On-Chip Proteolytic Digestion and Analysis Using “Wrong-Way-Round” Electrospray Time-of-Flight Mass Spectrometry

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 enzym...

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Bibliographic Details
Published in:Analytical chemistry (Washington) 2001-04, Vol.73 (8), p.1733-1739
Main Authors: Lazar, Iulia M, Ramsey, Roswitha S, Ramsey, J. Michael
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Analysis
Analytical, structural and metabolic biochemistry
Animals
Biological and medical sciences
Cattle
Cytochrome c Group - analysis
Cytochrome c Group - chemistry
Fundamental and applied biological sciences. Psychology
General aspects, investigation methods
Glucagon - analysis
Glucagon - chemistry
Hemoglobin
Hemoglobins - analysis
Hemoglobins - chemistry
Humans
Hydrolysis
Ions
Microcomputers
Molecular Sequence Data
Proteins
Proteins - analysis
Proteins - chemistry
Scientific imaging
Spectrometry, Mass, Electrospray Ionization
Trypsin - chemistry
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Summary: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/μL 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.
ISSN:0003-2700
1520-6882
DOI:10.1021/ac001420+