Integration of an on-line protein digestion microreactor to a nanoelectrospray emitter for peptide mapping.
A method for integrating nanoelectrospray mass spectrometry with a microreactor for on-line digestion and fast peptide mass mapping from dilute protein samples is presented. Fused silica capillaries (i.d. 50 microm, o.d. 360 microm) are employed as the digestion microreactor and the nanoelectrospray emitter by immobilizing trypsin onto the surface of the inner wall of the fused silica capillary tubing. The procedure is demonstrated using solutions of 1pmol/mul angiotensin II, cytochrome c, hemoglobin, and beta-casein. Because the inner walls of the capillaries are modified by covalent chemical bonds, the adsorption of peptides and proteins to the inner walls of the capillaries is suppressed. This procedure was performed with solutions as dilute as 1fmol/mul (1nM) cytochrome c. This method shows generation of tryptic peptides with sequence coverage up to 90% within minutes; trypsin autolysis products are not detected. In addition, the immobilized enzyme can be cleaned easily, enabling the microreactor to be reused for nanoelectrospray.
Zhao
Cheng
C
Department of Chemistry, University at Buffalo, State University of New York, Buffalo, NY 14260-3000, USA.
Jiang
Honghai
H
Smith
Douglas R
DR
Bruckenstein
Stanley
S
Wood
Troy D
TD
D000595:Amino Acid Sequence; D000809:Angiotensins; D000818:Animals; D019149:Bioreactors; D002364:Caseins; D002417:Cattle; D045304:Cytochromes c; D004063:Digestion; D004800:Enzymes, Immobilized / Q000378:metabolism; D006801:Humans; D008832:Microchemistry / Q000379:methods; D009211:Myoglobin; D036103:Nanotechnology / Q000295:instrumentation* / Q000379:methods; D010446:Peptide Fragments; D010449:Peptide Mapping / Q000295:instrumentation* / Q000379:methods*; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D012822:Silicon Dioxide / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization / Q000295:instrumentation* / Q000379:methods; D014357:Trypsin / Q000378:metabolism
D000809:Angiotensins; D002364:Caseins; D004800:Enzymes, Immobilized; D009211:Myoglobin; D010446:Peptide Fragments; D011506:Proteins; D012822:Silicon Dioxide; D045304:Cytochromes c; D014357:Trypsin