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Coupling of TiO 2 ‐mediated enrichment and on‐bead guanidinoethanethiol labeling for effective phosphopeptide analysis by matrix‐assisted laser desorption/ionization mass spectrometry
Author(s) -
Ahn Yeong Hee,
Ji Eun Sun,
Lee Ji Yeon,
Cho Kun,
Yoo Jong Shin
Publication year - 2007
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.3278
Subject(s) - phosphopeptide , chemistry , mass spectrometry , chromatography , matrix assisted laser desorption/ionization , desorption , combinatorial chemistry , peptide , adsorption , organic chemistry , biochemistry
Abstract Titanium dioxide (TiO 2 )‐mediated phosphopeptide enrichment has been introduced as an effective method for extracting phosphopeptides from highly complex peptide mixtures. Chemical labeling by β ‐elimination/Michael addition is also useful for increasing mass intensity in phosphopeptide analysis. Both of these methods were coupled in order to simultaneously enrich phosphopeptides and allow for detection and sequencing of the enriched peptides with high mass sensitivity. Phosphopeptides were successfully enriched on TiO 2 beads without the use of any hydroxy acid additives like 2,5‐dihydroxybenzoic acid. Labeling was accomplished on‐bead with a guanidinoethanethiol (GET) tag containing a guanidine moiety. These GET‐labeled derivatives were detected by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS). GET labeling converted phosphoserine into guanidinoethylcysteine, a structural arginine‐mimic. In particular, GET‐labeled lysine‐terminated phosphopeptides showed dramatically increased peak intensities compared to those of the corresponding intact phosphopeptides. Additionally, the on‐bead labeling minimized manipulation steps and sample loss. The coupled technique was also further validated by applying to the analysis of phosphopeptides from complex tryptic digests of phosphoprotein mixtures. Copyright © 2007 John Wiley & Sons, Ltd.