title: Selective sampling of phosphopeptides for detection by MALDI mass spectrometry.
abstract: The analysis of phosphopeptides by mass spectrometry (MS) is one of the most challenging tasks in proteomics. This is due to the lower isoelectric point (pI) of phosphopeptides, which leads to inefficient sample ionization in MS, particularly when competing with other peptides. The problem is compounded by the typical low abundance of phosphopeptides in biological samples. We describe here a simple nonsorptive method to isolate phosphopeptides based on their pI. A voltage is applied to selectively migrate the phosphopeptides into a capillary, which are negatively charged at acidic pH. The selectively sampled fraction is directly deposited onto MALDI sample target in nanoliter volumes (7-35 nL) for highly sensitive MS detection. No significant sample loss is evident in this procedure; hence, the MS was able to detect the isolated phosphopeptides at trace quantity. In this case, attomole-level detection limit is achieved for synthetic phosphopeptides (nM concentration and nL volume), from a mixture containing other peptides at up to 1 million times higher in concentration. Selective sampling was also applied to the tryptic digest of beta- and alpha-caseins to reveal the multiple phosphorylated peptides at the low-femtomole level using MALDI MS. Knowledge of pI based on the rejection/injection of peptides was found to be useful in peak assignment. To confirm the sequence of the selectively sampled peptides, fraction collection was performed for offline ESI MS/MS analysis.
authors.affiliation: Department of Chemistry and Department of Biochemistry, The University of Western Ontario, London, Ontario, Canada.
authors.forename: Haixia
authors.initials: H
authors.lastname: Zhang
authors.forename: Cunjie
authors.initials: C
authors.lastname: Zhang
authors.forename: Gilles A
authors.initials: GA
authors.lastname: Lajoie
authors.forename: Ken K-C
authors.initials: KK
authors.lastname: Yeung
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002364:Caseins / Q000737:chemistry / Q000378:metabolism; D002417:Cattle; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000737:chemistry* / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014357:Trypsin / Q000378:metabolism
chemical_list: D002364:Caseins; D010748:Phosphopeptides; D014357:Trypsin