title: On-chip proteolytic digestion and analysis using "wrong-way-round" electrospray time-of-flight mass spectrometry.
abstract: Rapid protein digestion and analysis using a hybrid microchip nanoelectrospray device and time-of-flight mass spectrometry detection are reported. The device consists of a planar glass chip with microfabricated channels coupled to a disposable nanospray emitter. Reactions between substrate and enzyme (trypsin), mixed off-chip and then immediately loaded into a sample reservoir on the device, are monitored in real time following the onset of electrospray. Protein cleavage products are determined at the optimum pH for generating tryptic fragments, directly from the digestion buffer using "wrong-way-round" electrospray, i.e., monitoring (MH)+ ions from basic solutions. Intense tryptic peptide ions are observed within a few minutes following sample loading on the microchip. Proteins were identified from low femtomole or even attomole quantities of analyte/spectrum using peptide mass fingerprinting, loading 0.1-2 pmol/microL of sample on the chip. The sequence coverage for analyzed proteins ranged from 70 to 95%. The rapid analysis of human hemoglobin is demonstrated using the technique.
authors.affiliation: Chemical and Analytical Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831-6365, USA.
authors.forename: I M
authors.initials: IM
authors.lastname: Lazar
authors.forename: R S
authors.initials: RS
authors.lastname: Ramsey
authors.forename: J M
authors.initials: JM
authors.lastname: Ramsey
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D003574:Cytochrome c Group / Q000032:analysis / Q000737:chemistry; D005934:Glucagon / Q000032:analysis / Q000737:chemistry; D006454:Hemoglobins / Q000032:analysis / Q000737:chemistry; D006801:Humans; D006868:Hydrolysis; D008838:Microcomputers; D008969:Molecular Sequence Data; D011506:Proteins / Q000032:analysis* / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization; D014357:Trypsin / Q000737:chemistry
chemical_list: D003574:Cytochrome c Group; D006454:Hemoglobins; D011506:Proteins; D005934:Glucagon; D014357:Trypsin