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- Samskog, Jenny, et al.
(författare)
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Miniaturized on-line proteolysis-packed capillary liquid chromatography-mass spectrometry for peptide mapping of lactate dehydrogenase
- 2003
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Ingår i: Journal of Chromatography A. - 0021-9673. ; 998:1/2, s. 83-91
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Tidskriftsartikel (refereegranskat)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.
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