Miniaturized on-line proteolysis-capillary liquid chromatography-mass spectrometry for peptide mapping of lactate...

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.