Phosphoproteome analysis of rat L6 myotubes using reversed-phase C18 prefractionation and titanium dioxide enrichment

Junjie Hou, Ziyou Cui, Zhensheng Xie, Peng Xue, Peng Wu, Xiulan Chen, Jing Li, Tanxi Cai, Fuquan Yang

Research output: Contribution to journalArticlepeer-review

31 Scopus citations

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 selfpacked RP C18 columns, phosphopeptide enrichment with TiO2, 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 ≥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.

Original languageEnglish (US)
Pages (from-to)777-788
Number of pages12
JournalJournal of Proteome Research
Volume9
Issue number2
DOIs
StatePublished - Feb 5 2010
Externally publishedYes

Keywords

  • 2D-LC-MS/MS
  • Phosphoproteome
  • RP-c18 Prefractionation
  • Rat L6 myotubes
  • Signaling pathway
  • TiO

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