Selective Enrichment and Quantification of N-Terminal Glycine Peptides via Sortase A Mediated Ligation

Abstract

The identification and quantification of low-abundant proteins are always impeded by high-abundant proteins in proteomic analysis because of the extreme complexity of peptide mixtures and wide dynamic range of protein abundances. Here, we developed a novel approach to enrich and quantify N-terminal glycine peptides through sortase A mediated ligation. This strategy was based on the formation of a covalent bond between the sortase A recognition motif LPXTG and a N-terminal glycine residue. Also, the quantification was achieved by introducing isotopically labeled threonine in the motif LPXTG. In this strategy, both the enrichment of N-terminal glycine peptides and the stable isotope labeling were achieved in a single step. We applied this approach for the proteome analysis of MCF-7 cell line. It was demonstrated a significant reduction in sample complexity via highly selective and efficient enrichment of N-terminal glycine peptides, thereby detecting lots of less abundant proteins and enhancing proteome coverage. In comparison to the untreated sample, an increase of 34% of proteins was additionally identified. Furthermore, 97% of proteins were successfully quantified with high accuracy. In summary, this quantitative N-terminal glycine peptides enrichment strategy is expected for high-throughput qualitative and quantitative proteomic analysis as a complementary approach to conventional shotgun proteomics.

A Systematic Investigation of Proteoforms with N‐Terminal Glycine and Their Dynamics Reveals Its Impacts on Protein Stability

“…Previously, it was thought that the substrates of sortase A require multiple consecutive glycine residues at the N‐terminus [13] . However, it was found that the ligation could also effectively happen on peptides with other amino acid residues immediately after N‐terminal glycine [9,11] . In the current datasets, only 8–10 % of the amino acid residues immediately after the terminal glycine are glycine (Figure 1D).…”

“…The enzyme was widely used for protein engineering and the labeling of cell‐surface proteins [9–10] . “X” can be any amino acid residue, and arginine was chosen because it provides a trypsin cleavable site in the sequence for eluting enriched peptides from the resins [11] . The mixture was incubated for only 1 h to minimize possible protein cleavages by endogenous proteases, which were inhibited by the addition of protease inhibitor cocktail.…”

“…[13] However, it was found that the ligation could also effectively happen on peptides with other amino acid residues immediately after N-terminal glycine. [9,11] In the current datasets, only 8-10 % of the amino acid residues immediately after the terminal glycine are glycine (Figure 1D). This is consistent with the previous reports in which sortase A can mediate the ligation with peptides containing any amino acid residues at the position next to N-terminal glycine.…”

“…[33] Cao et al selectively enriched tryptic peptides with N-terminal glycine using the sortase A-mediated ligation. [11] In our lab, we compared different types of sortase A (wild-type or mutated) from different sources for enriching N-terminal glycine containing peptides digested from the whole cell lysate. The method is highly effective in capturing peptides with N-terminal glycine.…”

A Systematic Investigation of Proteoforms with N‐Terminal Glycine and Their Dynamics Reveals Its Impacts on Protein Stability

“…This is consistent with the previous reports in which sortase A can mediate the ligation with peptides containing any amino acid residues at the position next to N-terminal glycine. [11] The occurrence of glycine in the human proteome is around 7 %. [14] A slightly higher frequency of glycine identified at the position next to Nterminal glycine using all three types of sortase A suggests that the enzyme may have a slightly higher efficiency in catalyzing the ligation of peptides with consecutive glycines at the N-terminus.…”