100% protein sequence coverage: a modern form of surrealism in proteomics.
This review intends not only to discuss the current possibilities to gain 100% sequence coverage for proteins, but also to point out the critical limits to such an attempt. The aim of 100% sequence coverage, as the review title already implies, seems to be rather surreal if the complexity and dynamic range of a proteome is taken into consideration. Nevertheless, established bottom-up shotgun approaches are able to roughly identify a complete proteome as exemplary shown by yeast. However, this proceeding ignores more or less the fact that a protein is present as various protein species. The unambiguous identification of protein species requires 100% sequence coverage. Furthermore, the separation of the proteome must be performed on the protein species and not on the peptide level. Therefore, top-down is a good strategy for protein species analysis. Classical 2D-electrophoresis followed by an enzymatic or chemical cleavage, which is a combination of top-down and bottom-up, is another interesting approach. Moreover, the review summarizes further top-down and bottom-up combinations and to which extent middle-down improves the identification of protein species. The attention is also focused on cleavage strategies other than trypsin, as 100% sequence coverage in bottom-up experiments is only obtainable with a combination of cleavage reagents.
Meyer
Bjoern
B
Institute of Pharmaceutical Chemistry, Goethe-University Frankfurt, Max-von-Laue-Str. 9, 60438, Frankfurt am Main, Germany. bjoern.meyer@pharmchem.uni-frankfurt.de
Papasotiriou
Dimitrios G
DG
Karas
Michael
M
D000595:Amino Acid Sequence; D000818:Animals; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000737:chemistry*; D040901:Proteomics / Q000379:methods*; D020539:Sequence Analysis, Protein / Q000379:methods*
D010446:Peptide Fragments; D011506:Proteins