Isolation Of N-terminal Peptides Research Articles

Novel functionalized nanomaterials for the effective enrichment of proteins and peptides with post-translational modifications

Low abundance proteins/peptides usually suffer strong interference with highly abundant proteins/peptides as well as other contaminants, resulting in low ionization efficiency in MS analysis. Hence, the enrichment and separation of proteins/peptides from complex mixtures are of great value to their successful identification.In this review we present an overview of the application of different nanocomposites for the specific enrichment of peptides/proteins with post-translational modifications (PTMs), such as phosphorylation and glycosylation. Furthermore, examples of the selective enrichment of cysteine-containing peptides, histidine-tagged proteins and the isolation of N-terminal peptides will be analyzed.

Quantitative proteomics analysis of the Arg/N-end rule pathway of targeted degradation in Arabidopsis roots.

According to the Arg/N-end rule pathway, proteins with basic N-termini are targeted for degradation by the Arabidopsis thaliana E3 ligase, PROTEOLYSIS6 (PRT6). Proteins can also become PRT6 substrates following post-translational arginylation by arginyltransferases ATE1 and 2. Here, we undertook a quantitative proteomics study of Arg/N-end rule mutants, ate1/2 and prt6, to investigate the impact of this pathway on the root proteome. Tandem mass tag labelling identified a small number of proteins with increased abundance in the mutants, some of which represent downstream targets of transcription factors known to be N-end rule substrates. Isolation of N-terminal peptides using terminal amine isotope labelling of samples (TAILS) combined with triple dimethyl labelling identified 1465 unique N-termini. Stabilising residues were over-represented among the free neo-N-termini, but destabilising residues were not markedly enriched in N-end rule mutants. The majority of free neo-N-termini were revealed following cleavage of organellar targeting signals, thus compartmentation may account in part for the presence of destabilising residues in the wild-type N-terminome. Our data suggest that PRT6 does not have a marked impact on the global proteome of Arabidopsis roots and is likely involved in the controlled degradation of relatively few regulatory proteins. All MS data have been deposited in the ProteomeXchange with identifier PXD001719 (http://proteomecentral.proteomexchange.org/dataset/PXD001719).

Probing the Efficiency of Proteolytic Events by Positional Proteomics

Several mass spectrometry-driven techniques allow to map the substrate repertoires and specificities of proteases. These techniques typically yield long lists of protease substrates and processed sites with (potential) physiological relevance, but in order to understand the primary function of a protease, it is important to discern bystander substrates from critical substrates. Because the former are generally processed with lower efficiency, data on the actual substrate cleavage efficiency could assist in categorizing protease substrates. In this study, quantitative mass spectrometry following metabolic proteome labeling (SILAC), combined with the isolation of N-terminal peptides by Combined Fractional Diagonal Chromatography, was used to monitor fluxes in the concentration of protease-generated neo-N-termini. In our experimental setup, a Jurkat cell lysate was treated with the human serine protease granzyme B (hGrB) for three different incubation periods. The extensive list of human granzyme B substrates previously catalogued by N-terminal Combined Fractional Diagonal Chromatography (1) was then used to assign 101 unique hGrB-specific neo-N-termini in 86 proteins. In this way, we were able to define several sites as getting efficiently cleaved in vitro and consequently recognize potential physiologically more relevant substrates. Among them the well-known hGrB substrate Bid was confirmed as being an efficient hGrB substrate next to several other potential regulators of hGrB induced apoptosis such as Bnip2 and Akap-8. Several of our proteomics results were further confirmed by substrate immunoblotting and by using peptide substrates incubated with human granzyme B.

Analysis of proteins inChlamydia trachomatisL2 outer membrane complex, COMC

The protein composition and N-terminal sequences of proteins in the outer membrane of Chlamydia trachomatis L2 were analysed following isolation of N-terminal peptides using combined fractional diagonal chromatography and identification by liquid chromatography tandem MS. Acetylation of primary amino groups of in vivo generated proteolytic cleavage sites facilitated identification of such sites in known outer membrane proteins (MOMPs). Our results further support a proposed prediction of the topology of the MOMPs. Furthermore, a previously unknown MOMP, CTL0626 (Ct372), was assigned as an MOMP with a carbohydrate-selective porin (OprB) family motif, and the presence of CTL0626 was confirmed using antibodies raised against the protein.