Simultaneous quantitation and size characterization of apolipoprotein(a) by ultra-performance liquid chromatography/mass spectrometry.

RATIONALE

Apolipoprotein(a) is a polymorphic glycoprotein covalently bound to apoB100 in Lp(a) particles and has been described to be both atherogenic and prothrombotic, although its exact mechanism of action is not well defined. Apolipoprotein(a) is routinely measured by immunoassays. Unfortunately, the accuracy of the measurement can be affected by the apolipoprotein(a) size (number of kringles) polymorphism in Lp(a) particles. Here we describe an ultra-performance liquid chromatography/mass spectrometry (UPLC/MS) assay that is capable of measuring apolipoprotein(a) concentrations while simultaneously determining the number of kringles present per protein.

METHODS

Plasma samples were diluted and proteins de-lipidated with deoxycholate prior to tryptic digestion. Distinct tryptic peptides from different regions of apolipoprotein(a) were measured to determine both concentration and the number of kringles present per protein. Separation and quantitation of tryptic peptides is carried out at 700 μL/min using a 1.7 µm C18 column (2.1 × 100 mm) coupled to a Thermo Vantage triple quadrupole (QQQ) mass spectrometer with a heated electrospray ionization (HESI) source.

RESULTS

This method was compared to established methods for measuring concentration (monoclonal antibody based ELISA) and size (gel-electrophoresis) using 80 plasma samples proved by NWLRL. The slope and r(2) value for the correlation of concentrations were determined to be 0.96 and 0.98, demonstrating excellent agreement of absolute values between the UPLC/MS and ELISA methods. As measured by UPLC/MS, the average kringle number or size is smaller than determined by the electrophoretic method.

CONCLUSIONS

A single UPLC/MS method was developed capable of measuring apolipoprotein(a) concentration and size (by measuring the number of kringles per protein). This assay passes criteria required for 'fit for purpose' assays including sensitivity, intra and interday reproducibility and freeze/thaw stability. While the agreement between UPLC/MS and ELISA is excellent for concentration and may provide researchers with additional tools for studying apolipoprotein(a), the dissimilarities between UPLC/MS and the electrophoretic method may also be exploited for understanding apolipoprotein(a) structure and function.