title: Miniaturized on-line proteolysis-capillary liquid chromatography-mass spectrometry for peptide mapping of lactate dehydrogenase.
abstract: In this study, methodology was developed for on-line and miniaturized enzymatic digestion with liquid chromatographic (LC) separation and mass spectrometric (MS) detection. A packed capillary LC-MS system was combined with on-line trypsin cleavage of a model protein, lactate dehydrogenase, to provide an efficient system for peptide mapping. The protein was injected onto an enzymatic capillary reactor and the resulting peptides were efficiently trapped on a capillary trapping column. Different trapping columns were evaluated to achieve a high binding capacity for the peptides generated in the enzyme reactor. The peptides were further eluted from the pre-column and separated on an analytical capillary column by a buffer more suitable for the following an electrospray ionisation (ESI) MS process. An important aspect of the on-line approach was the desalting of peptides performed in the trapping column to avoid detrimental signal suppression in the ESI process. The developed on-line system was finally compared to a classical digestion in solution, with reference to peptide sequence coverage and sensitivity. It was shown that the on-line system gave more than 100% higher peptide sequence coverage than traditional digestion methods.
authors.affiliation: Department of Analytical Chemistry, Uppsala University, P.O. Box 599, SE-751 24 Uppsala, Sweden.
authors.forename: Jenny
authors.initials: J
authors.lastname: Samskog
authors.forename: Dan
authors.initials: D
authors.lastname: Bylund
authors.forename: Sven P
authors.initials: SP
authors.lastname: Jacobsson
authors.forename: Karin E
authors.initials: KE
authors.lastname: Markides
mesh_terms: D000818:Animals; D002853:Chromatography, Liquid / Q000379:methods*; D006868:Hydrolysis; D007770:L-Lactate Dehydrogenase / Q000737:chemistry*; D008904:Miniaturization; D010449:Peptide Mapping / Q000379:methods*; D015203:Reproducibility of Results; D013552:Swine
chemical_list: D007770:L-Lactate Dehydrogenase