Detection of tyrosine phosphorylated peptides via skimmer collision-induced dissociation/ion trap mass spectrometry.
Phosphorylation of proteins is an important post-translational protein modification in cellular response to environmental change and occurs in both prokaryotes and eukaryotes. Identification of the amino acid on individual proteins that become phosphorylated in response to extracellular stimulus is essential for understanding the mechanisms involved in the intracellular signals that these modifications facilitate. Most protein kinases catalyze the phosphorylation of proteins on serine, threonine or tyrosine. Although tyrosine phosphorylation is often the least abundant of the three major phosphorylation sites, it is important owing to its role in signal pathways. Currently available methods for the identification of phosphorylation sites can often miss low levels of tyrosine phosphorylations. This paper describes a method for the identification of phosphotyrosine-containing peptides using electrospray ionization on an ion trap mass spectrometer. Skimmer-activated collision-induced dissociation (CID) was used to generate the phosphotyrosine immonium ion at m/z 216. This method is gentle enough that the protonated molecule of the intact peptide is still observed. In-trap CID was employed for the verification of the phosphotyrosine immonium ion. Using this technique, low levels of phosphotyrosine-containing peptides can be identified from peptide mixtures separated by nanoflow micro liquid chromatography/mass spectrometry.
Zolodz
Melissa D
MD
Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, 1333 RHPH Building, West Lafayette, Indiana 47907, USA.
Wood
Karl V
KV
D002364:Caseins / Q000737:chemistry; D003574:Cytochrome c Group / Q000737:chemistry; D007477:Ions; D010455:Peptides / Q000737:chemistry*; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000032:analysis*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000295:instrumentation / Q000379:methods*; D014357:Trypsin
D002364:Caseins; D003574:Cytochrome c Group; D007477:Ions; D010455:Peptides; D019000:Phosphotyrosine; D014357:Trypsin