Combination of Edman degradation of peptides with liquid chromatography/mass spectrometry workflow for peptide identification in bottom-up proteomics.

RATIONALE

High-throughput methods of proteomics are essential for identification of proteins in a cell or tissue under certain conditions. Most of these methods require tandem mass spectrometry (MS/MS). A multidimensional approach including predictive chromatography and partial chemical degradation could be a valuable alternative and/or addition to MS/MS.

METHODS

In the proposed strategy peptides are identified in a three-dimensional (3D) search space consisting of retention time (RT), mass, and reduced mass after one-step partial Edman degradation. The strategy was evaluated in silico for two databases: baker's yeast and human proteins. Rates of unambiguous identifications were estimated for mass accuracies from 0.001 to 0.05 Da and RT prediction accuracies from 0.1 to 5 min. Rates of Edman reactions were measured for test peptides.

RESULTS

A 3D description of proteolytic peptides allowing unambiguous identification without employing MS/MS of up to 95% and 80% of tryptic peptides from the yeast and human proteomes, respectively, was considered. Further extension of the search space to a four-dimensional one by incorporating the second N-terminal amino acid residue as the fourth dimension was also considered and was shown to result in up to 90% of human peptides being identified unambiguously.

CONCLUSIONS

The proposed 3D search space can be a useful alternative to MS/MS-based peptide identification approach. Experimental implementations of the proposed method within the on-line liquid chromatography/mass spectrometry (LC/MS) and off-line matrix-assisted laser desorption/ionization (MALDI) workflows are in progress.