Dynamic identification of phosphopeptides using immobilized metal ion affinity chromatography enrichment, subsequent partial beta-elimination/chemical tagging and matrix-assisted laser desorption/ionization mass spectrometric analysis.
The enrichment of phosphopeptides using immobilized metal ion affinity chromatography (IMAC) and subsequent mass spectrometric analysis is a powerful protocol for detecting phosphopeptides and analyzing their phosphorylation state. However, nonspecific binding peptides, such as acidic, nonphosphorylated peptides, often coelute and make analyses of mass spectra difficult. This study used a partial chemical tagging reaction of a phosphopeptide mixture, enriched by IMAC and contaminated with nonspecific binding peptides, following a modified beta-elimination/Michael addition method, and dynamic mass analysis of the resulting peptide pool. Mercaptoethanol was used as a chemical tag and nitrilotriacetic acid (NTA) immobilized on Sepharose beads was used for IMAC enrichment. The time-dependent dynamic mass analysis of the partially tagged reaction mixture detected intact phosphopeptides and their mercaptoethanol-tagged derivatives simultaneously by their mass difference (-20 Da for each phosphorylation site). The number of new peaks appearing with the mass shift gave the number of multiply phosphorylated sites in a phosphopeptide. Therefore, this partial chemical tagging/dynamic mass analysis method can be a powerful tool for rapid and efficient phosphopeptide identification and analysis of the phosphorylation state concurrently using only MS analysis data.
Ahn
Yeong Hee
YH
Proteome Analysis Team, Korea Basic Science Institute, Daejon 305-333, Korea.
Park
Eun Jung
EJ
Cho
Kun
K
Kim
Jin Young
JY
Ha
Sang Hoon
SH
Ryu
Sung Ho
SH
Yoo
Jong Shin
JS
D000327:Adsorption; D000345:Affinity Labels / Q000737:chemistry; D000595:Amino Acid Sequence; D002846:Chromatography, Affinity / Q000379:methods*; D008670:Metals / Q000737:chemistry; D008969:Molecular Sequence Data; D009862:Online Systems; D010748:Phosphopeptides / Q000032:analysis* / Q000737:chemistry*; D010766:Phosphorylation; D020539:Sequence Analysis, Protein / Q000379:methods*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D000345:Affinity Labels; D008670:Metals; D010748:Phosphopeptides