Somasundaram, P., Koudelka, T., Linke, D. and Tholey, Andreas (2016) C-Terminal Charge-Reversal Derivatization and Parallel Use of Multiple Proteases Facilitates Identification of Protein C-Termini by C-Terminomics. Journal of Proteome Research, 15 (4). pp. 1369-1378. DOI 10.1021/acs.jproteome.6b00146.

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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.

Document Type:	Article
Additional Information:	Times Cited: 1 Somasundaram, Prasath Koudelka, Tomas Linke, Dennis Tholey, Andreas
Keywords:	C-Terminus, modification, derivatization, mass spectrometry, EDC, NHS, HCD, CID, ETD, E. coli
Research affiliation:	Kiel University > Kiel Marine Science OceanRep > The Future Ocean - Cluster of Excellence
Refereed:	Yes
Open Access Journal?:	No
Publisher:	American Chemical Society
Projects:	Future Ocean
Date Deposited:	24 Feb 2017 14:18
Last Modified:	01 Jun 2019 16:30
URI:	https://oceanrep.geomar.de/id/eprint/36324

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