Molecular & Cellular Toxicology

  • 제1권2호
  • /
  • Pages.87-91
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  • 2005
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  • 1738-642X(pISSN)
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  • 2092-8467(eISSN)
대한독성유전 단백체학회 (The Korean Society of Toxicogenomics and Toxicoproteomics)
권호 표지

Capillary Size-exclusion Chromatography as a Gel-free Strategy in Plasma Proteomics

  • Cho, Man-Ho (Life Sciences Division, Korea Institute of Science and Technology) ;
  • Wishnok, John S. (Biological Engineering Division and Department of Chemistry, Massachusetts Institute of Technology) ;
  • Tannenbaum, Steven R. (Biological Engineering Division and Department of Chemistry, Massachusetts Institute of Technology)
  • 발행 : 2005.06.30
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초록

Although 2D-PAGE has been widely used as the primary method for protein separation, difficulties in displaying proteins with an extreme values of isoelectric paint (pI), molecular size and hydrophobicity limit the technique. In addition, time consuming steps involving protein transfer and extraction from the gel-pieces can result in sample loss. Here, we describe a novel protein separation technique with capillary size-exclusion chromatography (CSEC) for rapid protein identification from human plasma. The method includes protein fractionation along with molecular size followed by in-solution tryptic digestion and peptide analysis through reversed phase liquid chromatography (RPLC) coupled to nanoflow electrospray-tandem mass spectrometry (ESI-MS/MS). Tryptic peptides are applied an a $100\;{\mu}m\;i.d.{\times}10mm$ length pre-column and then separated on a $75\;{\mu}m{\times}200mm$ analytical column at -100 nL/min flaw rate. Proteins were identified over the wide ranges of pI (3.7-12.3) when this technique was applied to the analysis of $1-2\;{\mu}L$ of human plasma. This gel-free system provides fast fractionation and may be considered a complementary technique to SDS-PAGE in proteomics.

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