Tags for labeling protein N-termini with subtiligase for proteomics

Abstract

The peptide ligase subtiligase, derived from subtilisin, has been employed in the identification of protein N-termini in complex mixtures. Here, the peptide ester substrates for the ligation reaction were optimized with respect to solubility, resulting in greater incorporation of the N-terminal tags. Additionally, the quantitation of the incorporated tags was explored, and a "click" chemistry-based derivatization provided the ability to quantitate the tag to low nanomolar concentrations by sandwich ELISA. These new tags should expand the utility of subtiligase for the proteomic study of N-termini.The study of the proteome is aided by tools that can separate specific subsets of proteins. For example, in studying post-translational modifications (PTM), methods for enriching phosphoproteins and phosphopeptides have been developed. 1 One common PTM in biology is the production of new N-termini by proteolysis; another is the modification of N-termini through acetylation 3 or myristoylation. 4 Several labs have described indirect means for isolation or enrichment of N-terminal peptides. These techniques include strong cation exchange chromatography of tryptic peptides, 5 and the chemical labeling of amine-containing functionality in the proteome 6 followed by either diagonal chromatography 7 or affinity purification. 8 Alternatively, our laboratory has developed a direct enzymatic method to affinity tag and enrich for the subset of proteins with free N-termini in order to characterize cellular substrates of proteolysis, or, more generally, characterize the ensemble of native N-termini. A recent study using this method identified nearly three hundred caspase substrates and their precise cleavage sites from apoptotic Jurkat cells. 9This method utilizes subtiligase, a variant of the protease subtilisin BPN' that contains a cysteine residue in place of the active site serine-221 and an alanine residue replacing proline-225. 10 Unlike the wild-type enzyme, the resulting double mutant has negligible protease activity, yet it can efficiently acylate free N-termini of peptides or proteins using ester substrates. The workflow for the application of subtiligase to the study of the proteome is outlined in Scheme 1. A complex mixture of proteins is N-terminally labeled with subtiligase using a peptide ester containing a biotin affinity tag and a TEV protease 11,12 cleavage site. Tagged proteins are separated from the unincorporated tag and digested with trypsin. Tagged N-terminal peptides are captured on avidin beads, cleaved off the beads by TEV protease treatment and analyzed by LC-MS/MS. While approximately 80% of N-termini are blocked in mammalian cells, mostly due to acetylation, 13,14 certain populations of proteins are believed to have a greater fraction of free N-termini. These include signal-sequence-dependent proteins transported across membranes, such as nuclear-encoded mitochondrial proteins. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptWhile this approach is a powerful and sensiti...

Proteolytic Post-translational Modification of Proteins: Proteomic Tools and Methodology

“…Mahrus et al developed the most useful such approach with an elegant method using an engineered subtiligase to selectively label unblocked ␣-amines with a biotinylated peptide ester substrate, with labeling followed by trypsinization, avidin capture, and LC-MS/MS (32,33) (Fig. 1A).…”

Global Analysis of Cellular Proteolysis by Selective Enzymatic Labeling of Protein N-Termini

“…The enzyme is stored at −80 °C and retains activity for at least 2 years after purification. Activity can be tested and quantified using FRET ester reporters (Shimbo et al, 2012; Yoshihara, Mahrus, & Wells, 2008). …”

“…The synthetic ester used for labeling is customizable for different experimental goals (Yoshihara et al, 2008). The current version contains four distinct features: (i) an ester linkage for subtiligase acylation and transfer to the free peptide α-amine, (ii) the unique Abu-tag to facilitate MS identification, (iii) the TEV protease cleavage site for elution from avidin beads, and (iv) biotin for initial capture (Fig.…”

Methods for the proteomic identification of protease substrates

“…Identification of N-terminal peptides was accomplished by treating cell lysates with subtiligase and a biotinylated-peptide ester (Figure 2c). [36,37] The resulting peptide mixture was trypsinized, N-terminal peptides were captured on streptavidin beads, and the desired peptides were released by cleavage of a TEV protease site in the original biotinylated peptide. TEV protease cleavage leaves a dipeptide tag that can be used to confirm true positives.…”