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

Multiple reaction monitoring as a method for identifying protein posttranslational modifications.

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Product Application Laboratory, MDS Sciex, 71 Four Valley Drive, Concord, Ontario, Canada L4K 4V8.


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.

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