Structurally speaking, all proteins have a beginning and an end, and proteomically speaking, the study of each end has its own –omic. Tanco et al. (2015) provide a handy review of C-terminomics, the study of modifications that impact the protein’s C-terminus.1
The C-terminal tail of a protein plays an important role in regulating activity and cellular trafficking, acting as a recognition signal for many different cell processes. Modification arises either at the pre-translational level through gene events such as alternative splicing, or during translation itself. The C-terminus is also susceptible to post-translational modification (PTM), with phosphorylation, glycosylation and acetylation commonly affecting this area. The C-terminal tail is also open to proteolysis via specific carboxypeptidases, and to the action of ligases such as RimK that add extra amino acids to the area.
Tanco et al. review current proteomic methodologies available to researchers for studying these C-terminal events, noting that mass spectrometry (MS)-based techniques have largely supplanted ladder sequencing and chromatography with faster, more sensitive workflows. These approaches usually involve post-source decay (PSD) and collision-induced dissociation (CID) tandem MS (MS/MS), which give better results with lower sample volumes and deal well with complex analytes.
Although examination of intact proteins is preferable for C-terminal tails, top-down proteomics strategies are used less commonly than bottom-up approaches, as the latter affords better workflows. Top-down characterization is hampered by low throughput and inefficient fragmentation, among other factors. However, as the authors also note, C-terminal peptides ionize poorly for MS-based investigation, and current techniques usually involve additional processes that optimize data acquisition. For this reason, bottom-up strategies usually include a differential labeling protocol.
The authors cover various strategies useful in C-terminomics workflows in the review, discussing the advantages and drawbacks of each methodology. They separate the methods into two groups, labeling strategies and enrichment techniques, based on the primary method used to prepare the samples for analysis. These methods can be further categorized as either positive for C-terminal peptides isolation, or negative when N-terminal tails are selected and removed.
- Labeling strategies, which include 18O enzymatic labeling, can then be followed by differential digestion using Lys-C and Lys-N, which each target either the N-terminal or the C-terminal end of the protein for digestion. Other methods include cyanogen bromide digestion and oxazolone-based C-terminal derivatization.
- Enrichment strategies selectively purify the low-abundance C-terminal fragments for analysis. This selection process can be either positive or negative. Methods include the following:
o C-terminal COFRADIC (COmbined FRActional DIagonal Chromatography) uses peptide chromatography and enrichment techniques to purify both N-terminal and C-terminal analytes.
o C-TAILS (C-Terminal Amino-based Isotope Labeling of Substrates) negatively enriches for C-terminals.
o N-terminal tails can be captured by focusing on COOH-containing peptide fragments.
o Discriminatory trypsin digestion exploits the stoichiometry of proteolysis coupled with anhydrotrypsin-agarose conjugates that remove all other peptide fragments from the digest.
o C-terminal peptides can be isolated from cyanogen bromide gels.
o ProC-TEL (Profiling Protein C-Termini by Enzymatic Labeling) is an enzymatic labeling procedure that positively selects C-terminal analytes. The method uses reagents such as carboxypeptidase Y to add an affinity tag to C-terminals. This tag can then be used to pull out the C-terminal regions for MS analysis.
In drawing the review to a conclusion, Tanco et al. specify that no single method is suitable for overall C-terminomics study, and that researchers should select the best approach for the experiment in question. Although improvements in techniques are possible, the authors believe that studying C-terminomics uncovers PTMs that affect this region and are a valuable addition to proteomics research.
1. Tanco, S., et al. (2015) “C-terminomics: Targeted analysis of natural and posttranslationally modified protein and peptide C-termini,” Proteomics 15 (pp.903–14).
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