Single Reaction Monitoring/Multiple Reaction Monitoring

SRM is typically performed using triple quadrupole MS, where specificity is achieved by monitoring of precursor-product transition [55]. SRM/MRM utilizes peptides unique to the protein or a post-translationally modified peptide. Best practice assay design principles and implementation have been described [56]. Optimal transitions and retention time information are typically determined from laboratory-acquired data: prior data-dependent acquisition (DDA) or analysis of synthetic peptides can also be designed computationally [57]. SRM/MRM plots typically contain a single peak, from which integrated peak area provides a quantitative measure. Internal reference unmodified peptides from the target protein can be used for signal normalization for label-free quantification. Pseudo-SRM is a variant of this technique, where a target inclusion list directs acquisition of full-scan MS2 spectra for selected precursors. The resulting extracted ion chromatograms are used to generate peak areas for transitions of interest and are used for quantitation [58]. The MRM method has a wide linear dynamic range of up to five orders of magnitude and high precision, making it well suited for the analysis of low- abundance peptides, which typically include post-translationally modified peptides.

For absolute quantification, a selected analyte-AQUA peptide pair is analyzed using two alternating SRM experiments performed during a single LC-SRM analysis. The SRM transition and LC retention time information are determined using synthetic peptides. This method is applicable to post-translationally modified and unmodified peptides [54], including acetylation and methylation as exemplified by a library of 93 reference peptides, which include lysine mono-, di-, and trimethylation; arginine monomethylation; serine/threonine phosphorylation; lysine acetylation; and N-terminal acetylation [59]. Analysis of this reference set demonstrated that detection efficiency did not correlate with molecular weight, hydrophobicity, basicity, or modification type. However, differences in detection efficiencies suggested a positional effect of lysine acetylation within a given peptide sequence [59].

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