Designing a multiple reaction monitoring method for protein quantification in a complex sample

A method for selecting peptides to build a multiple reaction monitoring (MRM) method for protein quantification was presented. Both a1 and yn-1 ions were monitored in the MRM method since the amine-specific reductive methylation labeling technique used been shown to enhance the a 1 and yn-1 ion signals in the MS/MS spectra of doubly charged tryptic peptides. The present experiment involved using empirical data to correlate ion intensities with several peptide characteristics to determine which characteristics cause certain peptides to produce more intense MRM peaks than others. The results were applied to a theoretical digest of the genome of Aeromonas salmonicida, a known fish pathogen which causes furunculosis, to determine which peptides were expected to produce observable MRM signals; these peptides were then used to build the MRM method. The MRM method was shown to be adequate for protein quantification of differentially labeled protein samples originating from different samples.