Federation of Clinical Chemistry standardization project for measurements of apolipoproteins A-I and B. III. Comparability of apolipoprotein A-I values by use of international reference material. , 1993 .

[12]  H. R. Bergen,et al.  Simultaneous phenotyping and quantification of α-1-antitrypsin by liquid chromatography-tandem mass spectrometry. , 2011, Clinical chemistry.

[13]  Steven Hawken,et al.  Preventive cardiologyAbstractsEffect of potentially modifiable risk factors associated with myocardial infarction in 52 countries (the INTERHEART study): Case-control study , 2004 .

[14]  Andrew N Hoofnagle,et al.  Simultaneous quantification of apolipoprotein A-I and apolipoprotein B by liquid-chromatography-multiple- reaction-monitoring mass spectrometry. , 2010, Clinical chemistry.

[15]  Michael E. Lassman,et al.  A rapid method for cross-species quantitation of apolipoproteins A1, B48 and B100 in plasma by ultra-performance liquid chromatography/tandem mass spectrometry. , 2012, Rapid communications in mass spectrometry : RCM.

[16]  Mary F. Lopez,et al.  Assessment of peptide chemical modifications on the development of an accurate and precise multiplex selected reaction monitoring assay for apolipoprotein e isoforms. , 2014, Journal of proteome research.

[17]  G. Walldius,et al.  The apoB/apoA‐I ratio: a strong, new risk factor for cardiovascular disease and a target for lipid‐lowering therapy – a review of the evidence , 2006, Journal of internal medicine.

[18]  J. Borén,et al.  Clinical impact of direct HDLc and LDLc method bias in hypertriglyceridemia. A simulation study of the EAS-EFLM Collaborative Project Group. , 2014, Atherosclerosis.

[19]  Andrew N Hoofnagle,et al.  Multiple-reaction monitoring-mass spectrometric assays can accurately measure the relative protein abundance in complex mixtures. , 2012, Clinical chemistry.

[20]  R. Bateman,et al.  Method for the simultaneous quantitation of apolipoprotein E isoforms using tandem mass spectrometry. , 2009, Analytical biochemistry.

[21]  Andrew N Hoofnagle,et al.  Quantitative clinical proteomics by liquid chromatography-tandem mass spectrometry: assessing the platform. , 2010, Clinical chemistry.

[22]  Richard G Kay,et al.  The application of ultra-performance liquid chromatography/tandem mass spectrometry to the detection and quantitation of apolipoproteins in human serum. , 2007, Rapid communications in mass spectrometry : RCM.

[23]  Leigh Anderson,et al.  Quantitative Mass Spectrometric Multiple Reaction Monitoring Assays for Major Plasma Proteins* , 2006, Molecular & Cellular Proteomics.

[24]  A. Van der Laarse,et al.  Quantifying protein measurands by peptide measurements: where do errors arise? , 2015, Journal of proteome research.

[25]  G. Watts,et al.  An ABC of apolipoprotein C‐III: a clinically useful new cardiovascular risk factor? , 2008, International journal of clinical practice.

[26]  R. Nelson,et al.  Development of multiplex mass spectrometric immunoassay for detection and quantification of apolipoproteins C-I, C-II, C-III and their proteoforms. , 2015, Methods.

[27]  A. Kei,et al.  A review of the role of apolipoprotein C-II in lipoprotein metabolism and cardiovascular disease. , 2012, Metabolism: clinical and experimental.

[28]  J. Lambert,et al.  Total ApoE and ApoE4 Isoform Assays in an Alzheimer's Disease Case-control Study by Targeted Mass Spectrometry (n = 669): A Pilot Assay for Methionine-containing Proteotypic Peptides* , 2012, Molecular & Cellular Proteomics.

[29]  L. Havekes,et al.  Role of ApoCs in lipoprotein metabolism: functional differences between ApoC1, ApoC2, and ApoC3. , 1999, Arteriosclerosis, thrombosis, and vascular biology.

[30]  Anders Kallner,et al.  Measurement Procedure Comparison and Bias Estimation Using Patient Samples; Approved Guideline—Third Edition , 2013 .

[31]  Yadong Huang.  Mechanisms linking apolipoprotein E isoforms with cardiovascular and neurological diseases , 2010, Current opinion in lipidology.

[32]  U. Ceglarek,et al.  Quantification of seven apolipoproteins in human plasma by proteotypic peptides using fast LC‐MS/MS , 2013, Proteomics. Clinical applications.

[33]  M. Caslake,et al.  Apolipoproteins: metabolic role and clinical biochemistry applications , 2011, Annals of clinical biochemistry.

[34]  I. Turko,et al.  15N-labeled full-length apolipoprotein E4 as an internal standard for mass spectrometry quantification of apolipoprotein E isoforms. , 2012, Analytical chemistry.