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

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...