Multiple Reaction Monitoring

Multiple reaction monitoring (MRM), also known as selected reaction monitoring (SRM), is a targeted mass spectrometric methodology that is not limited to the analysis of PTM modified peptides but has been used extensively as a sensitive method to analyze various types of peptides. In MRM analyses MS/MS is applied to detect and quantify selected peptides of interest, such as those previously identified in differential discovery studies or specific post-translationally modified forms of a known peptide. Here, the specificity of precursor to product transitions is harnessed for quantitative analysis of multiple proteins in a single sample. Software tools such as MRMaid [41] or Skyline [42] allow rapid MRM transition generation and method construction for targeted analyses.

Multiple Reaction Monitoring Initiated Detection and Sequencing

Multiple reaction monitoring-initiated detection and sequencing (MIDAS™)

[43] has been a well-utilized method for the analysis of PTM modified peptides with application to acetylated [44], phosphorylated [45], and ubiquitinated [46] species. MIDAS is a hypothesis-driven approach that requires the primary sequence of the target protein to be known and a proteolytic digest of this protein to be performed. MIDAS allows one to perform a targeted search for the presence of post-translationally modified peptides with detection based on the combination of the predicted molecular weight (measured as mass-charge ratio) of the PTM modified proteolytic peptide and a diagnostic fragment which is generated by specific fragmentation of modified peptides during CID performed in MS/MS analysis. Sequence information is subsequently obtained which enables PTM site assignment.

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