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On-chip microextraction for proteomic sample preparation of in-gel digests

Ekström, Simon LU ; Malmström, Johan LU ; Wallman, Lars LU ; Löfgren, Mikael; Nilsson, Johan LU ; Laurell, Thomas LU and Marko-Varga, György LU (2002) In Proteomics 2(4). p.413-421
Abstract

Despite the high sensitivity and relatively high tolerance for contaminants of matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) there is often a need to purify and concentrate the sample solution, especially after in-gel digestion of proteins separated by two-dimensional gel electrophoresis (2-DE). A silicon microextraction chip (SMEC) for sample clean-up and trace enrichment of peptides was manufactured and investigated. The microchip structure was used to trap reversed-phase chromatography media (POROS R2 beads) that facilitates sample purification/enrichment of contaminated and dilute samples prior to the MALDI-TOF MS analysis. The validity of the SMEC sample preparation technique was... (More)

Despite the high sensitivity and relatively high tolerance for contaminants of matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) there is often a need to purify and concentrate the sample solution, especially after in-gel digestion of proteins separated by two-dimensional gel electrophoresis (2-DE). A silicon microextraction chip (SMEC) for sample clean-up and trace enrichment of peptides was manufactured and investigated. The microchip structure was used to trap reversed-phase chromatography media (POROS R2 beads) that facilitates sample purification/enrichment of contaminated and dilute samples prior to the MALDI-TOF MS analysis. The validity of the SMEC sample preparation technique was successfully investigated by performing analysis on a 10 nM peptide mixture containing 2 m urea in 0.1 m phosphate-buffered saline with MALDI-TOF MS. It is demonstrated that the microchip sample clean-up and enrichment of peptides can facilitate identification of proteins from 2-DE separations. The microchip structure was also used to trap beads immobilized with trypsin, thereby effectively becoming a microreactor for enzymatic digestion of proteins. This microreactor was used to generate a peptide map from a 100 nM bovine serum albumin sample.

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@article{3d964ccc-c2d8-4673-9775-29ee38ea3ac4,
  abstract     = {<p>Despite the high sensitivity and relatively high tolerance for contaminants of matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) there is often a need to purify and concentrate the sample solution, especially after in-gel digestion of proteins separated by two-dimensional gel electrophoresis (2-DE). A silicon microextraction chip (SMEC) for sample clean-up and trace enrichment of peptides was manufactured and investigated. The microchip structure was used to trap reversed-phase chromatography media (POROS R2 beads) that facilitates sample purification/enrichment of contaminated and dilute samples prior to the MALDI-TOF MS analysis. The validity of the SMEC sample preparation technique was successfully investigated by performing analysis on a 10 nM peptide mixture containing 2 m urea in 0.1 m phosphate-buffered saline with MALDI-TOF MS. It is demonstrated that the microchip sample clean-up and enrichment of peptides can facilitate identification of proteins from 2-DE separations. The microchip structure was also used to trap beads immobilized with trypsin, thereby effectively becoming a microreactor for enzymatic digestion of proteins. This microreactor was used to generate a peptide map from a 100 nM bovine serum albumin sample.</p>},
  author       = {Ekström, Simon and Malmström, Johan and Wallman, Lars and Löfgren, Mikael and Nilsson, Johan and Laurell, Thomas and Marko-Varga, György},
  issn         = {1615-9861},
  keyword      = {Electrophoresis, Gel, Two-Dimensional,Enzymes, Immobilized,Peptides,Protein Array Analysis,Proteome,Reproducibility of Results,Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization,Evaluation Studies,Journal Article,Research Support, Non-U.S. Gov't},
  language     = {eng},
  number       = {4},
  pages        = {413--421},
  publisher    = {John Wiley & Sons},
  series       = {Proteomics},
  title        = {On-chip microextraction for proteomic sample preparation of in-gel digests},
  url          = {http://dx.doi.org/10.1002/1615-9861(200204)2:4<413::AID-PROT413>3.0.CO;2-1http://dx.doi.org/10.1002/1615-9861(200204)2:4<413::AID-PROT413>3.0.CO;2-1},
  volume       = {2},
  year         = {2002},
}