Multiple and Single Reaction Monitoring Mass Spectrometry for Absolute Quantitation of Proteins

Abstract

The use of mass spectrometry (MS) to measure proteins has grown exponentially over the past 25 years. This growth has been primarily driven by the advent of proteomics in which scientists have developed methods to identify and quantitate as many proteins in a complex mixture as possible. Early studies trended towards the development of techniques that enabled greater quantitative coverage of the proteome. Many of these developments focused on relative quantitation in which the change in the abundances of proteins in comparative samples was measured. However, relative quantitation only allows a limited number of samples to be compared. This deficiency led to the development of technologies that allowed the relative quantitation of an unlimited number of samples to be measured, but what was still lacking was an emphasis on the ability of MS to measure the absolute abundance of proteins. A more recent technology trend has taken full advantage of the analytical attributes afforded in the use of MS for protein measurements. This trend utilizes the accuracy, sensitivity, specificity, and multiplexed capabilities of MS to quantity specific proteins within complex mixtures. Combined with the use of stable isotope-labeled internal standards, MS assays are now being developed to quantitate key diagnostic and prognostic proteins within clinical samples such as serum, plasma, urine, and cerebrospinal fluid. This chapter describes the technology behind the development of MS-based clinical protein assays and provides examples of where these assays are being used in diagnostic and prognostic settings.