Immuno-Mass Spectrometry: Quantification of Low-Abundance Proteins in Biological Fluids

Abstract

Mass spectrometry is emerging as one of the most promising analytical techniques to examine simultaneously hundreds of analytes quickly, precisely, and accurately, using minute sample volumes. Currently, a major bottleneck in the verification phase of putative biomarkers is the lack of methods/reagents to quantify low levels of analytes in biological fluids. A major objective is to establish a high-throughput multiple reaction monitoring (MRM) assay capable of quantifying low-abundance proteins or peptides in biological fluids (low μg/L range) using mass spectrometry. The experimental procedure we propose, called immuno-mass spectrometry, consists of immuno-capturing analytes of interest from relevant biological fluids in 96-well microtiter plates and performing in-well tryptic digestion, with subsequent MRM of digested peptides on a triple quadrupole mass spectrometer. With such a strategy, limits of detection of 0.1–1 μg/L proteins in serum with a coefficient of variation of <20% can be obtained. This methodology could be adapted quickly and easily to potential candidates of interest, thus providing a much needed technology to bridge the gap between discovery and validation platforms.