title: Integration of an on-line protein digestion microreactor to a nanoelectrospray emitter for peptide mapping.
abstract: 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.
authors.affiliation: Department of Chemistry, University at Buffalo, State University of New York, Buffalo, NY 14260-3000, USA.
authors.forename: Cheng
authors.initials: C
authors.lastname: Zhao
authors.forename: Honghai
authors.initials: H
authors.lastname: Jiang
authors.forename: Douglas R
authors.initials: DR
authors.lastname: Smith
authors.forename: Stanley
authors.initials: S
authors.lastname: Bruckenstein
authors.forename: Troy D
authors.initials: TD
authors.lastname: Wood
mesh_terms: 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
chemical_list: D000809:Angiotensins; D002364:Caseins; D004800:Enzymes, Immobilized; D009211:Myoglobin; D010446:Peptide Fragments; D011506:Proteins; D012822:Silicon Dioxide; D045304:Cytochromes c; D014357:Trypsin