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 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.
Davis
M T
MT
Division of Immunology, Beckman Research Institute of The City of Hope, Duarte, California 91010.
Lee
T D
TD
Ronk
M
M
Hefta
S A
SA
D000595:Amino Acid Sequence; D002853:Chromatography, Liquid; D006446:Hemoglobin E / Q000737:chemistry; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010449:Peptide Mapping / Q000379:methods*; D014357:Trypsin / Q000494:pharmacology*
D006446:Hemoglobin E; D014357:Trypsin