Development of non-gel-based two-dimensional separation of intact proteins by an on-line hyphenation of capillary isoelectric focusing and hollow fiber flow field-flow fractionation

Dukjin Kang, Myeong Hee Moon

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45 Citations (Scopus)

Abstract

A rapid, non-gel-based, on-line, two-dimensional separation method is introduced for proteome analysis. Protein fractionation was carried out by first exploiting the differences in their respective isoelectric points (pI) in a Teflon capillary using isoelectric focusing (IEF), followed by a molecular weight (MW)-based separation in a hollow fiber by flow field-flow fractionation (F1FFF). The method developed here (CIEF-HFF1FFF) may be a powerful alternative to two-dimensional polyacrylamide gel electrophoresis, which is currently used for the separation and purification of proteins. In CIEF-HFF1FFF, proteins can be collected as a fraction of a certain pI and MW interval without being denatured. Additionally, the ampholyte solution is simultaneously removed during separation in the hollow fiber, and the overall process time is significantly reduced. This method was applied to a human urinary proteome sample, leading to the identification of 114 proteins with the subsequent off-line use of nanoflow liquid chromatography-tandem mass spectrometry after the tryptic digestion of each collected protein fraction.

Original languageEnglish
Pages (from-to)5789-5798
Number of pages10
JournalAnalytical Chemistry
Volume78
Issue number16
DOIs
Publication statusPublished - 2006 Aug 15

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Fractionation
Flow fields
Fibers
Proteome
Proteins
Molecular weight
Liquid chromatography
Polytetrafluoroethylene
Electrophoresis
Purification
Mass spectrometry
Buffers
Isoelectric Focusing

All Science Journal Classification (ASJC) codes

  • Analytical Chemistry

Cite this

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abstract = "A rapid, non-gel-based, on-line, two-dimensional separation method is introduced for proteome analysis. Protein fractionation was carried out by first exploiting the differences in their respective isoelectric points (pI) in a Teflon capillary using isoelectric focusing (IEF), followed by a molecular weight (MW)-based separation in a hollow fiber by flow field-flow fractionation (F1FFF). The method developed here (CIEF-HFF1FFF) may be a powerful alternative to two-dimensional polyacrylamide gel electrophoresis, which is currently used for the separation and purification of proteins. In CIEF-HFF1FFF, proteins can be collected as a fraction of a certain pI and MW interval without being denatured. Additionally, the ampholyte solution is simultaneously removed during separation in the hollow fiber, and the overall process time is significantly reduced. This method was applied to a human urinary proteome sample, leading to the identification of 114 proteins with the subsequent off-line use of nanoflow liquid chromatography-tandem mass spectrometry after the tryptic digestion of each collected protein fraction.",
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N2 - A rapid, non-gel-based, on-line, two-dimensional separation method is introduced for proteome analysis. Protein fractionation was carried out by first exploiting the differences in their respective isoelectric points (pI) in a Teflon capillary using isoelectric focusing (IEF), followed by a molecular weight (MW)-based separation in a hollow fiber by flow field-flow fractionation (F1FFF). The method developed here (CIEF-HFF1FFF) may be a powerful alternative to two-dimensional polyacrylamide gel electrophoresis, which is currently used for the separation and purification of proteins. In CIEF-HFF1FFF, proteins can be collected as a fraction of a certain pI and MW interval without being denatured. Additionally, the ampholyte solution is simultaneously removed during separation in the hollow fiber, and the overall process time is significantly reduced. This method was applied to a human urinary proteome sample, leading to the identification of 114 proteins with the subsequent off-line use of nanoflow liquid chromatography-tandem mass spectrometry after the tryptic digestion of each collected protein fraction.

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