Simultaneous Quantification of Apolipoprotein A-I and Apolipoprotein B by Liquid-Chromatography–Multiple- Reaction–Monitoring Mass Spectrometry

Abstract

BACKGROUND If liquid-chromatography–multiple-reaction–monitoring mass spectrometry (LC-MRM/MS) could be used in the large-scale preclinical verification of putative biomarkers, it would obviate the need for the development of expensive immunoassays. In addition, the translation of novel biomarkers to clinical use would be accelerated if the assays used in preclinical studies were the same as those used in the clinical laboratory. To validate this approach, we developed a multiplexed assay for the quantification of 2 clinically well-known biomarkers in human plasma, apolipoprotein A-I and apolipoprotein B (apoA-I and apoB). METHODS We used PeptideAtlas to identify candidate peptides. Human samples were denatured with urea or trifluoroethanol, reduced and alkylated, and digested with trypsin. We compared reversed-phase chromatographic separation of peptides with normal flow and microflow, and we normalized endogenous peptide peak areas to internal standard peptides. We evaluated different methods of calibration and compared the final method with a nephelometric immunoassay. RESULTS We developed a final method using trifluoroethanol denaturation, 21-h digestion, normal flow chromatography-electrospray ionization, and calibration with a single normal human plasma sample. For samples injected in duplicate, the method had intraassay CVs <6% and interassay CVs <12% for both proteins, and compared well with immunoassay (n = 47; Deming regression, LC-MRM/MS = 1.17 × immunoassay – 36.6; Sx|y = 10.3 for apoA-I and LC-MRM/MS = 1.21 × immunoassay + 7.0; Sx|y = 7.9 for apoB). CONCLUSIONS Multiplexed quantification of proteins in human plasma/serum by LC-MRM/MS is possible and compares well with clinically useful immunoassays. The potential application of single-point calibration to large clinical studies could simplify efforts to reduce day-to-day digestion variability.

Quantitative Clinical Chemistry Proteomics (qCCP) using mass spectrometry: general characteristics and application

“…In at least one study, quantification of clinically useful biomarker proteins in plasma by multiplexed LC-MRM/MS has demonstrated promise in the absolute quantification of biomarkers in plasma [15 ]. Using external calibration and comparison with existing clinical immunoassays for ApoA-I and ApoB, the authors confirmed that the direct analysis of tryptic digests is a viable approach, and may someday offer a cost-efficient and sample-saving analysis method for the clinical laboratory.…”

Advanced lipoprotein testing for cardiovascular diseases risk assessment: a review of the novel approaches in lipoprotein profiling

“…Apolipoprotein quantification by liquid chromatography ID/MS (LC-ID/MS) was first reported in the late 1990s by Barr et al for apoA-I [58] and was further applied to other apolipoproteins (apoB, C and E) in the following years [59][60][61].…”

“…After digestion, apolipoprotein-specific tryptic peptides were identified for each major class of apolipoproteins, and some of them were selected for quantification by ID/MS [60][61][62]. ID/MS quantification uses synthetic labeled entities with 13 C, 15 N or deuterium as internal standards (IS) to spike the samples.…”

“…These materials were endorsed by the World Health Organization (WHO) and widely used to recalibrate routine apolipoprotein immunoassays but were never intended for standardization purposes. It appears nevertheless that most ID-LC/MS methods for apolipoprotein quantification use these WHO RMs as external calibrators [60][61][62].…”

“…Given its high accuracy, good comparability with IN assays [59,60], possible SI traceability and high throughput, ID-LC/MS is one of the candidate reference methods for apoB and apoA-I quantification in serum. However, this method uses expensive materials, and although "turn-key" approaches have been reported [62], ID-LC/MS requires trained technical staff and dedicated instrumentation.…”