LAUSR

To the Editor: Apolipoprotein A1 (apoA1) is the major protein component of high-density lipoprotein particles in blood, and its concentration in serum and plasma is a marker of atherosclerotic cardiovascular diseases. Clinical laboratories commonly use immunometric methods to measure apoA1; however, an alternative approach, LC-MS/MS, has been proposed (1–3). Uptake of these LC-MS/MS methods for routine testing in a clinical setting has been challenging owing to complex and time-intensive sample preparation work flows. Overnight digestion has been previously applied for quantification of apoA1 by use of proteolytic peptides VQPYLDDFQK, DYVSQFEGSALGK, and THLAPYSDELR (1, 2) and a 3-h digest using peptide VQPYLDDFQK (3). Toward the design of a streamlined work flow for implementation in a clinical laboratory, we simplified sample preparation by using only additive steps and eliminating the use of chemical denaturants, reduction, and alkylation. Herein we demonstrate that such a simple and rapid work flow, targeting a fast-forming proteolytic peptide, can be used to develop a quantitative apoA1 LC-MS/MS assay in agreement with an established immunonephelometric method. Our optimized protocol generated several proteotypic peptides suitable for apoA1 quantification by using a brief 20-min digestion without reduction or alkylation steps or the use of chemical denaturants. On the basis of several factors including the 24-h digestion profile (4), THLAPYSDELR was selected for further development. To generate the external 6-point calibration curve, the unlabeled THLAPYSDELR peptide was synthesized, with purity determined by high-performance LC and amino acid analysis (New England Peptide). The lyophilized peptide stocks were initially solubilized in a 5% acetonitrile and 0.1% formic acid solution and further diluted with …