title: C-Terminal Charge-Reversal Derivatization and Parallel Use of Multiple Proteases Facilitates Identification of Protein C-Termini by C-Terminomics.
abstract: The identification of protein C-termini in complex proteomes is challenging due to the poor ionization efficiency of the carboxyl group. Amidating the negatively charged C-termini with ethanolamine (EA) has been suggested to improve the detection of C-terminal peptides and allows for a directed depletion of internal peptides after proteolysis using carboxyl reactive polymers. In the present study, the derivatization with N,N-dimethylethylenediamine (DMEDA) and (4-aminobutyl)guanidine (AG) leading to a positively charged C-terminus was investigated. C-terminal charge-reversed peptides showed improved coverage of b- and y-ion series in the MS/MS spectra compared to their noncharged counterparts. DMEDA-derivatized peptides resulted in many peptides with charge states of 3+, which benefited from ETD fragmentation. This makes the charge-reversal strategy particularly useful for the analysis of protein C-termini, which may also be post-translationally modified. The labeling strategy and the indirect enrichment of C-termini worked with similar efficiency for both DMEDA and EA, and their applicability was demonstrated on an E. coli proteome. Utilizing two proteases and different MS/MS activation mechanisms allowed for the identification of >400 C-termini, encompassing both canonical and truncated C-termini.
authors.affiliation: AG Systematische Proteomforschung & Bioanalytik, Institut für Experimentelle Medizin, Christian-Albrechts-Universität zu Kiel , Niemannsweg 11, 24105 Kiel, Germany.
authors.forename: Prasath
authors.initials: P
authors.lastname: Somasundaram
authors.affiliation: AG Systematische Proteomforschung & Bioanalytik, Institut für Experimentelle Medizin, Christian-Albrechts-Universität zu Kiel , Niemannsweg 11, 24105 Kiel, Germany.
authors.forename: Tomas
authors.initials: T
authors.lastname: Koudelka
authors.affiliation: AG Systematische Proteomforschung & Bioanalytik, Institut für Experimentelle Medizin, Christian-Albrechts-Universität zu Kiel , Niemannsweg 11, 24105 Kiel, Germany.
authors.forename: Dennis
authors.initials: D
authors.lastname: Linke
authors.affiliation: AG Systematische Proteomforschung & Bioanalytik, Institut für Experimentelle Medizin, Christian-Albrechts-Universität zu Kiel , Niemannsweg 11, 24105 Kiel, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Tholey
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification*; D004926:Escherichia coli / Q000737:chemistry; D019856:Ethanolamine / Q000737:chemistry*; D005029:Ethylenediamines / Q000737:chemistry*; D006146:Guanidines / Q000737:chemistry*; D010447:Peptide Hydrolases / Q000737:chemistry / Q000302:isolation & purification*; D010455:Peptides / Q000032:analysis* / Q000737:chemistry; D000072417:Protein Domains; D059748:Proteolysis; D055672:Static Electricity; D053719:Tandem Mass Spectrometry
chemical_list: D001426:Bacterial Proteins; D005029:Ethylenediamines; D006146:Guanidines; D010455:Peptides; C027450:dimethylethylenediamine; D019856:Ethanolamine; D010447:Peptide Hydrolases
keywords: C-terminus; CID; E. coli; EDC; ETD; HCD; NHS; derivatization; mass spectrometry; modification