Multiple reaction monitoring as a method for identifying protein posttranslational modifications.
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.
Cox
David M
DM
Product Application Laboratory, MDS Sciex, 71 Four Valley Drive, Concord, Ontario, Canada L4K 4V8. david.cox@sciex.com
Zhong
F
F
Du
M
M
Duchoslav
E
E
Sakuma
T
T
McDermott
J C
JC
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D002853:Chromatography, Liquid; D064326:MEF2 Transcription Factors; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D018006:Myogenic Regulatory Factors / Q000737:chemistry / Q000378:metabolism; D010748:Phosphopeptides / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational
D064326:MEF2 Transcription Factors; D018006:Myogenic Regulatory Factors; D010748:Phosphopeptides