Microscale Immobilized Protease Reactor Columns for Peptide Mapping by Liquid Chromatography/Mass Spectral Analyses

Methods and procedures for the construction and operation of a microscale immobilized protease (trypsin) reactor are described. Optimization of reactor efficiency with regard to reactor flowrate, length, and temperature as well as sample concentration has been examined, and a novel system for sample...

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Bibliographic Details
Published in:	Analytical biochemistry Vol. 224; no. 1; pp. 235 - 244
Main Authors:	Davis, M.T., Lee, T.D., Ronk, M., Hefta, S.A.
Format:	Journal Article
Language:	English
Published:	United States Elsevier Inc 1995
Subjects:	Amino Acid Sequence Chromatography, Liquid Hemoglobin E - chemistry Mass Spectrometry Molecular Sequence Data Peptide Mapping - methods Trypsin - pharmacology
Online Access:	Get full text
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Description
Summary:	Methods and procedures for the construction and operation of a microscale immobilized protease (trypsin) reactor are described. Optimization of reactor efficiency with regard to reactor flowrate, length, and temperature as well as sample concentration has been examined, and a novel system for sample preconcentration is presented. The structural analysis of a standard protein (cytochrome c) by a combination of trypsin microreactor digestion followed by off-line electrospray ionization MS analysis is demonstrated at the 10-pmol level. The application of these techniques for the rapid confirmation (1-2 h) of a genetic variant (hemoglobin E) including MS/MS analysis of the variant peptide is included.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	0003-2697 1096-0309
DOI:	10.1006/abio.1995.1035