Coupling of TiO(2)-mediated enrichment and on-bead guanidinoethanethiol labeling for effective phosphopeptide analysis by matrix-assisted laser desorption/ionization mass spectrometry.
Titanium dioxide (TiO2)-mediated phosphopeptide enrichment has been introduced as an effective method for extracting phosphopeptides from highly complex peptide mixtures. Chemical labeling by beta-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 TiO2 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.
Ahn
Yeong Hee
YH
Research Center for Drug Metabolism, College of Life Science, Jilin University, Changchun, PR China.
Ji
Eun Sun
ES
Lee
Ji Yeon
JY
Cho
Kun
K
Yoo
Jong Shin
JS
D000595:Amino Acid Sequence; D000818:Animals; D002364:Caseins / Q000737:chemistry; D019791:Guanidine / Q000737:chemistry*; D008969:Molecular Sequence Data; D010047:Ovalbumin / Q000737:chemistry; D010748:Phosphopeptides / Q000032:analysis / Q000737:chemistry*; D010750:Phosphoproteins / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013438:Sulfhydryl Compounds / Q000737:chemistry*; D014025:Titanium / Q000737:chemistry*
D002364:Caseins; D010748:Phosphopeptides; D010750:Phosphoproteins; D013438:Sulfhydryl Compounds; C009495:titanium dioxide; D010047:Ovalbumin; D014025:Titanium; D019791:Guanidine; C007638:ethanethiol