title: Phosphoproteome analysis of rat L6 myotubes using reversed-phase C18 prefractionation and titanium dioxide enrichment.
abstract: The rat L6 myotubes is an important in vitro model system for studying signaling pathways in skeletal muscle. Exploring phosphorylation events involved in the skeletal muscle is very significant for elucidating the kinase-substrate relationship, understanding regulatory mechanisms involved in signaling pathways and providing insights into numerous cell processes. Here, we used mass spectrometry-based proteomics to conduct global phosphoproteome profiling of rat L6 myotubes. Using an efficient phosphoproteomic strategy including prefractionation of tryptic peptide mixtures with self-packed RP C18 columns, phosphopeptide enrichment with TiO(2), and 2D-LC (SCX/RP)-MS/MS analysis, a total of 2230 unique phosphopeptides from 1195 proteins were identified with a false-discovery rate of less than 1.0% using a target/decoy database searching strategy. After determining the degree of certainty of the phosphorylation site location (Ascore value >or=19), 11 Ser motifs and one Thr motif were derived from our data set using the Motif-X algorithm. Several potential signaling pathways were found in our myotubes phosphoproteome, such as the MAPK signaling pathway and the IGF-1/Insulin signaling pathway.
authors.affiliation: Proteomic Platform, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China.
authors.forename: Junjie
authors.initials: J
authors.lastname: Hou
authors.forename: Ziyou
authors.initials: Z
authors.lastname: Cui
authors.forename: Zhensheng
authors.initials: Z
authors.lastname: Xie
authors.forename: Peng
authors.initials: P
authors.lastname: Xue
authors.forename: Peng
authors.initials: P
authors.lastname: Wu
authors.forename: Xiulan
authors.initials: X
authors.lastname: Chen
authors.forename: Jing
authors.initials: J
authors.lastname: Li
authors.forename: Tanxi
authors.initials: T
authors.lastname: Cai
authors.forename: Fuquan
authors.initials: F
authors.lastname: Yang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002853:Chromatography, Liquid; D008969:Molecular Sequence Data; D018485:Muscle Fibers, Skeletal / Q000378:metabolism*; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D020543:Proteome; D051381:Rats; D053719:Tandem Mass Spectrometry; D014025:Titanium / Q000737:chemistry*
chemical_list: D010750:Phosphoproteins; D020543:Proteome; C009495:titanium dioxide; D014025:Titanium