Positive Enrichment of C-Terminal Peptides Using Oxazolone Chemistry and Biotinylation

Abstract

Selective capture of protein C-termini is still challenging in view of the lower reactivity of the carboxyl group relative to amino groups and difficulties in site-specifically labeling the carboxyl group on the C-terminus rather than that on the side chains of acidic amino acids. For highly efficient purification of C-terminus peptides, a novel positive enrichment approach based on the oxazolone chemistry has been developed in this study. A bifunctional group reagent containing biotin and arginine was incorporated into the C-terminus of protein. Together with a streptavidin affinity strategy, the C-terminal peptides could be readily purified and analyzed by mass spectrometry (MS). Unlike the negative enrichment approach, C-terminal peptides, other than non-C-terminal peptides, were captured directly from the peptide mixture in this new method. The labeling efficiency (higher than 90%), enrichment selectivity (purifying C-terminal peptides from mixtures of non-C-terminal peptides at a 1:50 molar ratio), and ionization efficiencies in MS were dramatically improved. Moreover, the highly efficient identification of C-terminal peptides was further achieved by defining biotin as the 21st amino acid and optimizing the database search strategy. We have successfully identified 183 C-terminal peptides from Thermoanaerobacter tengcongensis using this creative method, which affords a highly selective and efficient purification approach for C-terminomics study.

Discover the Post‐Translational Modification Proteome Using Mass Spectrometry

“…[ 66 ] Based on our previous work, we further developed a positive enrichment workflow for protein C‐termini by introducing biotin‐derivatized arginine into protein C‐terminus. [ 67 ] The high affinity between biotin and commercially available streptavidin beads enabled the effective separation of C‐terminal peptides from internal peptides. As a result, a total of 182 C‐terminal peptides were identified.…”

New beginnings and new ends: methods for large-scale characterization of protein termini and their use in plant biology

“…A method for selective chemical biotinylation of protein C-termini has also been developed (Liu et al , 2013, 2015). This method employs the established oxazolone chemistry (Liu et al , 2013) to conjugate Arg–Arg dipeptide-linked biotin to protein C-termini.…”

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

“…Modification of the activated C termini with a bifunctional peptide containing an N-terminal Arginine and a C-terminal Biotin allowed selective enrichment with streptavidin-coupled magnetic beads (Fig. 4B) [62].…”