Increased Throughput for Low-Abundance Protein Biomarker Verification by Liquid Chromatography/Tandem Mass Spectrometry

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Abstract
Low-abundance protein quantification has historically been performed using ligand binding techniques. However, due to the time and cost associated with developing enzyme-linked immunosorbent assay (ELISA), mass spectrometric approaches are playing an increasingly important role. Protein quantification at or below the nanogram per milliliter level using liquid chromatography/tandem mass spectrometry (LC/MS/MS) typically utilizes an immunoaffinity (IA) enrichment step such as immunoprecipitation. In order to maximize mass spectrometry (MS) sensitivity, protein enrichment is followed by a proteolytic cleavage step used to generate a surrogate peptide with better mass spectrometric properties. Unlike ELISA, IA−LC/MS/MS is a serial technique that can require up to 3 days for a single batch analysis due to lengthy incubation and digestion steps. This report describes the use of immunoprecipitation in 96-well ELISA format (IPE) and microwave-assisted protein digestion to reduce the time required to perform LC/MS/MS protein analyses to within a single day. The utility of this approach was investigated through its application to previously published LC/MS/MS protein assays from our laboratory for two cardiotoxicity biomarkers, Myl3 and NTproBNP. Using commercially available antibodies, IPE and microwave-assisted digestion were used to repeat intraday validations for these markers, and intraday precision (%CV) and accuracy (%RE) did not exceed 11% or 3% for either assay, respectively. Additionally, lower limits of quantification of 100 pg/mL (NTproBNP) and 0.95 ng/mL (Myl3) were achieved.

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