Many cellular processes are regulated by reversible
protein phosphorylation, and the ability to broadly identify and quantify
phosphoproteins from
proteomes would provide a basis for gaining a better understanding of these dynamic cellular processes. However, such a sensitive, efficient, and global method capable of addressing the phosphoproteome has yet to be developed. Here we describe an improved stable-
isotope labeling method using a
phosphoprotein isotope-coded solid-phase tag (PhIST) for isolating and measuring the relative abundances of phosphorylated
peptides from complex
peptide mixtures resulting from the enzymatic digestion of extracted
proteins. The PhIST approach is an extension of the previously reported
phosphoprotein isotope-coded affinity tag (PhIAT) approach developed by our laboratory, where phosphoseryl and phosphothreonyl residues were derivatized by
hydroxide ion-mediated beta-elimination followed by the Michael addition of
1,2-ethanedithiol (EDT). Instead of using the
biotin affinity tag,
peptides containing the EDT moiety were captured and labeled in one step using
isotope-coded solid-phase
reagents containing either light (12C6, 14N) or heavy (13C6, 15N) stable
isotopes. The captured
peptides labeled with the
isotope-coded tags were released from the solid-phase support by UV photocleavage and analyzed by capillary liquid chromatography-tandem mass spectrometry. The efficiency and sensitivity of the PhIST labeling approach for identification of
phosphopeptides from mixtures were determined using
casein proteins. Its utility for proteomic applications was demonstrated by the labeling of soluble
phosphoproteins from a human
breast cancer cell line.