Abstract
The analysis by liquid chromatography coupled to tandem mass spectrometry of complex peptide mixtures, generated by proteolysis of protein samples, is the main proteomics method used today. The approach is based on the assumption that each protein present in a sample reproducibly and predictably generates a relatively small number of peptides that can be identified by mass spectrometry. In this study this assumption was examined by a targeted peptide sequencing strategy using inclusion lists to trigger peptide fragmentation attempts. It was found that the number of peptides observed from a single protein is at least one order of magnitude greater than previously assumed. This unexpected complexity of proteomics samples implies substantial technical challenges, explains some perplexing results in the proteomics literature, and prompts the need for developing alternative experimental strategies for the rapid and comprehensive analysis of proteomes.
Highlights
The analysis by liquid chromatography coupled to tandem mass spectrometry of complex peptide mixtures, generated by proteolysis of protein samples, is the main proteomics method used today
The resulting peptide mixtures are subjected to one, two, or three-dimensional fractionation, and the peptides eluting from the last separation step, typically reverse-phase chromatography, are analyzed by MS/MS
The complete analysis of even moderately complex samples such as isolated organelles [8] or macromolecular complexes [9] has required enormous efforts. All these considerations suggest that the comprehensive analysis of a complex sample is more difficult than initially anticipated, and one of the reasons for this could be a degree of complexity resulting from proteolysis of the protein sample that is higher than expected
Summary
The analysis by liquid chromatography coupled to tandem mass spectrometry of complex peptide mixtures, generated by proteolysis of protein samples, is the main proteomics method used today. The approach is based on the assumption that each protein present in a sample reproducibly and predictably generates a relatively small number of peptides that can be identified by mass spectrometry. In this study this assumption was examined by a targeted peptide sequencing strategy using inclusion lists to trigger peptide fragmentation attempts. The studies in which the proteins constituting a proteome are identified and quantified are informative and relevant from a biological and biomedical point of view At present this is most frequently attempted by a shotgun, tandem mass spectrometry-based strategy [1]. All these considerations suggest that the comprehensive analysis of a complex sample is more difficult than initially anticipated, and one of the reasons for this could be a degree of complexity resulting from proteolysis of the protein sample that is higher than expected
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