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

• Published in: Rapid communications in mass spectrometry 2013-02, Vol.27 (3), p.391-400
• Main Authors: Lobas, Anna A., Verenchikov, Anatoly N., Goloborodko, Anton A., Levitsky, Lev I., Gorshkov, Mikhail V.
• Format: Article
• Language: English
• Subjects: Chromatography, High Pressure Liquid - methods; Computer Simulation; Databases, Protein; Degradation; Human; Humans; Liquid chromatography; Mass spectrometry; Organophosphorus Compounds - chemistry; Peptide Fragments - analysis; Peptide Fragments - chemistry; Peptides; Proteins - analysis; Proteins - chemistry; Proteomics - methods; Saccharomyces cerevisiae Proteins - analysis; Saccharomyces cerevisiae Proteins - chemistry; Searching; Tandem Mass Spectrometry - methods; Three dimensional; Yeast
• ISSN: 0951-4198; 1097-0231
• DOI: 10.1002/rcm.6462

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. Copyright © 2012 John Wiley & Sons, Ltd.