On-chip microextraction for proteomic sample preparation of in-gel digests.
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.
Ekström
Simon
S
Department of Electrical Measurements, University of Lund, Sweden.
Malmström
Johan
J
Wallman
Lars
L
Löfgren
Mikael
M
Nilsson
Johan
J
Laurell
Thomas
T
Marko-Varga
György
G
D015180:Electrophoresis, Gel, Two-Dimensional; D004800:Enzymes, Immobilized / Q000737:chemistry; D010455:Peptides / Q000032:analysis; D040081:Protein Array Analysis / Q000295:instrumentation / Q000379:methods; D020543:Proteome / Q000032:analysis*; D015203:Reproducibility of Results; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D004800:Enzymes, Immobilized; D010455:Peptides; D020543:Proteome