Two-dimensional HPLC on-line analysis of phosphopeptides using titania and monolithic columns.
Conventional and comprehensive two-dimensional (2D) HPLC systems using the combination of titania and monolithic columns were established for the on-line analysis of phosphopeptides. Compared with immobilized metal affinity chromatography of a general method for the analysis of phosphopeptides, the use of titania columns in the analysis permits the specific isolation of phosphopeptides in a higher yield. Using the current 2D HPLC systems, phosphopeptides were specifically isolated from nonphosphorylated peptides by the first-dimension titania column, followed by the high-speed separation of the phosphopeptides by the second-dimension monolithic column. Proteolytic digests of beta-casein were analyzed within 30 min using the comprehensive 2D HPLC system; all phosphopeptides from beta-casein could be efficiently isolated and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The comprehensive 2D HPLC system coupled with mass spectrometry will be useful for high-throughput and on-line phosphoproteome analyses.
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.
Koji
K
Hata
Hironobu
H
Morisaka
Keisuke
K
Hara
Joji
J
Mima
Noboru
N
Yumoto
Yoshiro
Y
Tatsu
Masahiro
M
Furuno
Norio
N
Ishizuka
Mitsuyoshi
M
Ueda
D002364:Caseins / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation* / Q000379:methods*; D009862:Online Systems / Q000295:instrumentation; D010748:Phosphopeptides / Q000032:analysis*; D012822:Silicon Dioxide; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014025:Titanium; D014357:Trypsin / Q000378:metabolism
D002364:Caseins; D010748:Phosphopeptides; C009495:titanium dioxide; D012822:Silicon Dioxide; D014025:Titanium; D014357:Trypsin