Phase transfer surfactant-aided trypsin digestion for membrane proteome analysis

Takeshi Masuda, Masaru Tomita, Yasushi Ishihama

Research output: Contribution to journalArticlepeer-review

286 Citations (Scopus)

Abstract

We have developed a new protocol for digesting hydrophobic proteins using trypsin with the aid of phase-transfer surfactants (PTS), such as sodium deoxycholate (SDC). SDC increases the solubility of hydrophobic proteins, enhances the activity of trypsin, and improves the accessibility to trypsin of proteins denatured during the extraction process. After digestion, SDC was successfully removed from the acidified solution containing tryptic peptides by adding a water-immiscible organic solvent, into which SDC was predominantly transferred, while the digested peptides remained in the aqueous phase. Compared with a protocol using an acid-labile surfactant, this PTS protocol increased the number of identified proteins and the recovery of hydrophobic peptides in the analysis of 400 ng of a membrane-enriched fraction of Escherichia coli. Application of the PTS protocol to 9.0 μg of a membrane-enriched pellet from human cervical cancer HeLa cells resulted in identification of a total of 1450 proteins, of which 764 (53%) were membrane proteins, by two-dimensional strong cation exchange (SCX)-C18 LC-MSMS with 5 SCX fractions. The distribution of the number of transmembrane domains in proteins identified in this study was in agreement with that in the IPI human database, suggesting that the PTS protocol can provide unbiased digestion of the membrane proteome.

Original languageEnglish
Pages (from-to)731-740
Number of pages10
JournalJournal of Proteome Research
Volume7
Issue number2
DOIs
Publication statusPublished - 2008 Feb 1

Keywords

  • HeLa cell
  • Hydrophobic peptides
  • Membrane proteome
  • Membrane-enriched fraction
  • Phase transfer surfactant
  • Sodium deoxycholate
  • Transmembrane domain
  • Trypsin digestion

ASJC Scopus subject areas

  • Biochemistry
  • Chemistry(all)

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