Trypsin-Based Monolithic Bioreactor Coupled On-Line with LC/MS/MS System for Protein Digestion and Variant Identification in Standard Solutions and Serum Samples
- 4 March 2005
- journal article
- research article
- Published by American Chemical Society (ACS) in Journal of Proteome Research
- Vol. 4 (2) , 481-490
- https://doi.org/10.1021/pr049796h
Abstract
The applicability of a trypsin-based monolithic bioreactor coupled on-line with LC/MS/MS for rapid proteolytic digestion and protein identification is here described. Dilute samples are passed through the bioreactor for generation of proteolytic fragments in less than 10 min. After digestion and peptide separation, electrospray ionization tandem mass spectrometry is used to generate a peptide map and to identify proteolytic peptides by correlating their fragmentation spectra with amino acid sequences from a protein database. By digesting picomoles of proteins sufficient data from ESI and MS/MS were obtained to unambiguously identify proteins alone and in serum samples. This approach was also extended to locate mutation sites in beta-lactoglobulin A and B variants. Keywords: immobilized trypsin • in-column digestion • LC−MS/MS • variant identificationKeywords
This publication has 29 references indexed in Scilit:
- Recent liquid chromatographic–(tandem) mass spectrometric applications in proteomicsJournal of Chromatography A, 2003
- Proteome analysis by two-dimensional gel electrophoresis and mass spectrometry: strengths and limitationsTrAC Trends in Analytical Chemistry, 2003
- pH and Ionic Strength Dependence of Protein (Un)Folding and Ligand Binding to Bovine β-Lactoglobulins A and BBiochemistry, 2002
- Evaluation of Multidimensional Chromatography Coupled with Tandem Mass Spectrometry (LC/LC−MS/MS) for Large-Scale Protein Analysis: The Yeast ProteomeJournal of Proteome Research, 2002
- Detection of transthyretin variants using immunoprecipitation and matrix-assisted laser desorption/ionization bioreactive probes: A clinical application of mass spectrometryJournal of the American Society for Mass Spectrometry, 2000
- Liquid chromatography and electrospray mass spectrometric mapping of peptides from human plasma filtrateJournal of the American Society for Mass Spectrometry, 1999
- Direct Analysis and Identification of Proteins in Mixtures by LC/MS/MS and Database Searching at the Low-Femtomole LevelAnalytical Chemistry, 1997
- Automated proteolytic mapping of proteinsJournal of Chromatography A, 1996
- Rapid Tryptic Mapping Using Enzymically Active Mass Spectrometer Probe TipsAnalytical Chemistry, 1995
- Intermolecular Disulfide Linkages Are Not Required for Transthyretin Amyloid Fibril Formation in VitroBiochemical and Biophysical Research Communications, 1993