Phase transfer surfactant-aided trypsin digestion for membrane proteome analysis

Takeshi Masuda, Masaru Tomita, Yasushi Ishihama

Research output: Contribution to journalArticle

250 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

Fingerprint

Proteome
Surface-Active Agents
Deoxycholic Acid
Trypsin
Digestion
Membranes
Proteins
Peptides
Cations
HeLa Cells
Uterine Cervical Neoplasms
Solubility
Membrane Proteins
Organic solvents
Escherichia coli
Databases
Acids
Water
Recovery

Keywords

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

ASJC Scopus subject areas

  • Genetics
  • Biotechnology
  • Biochemistry

Cite this

Phase transfer surfactant-aided trypsin digestion for membrane proteome analysis. / Masuda, Takeshi; Tomita, Masaru; Ishihama, Yasushi.

In: Journal of Proteome Research, Vol. 7, No. 2, 02.2008, p. 731-740.

Research output: Contribution to journalArticle

@article{839f60234dfa43c9b41a62c1dc6e2bc8,
title = "Phase transfer surfactant-aided trypsin digestion for membrane proteome analysis",
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.",
keywords = "HeLa cell, Hydrophobic peptides, Membrane proteome, Membrane-enriched fraction, Phase transfer surfactant, Sodium deoxycholate, Transmembrane domain, Trypsin digestion",
author = "Takeshi Masuda and Masaru Tomita and Yasushi Ishihama",
year = "2008",
month = "2",
doi = "10.1021/pr700658q",
language = "English",
volume = "7",
pages = "731--740",
journal = "Journal of Proteome Research",
issn = "1535-3893",
publisher = "American Chemical Society",
number = "2",

}

TY - JOUR

T1 - Phase transfer surfactant-aided trypsin digestion for membrane proteome analysis

AU - Masuda, Takeshi

AU - Tomita, Masaru

AU - Ishihama, Yasushi

PY - 2008/2

Y1 - 2008/2

N2 - 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.

AB - 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.

KW - HeLa cell

KW - Hydrophobic peptides

KW - Membrane proteome

KW - Membrane-enriched fraction

KW - Phase transfer surfactant

KW - Sodium deoxycholate

KW - Transmembrane domain

KW - Trypsin digestion

UR - http://www.scopus.com/inward/record.url?scp=39749125069&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=39749125069&partnerID=8YFLogxK

U2 - 10.1021/pr700658q

DO - 10.1021/pr700658q

M3 - Article

C2 - 18183947

AN - SCOPUS:39749125069

VL - 7

SP - 731

EP - 740

JO - Journal of Proteome Research

JF - Journal of Proteome Research

SN - 1535-3893

IS - 2

ER -