Arginine-mimic labeling with guanidinoethanethiol to increase mass sensitivity of lysine-terminated phosphopeptides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) generally shows better mass sensitivity for arginine-terminated peptides than for lysine-terminated peptides, presumed to arise from the higher proton affinity of the guanidine group in arginine. Here, we report a new method for analyzing phosphopeptides in which phosphopeptides are labeled with a novel chemical tag, guanidinoethanethiol (GET), by a beta-elimination/Michael addition before MS analysis. GET labeling converts phosphoserine into guanidinoethylcysteine (Gec) containing a guanidine moiety, along with an increase in mass of 21.1 Da. GET-labeled peptides are detected by MALDI MS with greatly increased peak intensities compared to those of intact phosphopeptides. In particular, GET labeling of lysine-terminated phosphopeptides dramatically increased peak intensity. GET labeling of lysine-terminated phosphopeptides improved sensitivity up to 22 times compared to that of the corresponding aminoethanethiol (AET) labeling, in which AET was used as a labeling tag containing an amino group instead of the guanidine group. These results show the guanidine group plays a very important role in increasing the observed sensitivity of MALDI MS for labeled peptide, derivatized from serine-phosphorylated peptides.
Ahn
Yeong Hee
YH
Division of Instrument Development, Korea Basic Science Institute, Daejon 305-333, Korea.
Ji
Eun Sun
ES
Lee
Ji Yeon
JY
Cho
Kun
K
Yoo
Jong Shin
JS
D001120:Arginine / Q000737:chemistry*; D019791:Guanidine / Q000737:chemistry*; D008239:Lysine / Q000032:analysis / Q000737:chemistry*; D010449:Peptide Mapping / Q000379:methods*; D010748:Phosphopeptides / Q000032:analysis / Q000737:chemistry*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013194:Staining and Labeling / Q000379:methods; D013438:Sulfhydryl Compounds / Q000737:chemistry*
D010748:Phosphopeptides; D013438:Sulfhydryl Compounds; D001120:Arginine; D019791:Guanidine; D008239:Lysine; C007638:ethanethiol