The sequence and nature of the amino (N) and carboxyl (C) termini of proteins provides crucial functional annotation of proteomes as their modification or truncation affects all proteins, often influencing protein fate and function. While protein N termini are relatively well studied [1-3] comparatively little is known about protein C termini. Techniques for proteome–wide characterization of C–terminal sequences have long been considered a fundamentally missing tool in functional proteomics. In contrast to commonly used techniques for C–terminal sequencing, which are restricted to samples consisting of only few proteins, the present protocol constitutes one of the first strategies for the targeted analysis of protein C termini from complex cellular proteomes .
With proteolytic processing being a key post-translational modification, interest in the focused analysis of protein N and C termini stems from research on proteolysis on a proteome-wide level [5, 6]. System–wide approaches to elucidate proteases and their substrates are commonly referred to as degradomics  and include more specialized techniques focusing on the N terminome, also called N terminomics [8-11]. Examples of important N-terminal cleavages are many [5, 6], but these have been less studied for C-terminal truncations generating neo C termini. In part this is because few techniques have been developed to target the carboxyl terminus of proteins. In turn, this is due to its lower chemical reactivity compared to primary amino groups. This is a serious deficit because proteolytic processing close to protein C termini and truncations by carboxypeptidases—increasingly recognized for their involvement in physiological and pathological processes [12-14]—can only be investigated with degradomic approaches focusing on neo C termini.
We present a strategy for the enrichment of C-terminal peptides from complex proteomes and their identification by liquid chromatography-tandem mass spectrometry (LC–MS/MS). We call this focused analysis of the C termini of proteomes on a global scale C-terminomics. Stable isotope labeling can be incorporated to compare C–terminal processing in different samples. The protocol focuses on commercially available, cost–efficient reagents. The essence of the approach is a negative selection strategy that involves binding and removal of internal tryptic peptides to enrich for the C- terminal peptides. To do this thiol groups of full-length proteins are first reduced and alkylated followed by reductive methylation of N-terminal α-amines and lysine ε-amines. Now one key step is performed: carboxyl groups—including protein carboxy-termini together with aspartate and glutamate side-chains—are protected by carbodiimide-mediated condensation with ethanolamine. At a concentration of 1.0 M, ethanolamine is used in large excess over the protein components, similar to other carboxyl-labeling approaches . Proteins are then trypsin digested, yielding internal tryptic peptides with free, newly formed neo N and C termini and original protein C-terminal peptides that have protected carboxyl groups from the previous step. To prevent cross-reactivity at the following selection step, newly formed N termini are also chemically protected by reductive methylation. Internal and N-terminal peptides are then covalently coupled through their free carboxyl groups to the primary amine groups of a linear polymer poly-allylamine by carbodiimide-mediated condensation. Poly-allylamine is used at a 1.0 mM concentration, which represents a primary amine concentration of ~1.0 M. The protected protein C-terminal peptides remain uncoupled and are separated from the high molecular weight polymer by ultrafiltration using a size cutoff of 10 kDa. Uncoupled protein C-terminal peptides remain in the flow-through and are analyzed by LC-MS/MS.
Chemical labeling of protein C termini performed at the protein level provides an essential validation step for natural and proteolytically generated C termini since it clearly distinguishes in vivo cleavage events from proteolysis during sample preparation. Similarly, incorporation of stable isotope labeling is essential for quantitative comparison of C–terminal processing in different samples in order to distinguish altered proteolysis (e.g. by protease over–expression or depletion) from ongoing background proteolysis. The present labeling strategy makes use of the amine protection steps that we call “carboxy-terminal amine incorporated labeling of substrates (C-TAILS)”.