Absolute quantification of proteins using standard peptides and multiple reaction monitoring.

Mass spectrometry (MS) is a powerful tool for protein identification and has therefore become indispensable for proteome studies. In recent years, simple protein identification by MS has become routine, and more attention has been devoted to the MS-based investigation of posttranslational modifications and the quantification of proteins and peptides. Numerous methods and techniques for relative quantification of proteins by MS have emerged and have been applied successfully to answer various questions of protein abundance. Absolute quantification is often applied in clinical research and biomarker discovery, but has also been used to determine protein stoichiometries in protein complexes. However, the number of methods available for absolute quantification is still restricted and often requires the generation of standard peptides containing amino acids labeled with stable isotopes, although label-free approaches are also gaining importance. Complete hydrolysis of the proteins to be quantified is known to be one of the prerequisites for reliable absolute quantification, and selection and suitability of the standard peptides are critical factors in the planning of a quantitative study. Along the different methods to read out quantitative signals by MS, multiple reaction monitoring (MRM) has proven to be most suitable, with a wide linear range. However, analysis by MRM is a targeted approach and each case requires the individual design of suitable assays, which is a time-consuming step during the preliminary analysis. In this chapter, we present various protocols for in-solution hydrolysis, manual selection of suitable standard peptides, and design of MRM transitions.