An Approach to Incorporate Multi‐Enzyme Digestion into C‐TAILS for C‐Terminomics Studies

Abstract

Protein C-termini study is still a challenging task and far behind its counterpart, N-termini study. MS based C-terminomics study is often hampered by the low ionization efficiency of C-terminal peptides and the lack of efficient enrichment methods. We previously optimized the C-terminal amine-based isotope labeling of substrates (C-TAILS) method and identified 369 genuine protein C-termini in Escherichia coli. A key limitation of C-TAILS is that the prior protection of amines and carboxylic groups at protein level makes Arg-C as the only specific enzyme in practice. Herein, we report an approach combining multi-enzyme digestion and C-TAILS, which significantly increases the identification rate of C-terminal peptides and consequently improves the applicability of C-TAILS in biological studies. We carry out a systematic study and confirm that the omission of the prior amine protection at protein level has a negligible influence and allows the application of multi-enzyme digestion. We successfully apply five different enzyme digestions to C-TAILS, including trypsin, Arg-C, Lys-C, Lys-N, and Lysarginase. As a result, we identify a total of 722 protein C-termini in E. coli, which is at least 66% more than the results using any single enzyme. Moreover, the favored enzyme and enzyme combination are discovered. Data are available via ProteomeXchange with identifier PXD004275.

The carboxy-terminus, a key regulator of protein function

“…It is challenging to biochemically identify the C-terminus of proteins biochemically as the C-terminal is relatively less reactive than the amino terminus (Marino et al 2015). Nevertheless, the TopFIND group improvised MS techniques, such as C-terminal amine-based isotope labeling of substrates (C-TAILS) and combined fractional diagonal chromatography to identify 130,182 neo C-termini (Bonetto et al 1997; Sechi and Chait 2000; Colaert et al 2013; Nika et al 2013; Fortelny et al 2015; Zhang et al 2018). Proteomic analysis of Escherichia coli , Mus musculus , and Homo sapiens identified that >10% of C-termini are derived from proteolysis, thus we estimate that endoproteolysis produces at least 2000 new human C-termini (Lange and Overall 2013).…”

Positional proteomics for identification of secreted proteoforms released by site-specific processing of membrane proteins

“…In efforts to optimize the protocol, modification of protein primary amines by acetylation rather than dimethylation appeared beneficial and inclusion of single charged ions during mass spectrometry analysis increased the number of identified C termini [69]. Furthermore, the first amine protection step may also be omitted without obvious detrimental effect, as the concentration of protein primary amines appeared negligible compared to the excess amidation reagent used for carboxyl modification [70].…”

“…This is exacerbated by the fact that C-terminal peptides in tryptic digest lack basic residues and thus are less likely to ionize. The alternative digestion protease LysargiNase, which mirrors trypsin specificity and thus results in C-terminal peptides with N-terminal of Lys and Arg residues [87], appears promising to overcome this challenge [68,88]. Also chemical charge-reversal by derivatization with basic amines such as N,N-dimethylethylenediamine [89] and inclusion of single-charged precursor ions during mass spectrometric analysis improved identification of C-terminal peptides from complex proteomes [69].…”

“…by adherence to reaction vessel surfaces, or impairs ionization, all of which renders identification by MS/MS unlikely. Hence, parallel digest with digestion enzymes differing in sequence specificity, for example trypsin, GluC, subtilisin and chymotrypsin, is necessary to achieve greater terminome coverage [35,70,81,92]. In addition, these replicate experiments provide an opportunity for validation of single terminal peptide identifications as the same terminus may be identified by peptides with distinct sequence in the different assays.…”

In-Depth Analysis of C Terminomes Based on LysC Digestion and Site-Selective Dimethylation

“…However, access to terminal peptides in shotgun proteomic studies is still challenging because of the highly dynamic proteome and low abundant terminal peptides . Compared with its counterpart N termini study, protein C termini study is far behind due to two main reasons. , First, reactivity of the carboxyl group is relatively lower than that of the amino group, and it is particularly challenging to achieve site-specific labeling of carboxyl groups in proteins. Second, protein C termini inherently lack basic residues and show low ionization efficiency.…”