title: A fully automated phosphopeptide purification system for large-scale phosphoproteome analysis.
abstract: 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.
authors.affiliation: Graduate School of Science and Technology, Department of Chemistry, Faculty of Science, Niigata University, Igarashi-2, Nishi-ku, Niigata 950-2181, Japan.
authors.forename: Yuka
authors.initials: Y
authors.lastname: Iwase
authors.forename: Satomi
authors.initials: S
authors.lastname: Honma
authors.forename: Megumi
authors.initials: M
authors.lastname: Matsuzaki
authors.forename: Yukiko
authors.initials: Y
authors.lastname: Miyakawa
authors.forename: Takuma
authors.initials: T
authors.lastname: Kanno
authors.forename: Kohei
authors.initials: K
authors.lastname: Ishii
authors.forename: Naotaka
authors.initials: N
authors.lastname: Furuichi
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Furukawa
authors.forename: Tsuneyoshi
authors.initials: T
authors.lastname: Horigome
mesh_terms: 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
chemical_list: D010748:Phosphopeptides; D010750:Phosphoproteins