Bringing LC to MALDI technology Delivers increased sequence coverage
Protein identification in complex mixtures is pivotal in the field of proteomics. A common approach comprises the separation of a cell extract by 2D-gel electrophoresis, excision and in-gel enzymatic digestion of a gel spot followed by identification using mass spectrometry.
Limitations to this approach include restrictions on the range of amenable proteins, together with difficulties arising from the differential detectability of proteolytic peptides. Coupling mass spectrometry with the chromatographic separation of proteolytic peptides from an in-gel digest of a 1D SDS PAGE protein band appears attractive for alleviating these problems.
Here, we evaluate the combination of single stage protein separation with LC-MALDI-TOF-MS of tryptic digests, in which eluate fractions are spotted automatically onto the MALDI target, and compare results from the conventional gel approach and those obtained using the LC-MALDI-TOF technique.
LC-MALDI-MS analysis of a 1D gel electrophoresis band is proven to offer a number of significant advantages over the conventional peptide mass fingerprinting approach widely used in proteomics:
- Increase in information rich data
- Improvement in proteome coverage of complex protein mixtures
- Improved confidence level in protein match with greater probability scores being achieved.
- LC-MALDI-MS target can be saved as a library of the gel digest where sample can be re-analysed in the future if required
- Reduced number of peptides in each MALDI spot leads to fewer ion suppression effects
MALDI alone. As a result of the complexity of the PMF spectrum and also suppression effects on ionisation caused by the large number of peptides present in the mixture, only two proteins could be unequivocally identified: Phosphoglycerate kinase and Enolase
LC-MALDI. In contrast, 109 peptide candidates from the LC-MALDI-TOF run were analysed by MS/MS. Fragment ion mass lists were compiled for each of these and assembled into a master mass list for MASCOT MS/MS ion search. 22 protein matches from the NCBInr database were found with ion scores indicating identity or significant homology.
The LC-MALDI system comprising a Prominence nano Nanoflow High-Performance Liquid Chromatograph, AccuSpot MALDI Plate Spotter, and AXIMA Performance MALDI-TOF MS performs automatic identification by MS/MS of samples separated by nanoflow LC from complex mixtures. It dramatically increases the analysis efficiency of proteome analysis.