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

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T1 - On-chip microextraction for proteomic sample preparation of in-gel digests

AU - Ekström, Simon

AU - Malmström, Johan

AU - Wallman, Lars

AU - Löfgren, Mikael

AU - Nilsson, Johan

AU - Laurell, Thomas

AU - Marko-Varga, György

PY - 2002/4

Y1 - 2002/4

N2 - 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.

AB - 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.

KW - Electrophoresis, Gel, Two-Dimensional

KW - Enzymes, Immobilized

KW - Peptides

KW - Protein Array Analysis

KW - Proteome

KW - Reproducibility of Results

KW - Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

KW - Evaluation Studies

KW - Journal Article

KW - Research Support, Non-U.S. Gov't

U2 - 10.1002/1615-9861(200204)2:4<413::AID-PROT413>3.0.CO;2-1

DO - 10.1002/1615-9861(200204)2:4<413::AID-PROT413>3.0.CO;2-1

M3 - Article

VL - 2

SP - 413

EP - 421

JO - Proteomics

T2 - Proteomics

JF - Proteomics

SN - 1615-9861

IS - 4

ER -