Multiple reaction monitoring as a method for identifying protein posttranslational modifications

J Biomol Tech. 2005 Jun;16(2):83-90.


The activity of many transcriptional regulators is significantly altered by posttranslational modifications of specific sites. For example, the activity of the muscle-restricted transcription factor family myocyte enhancer factor 2 (MEF2) is tightly controlled by phosphorylation. This modification is responsible for either an increase or a decrease in transcriptional activity, depending on the specific amino acid residues that are phosphorylated by signal-dependent kinases. Although mass spectrometry-based methods, such as precursor ion and neutral loss scans, are extremely useful for identifying unknown phosphopeptides from a complex mixture, they do not take advantage of any prior knowledge about the protein being investigated. Quite often a significant amount of information is available. This may include the primary sequence, type of phosphorylation (serine/threonine vs. tyrosine), or predicted phosphoacceptor sites (consensus peptide that is targeted by a kinase). This information can be used to predict precursor and fragment ion m/z values for a multiple reaction monitoring (MRM) experiment. By using these highly sensitive MRM experiments to trigger dependent product ion scans on a hybrid quadrupole linear ion-trap instrument, we were able to identify low levels of phosphorylation of MEF2A (a member of the MEF2 family), and alpha-casein. This method of monitoring protein phosphorylation at specific phosphoacceptor sites may prove useful in understanding the physiological regulation of protein function.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • COS Cells
  • Chlorocebus aethiops
  • Chromatography, Liquid
  • MEF2 Transcription Factors
  • Mass Spectrometry
  • Molecular Sequence Data
  • Myogenic Regulatory Factors / chemistry
  • Myogenic Regulatory Factors / metabolism
  • Phosphopeptides / chemistry
  • Phosphopeptides / metabolism*
  • Phosphorylation
  • Protein Processing, Post-Translational*


  • MEF2 Transcription Factors
  • Myogenic Regulatory Factors
  • Phosphopeptides