Development of a highly efficient 2-d system with a serially coupled long column and its application in identification of rat brain integral membrane proteins with ionic liquids-assisted solubilization and digestion

Dingyin Tao, Xiaoqiang Qiao, Liangliang Sun, Chunyan Hou, Liang Gao, Lihua Zhang, Yichu Shan, Zhen Liang, Yukui Zhang

Research output: Contribution to journalArticle

Abstract

Two dimensional high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (2D-HPLC-ESI-MS/MS) is one of the most powerful techniques for high resolution, efficiency, and throughput separation and identification of proteomes. For a bottom-up strategy-based proteome analysis, usually multistep salt elution was needed in the first dimension separation by SCX, to simplify the peptides for the further second dimensional separation by RPLC. Here, by using a 30 cm-long serially coupled long column (SCLC) in the second dimension, we reduced the salt steps of SCX from 13 to 5 to shorten the total analysis time. Compared to the commonly applied 2D-HPLC with over 10-step salt elution in SCX and microRPLC with a short column (SC), named as SC-2D, the peak capacity of 2D-HPLC with a SCLC column, named as SCLC-2D, was increased 3.3-folds while the analysis time was increased by only 1.17-folds. Therefore, the time-based protein identification efficiency was ∼55 protein groups/h, nearly 2-fold of that for SC-2D (∼28 protein groups/h). With the further combination of assisted solubilization by ionic liquids and SCLC-2D, 608 integral membrane proteins (IMPs) (27.66% of the total 2198 proteins, FDR < 1%) were identified from rat brain, more than those obtaind by the traditional urea method (252 unique IMPs, occupying 17.03% of total 1480 proteins). All of these results demonstrate the promise of the developed technique for large-scale proteome analysis.

Original languageEnglish (US)
Pages (from-to)732-738
Number of pages7
JournalJournal of proteome research
Volume10
Issue number2
DOIs
StatePublished - Feb 4 2011

Keywords

  • Integral membrane proteins
  • Ionic liquids
  • Multidimensional HPLC
  • Serially coupled column
  • Shotgun proteomics

ASJC Scopus subject areas

  • Biochemistry
  • Chemistry(all)

Fingerprint Dive into the research topics of 'Development of a highly efficient 2-d system with a serially coupled long column and its application in identification of rat brain integral membrane proteins with ionic liquids-assisted solubilization and digestion'. Together they form a unique fingerprint.

  • Cite this