A fully automated phosphopeptide purification system for large-scale phosphoproteome analysis.
For large-scale phosphoproteome analysis based on mass spectrometry, a fully automated phosphopeptide purification system is essential to obtain reproducible results. An automated system involving pre-cleaning of a sample with a polymer-based reversed-phase column, phosphopeptide purification with a titania column and analysis of the phosphopeptide fraction with a reversed-phase column was developed, and then the analytical conditions for a complex peptide mixture were optimized. A lower flow rate for application of samples to the titania column was essential to obtain high recoveries of phosphopeptides from complex protein digests. Washing with 1 M NaCl and 2-propanol, and two cycles of washing with four solvents for the titania column were necessary to minimize non-phosphorylated peptides in the phosphopeptide fraction. Using this system under the optimized conditions, a peptide fraction including >90% phosphopeptides could be obtained highly reproducibly from a tryptic digest of a complex protein mixture, i.e. a Xenopus egg cytosol fraction, without any pre-treatment.
Iwase
Yuka
Y
Graduate School of Science and Technology, Department of Chemistry, Faculty of Science, Niigata University, Igarashi-2, Nishi-ku, Niigata 950-2181, Japan.
Honma
Satomi
S
Matsuzaki
Megumi
M
Miyakawa
Yukiko
Y
Kanno
Takuma
T
Ishii
Kohei
K
Furuichi
Naotaka
N
Furukawa
Kazuhiro
K
Horigome
Tsuneyoshi
T
D000818:Animals; D001331:Automation; D003600:Cytosol / Q000737:chemistry; D013058:Mass Spectrometry; D010748:Phosphopeptides / Q000737:chemistry / Q000302:isolation & purification*; D010750:Phosphoproteins / Q000032:analysis / Q000737:chemistry*; D040901:Proteomics / Q000295:instrumentation / Q000379:methods*; D015203:Reproducibility of Results; D014981:Xenopus
D010748:Phosphopeptides; D010750:Phosphoproteins