Integrated sample processing system involving on-column protein adsorption, sample washing, and enzyme digestion for protein identification by LC-ESI MS/MS.
An automated system has been developed for protein identification using mass spectrometry that incorporates sample cleanup, preconcentration, and protein digestion in a single stage. The procedure involves the adsorption of a protein or a protein mixture from solution onto a hydrophobic medium that is contained within a microcolumn. The protein is digested while still bound to the hydrophobic support. The peptides are then eluted from surface digestion to an electrospray ionization mass spectrometer for detection and sequencing. The entire system is fully automated wherein the mass spectrometer is collecting data continuously. We demonstrate that this system is capable of identifying standard protein samples at concentrations down to 100 nM. Further development of this technique may offer a potential solution for proteomics applications that require unattended operation, such as on-line monitoring and identification of microorganisms on the basis of the detection of their protein biomarkers.
Craft
David
D
Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2.
Li
Liang
L
D000327:Adsorption; D002623:Chemistry Techniques, Analytical / Q000295:instrumentation / Q000379:methods; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D045304:Cytochromes c / Q000032:analysis; D004798:Enzymes / Q000737:chemistry / Q000378:metabolism; D009211:Myoglobin / Q000032:analysis; D011506:Proteins / Q000032:analysis / Q000378:metabolism; D040901:Proteomics / Q000295:instrumentation / Q000379:methods*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013696:Temperature
D004798:Enzymes; D009211:Myoglobin; D011506:Proteins; D045304:Cytochromes c