title: Phosphopeptide enrichment by aliphatic hydroxy acid-modified metal oxide chromatography for nano-LC-MS/MS in proteomics applications.
abstract: We developed novel methods for phosphopeptide enrichment using aliphatic hydroxy acid-modified metal oxide chromatography (MOC). Titania and zirconia were successfully applied to enrich phosphopeptides with the aid of aliphatic hydroxy acids, such as lactic acid and beta-hydroxypropanoic acid, to reduce the interaction between acidic non-phosphopeptides and the metal oxides. These methods removed the vast majority of non-phosphopeptides from phosphoprotein standard digests, and large numbers of phosphopeptides could be readily identified. The methods were coupled with nano-LC-MS/MS systems without difficulty. Recovery of phosphopeptides in MOC varied greatly from peptide to peptide, ranging from a few percent to 100%, and the average was almost 50%. Repeatability and linearity were satisfactory. In an examination of the cytoplasmic fraction of HeLa cells, more than 1000 phosphopeptides were identified using lactic acid-modified titania MOC and beta-hydroxypropanoic acid-modified zirconia MOC, respectively. The overlap between phosphopeptides enriched by these two methods was 40%, and the combined results provided 1646 unique phosphopeptides. To our knowledge, this is the first successful application of a single MOC-based approach to phosphopeptide enrichment from complex biological samples such as cell lysates.
authors.affiliation: Institute for Advanced Biosciences, Keio University, Tsuruoka 997-0017, Japan.
authors.forename: Naoyuki
authors.initials: N
authors.lastname: Sugiyama
authors.forename: Takeshi
authors.initials: T
authors.lastname: Masuda
authors.forename: Kosaku
authors.initials: K
authors.lastname: Shinoda
authors.forename: Akihiro
authors.initials: A
authors.lastname: Nakamura
authors.forename: Masaru
authors.initials: M
authors.lastname: Tomita
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Ishihama
mesh_terms: D000595:Amino Acid Sequence; D002457:Cell Extracts / Q000737:chemistry; D002853:Chromatography, Liquid; D003593:Cytoplasm / Q000737:chemistry / Q000187:drug effects; D006367:HeLa Cells; D006801:Humans; D006880:Hydroxy Acids / Q000737:chemistry* / Q000494:pharmacology; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D036103:Nanotechnology; D010748:Phosphopeptides / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification*; D040901:Proteomics / Q000295:instrumentation* / Q000379:methods*; D015203:Reproducibility of Results
chemical_list: D002457:Cell Extracts; D006880:Hydroxy Acids; D010748:Phosphopeptides