Tags for labeling protein N-termini with subtiligase for proteomics

ArticleinBioorganic & medicinal chemistry letters 18(22):6000-3 · September 2008with4 Reads
DOI: 10.1016/j.bmcl.2008.08.044 · Source: PubMed
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.
    • "by Chemical Labeling of the a-Amine of Proteins) [34] or subtiligase-based approaches [35]. Although the latter approaches cannot assess natural N-terminal modifications as a free a-amine is needed for tagging and pullout, both are suited to analyze proteasegenerated neo-N-termini [36e39]. "
    [Show abstract] [Hide abstract] ABSTRACT: Proteolytic processing is a pervasive and irreversible post-translational modification that expands the protein universe by generating new proteoforms (protein isoforms). Unlike signal peptide or prodomain removal, protease-generated proteoforms can rarely be predicted from gene sequences. Positional proteomic techniques that enrich for N- or C-terminal peptides from proteomes are indispensable for a comprehensive understanding of a protein's function in biological environments since protease cleavage frequently results in altered protein activity and localization. Proteases often process other proteases and protease inhibitors which perturbs proteolytic networks and potentiates the initial cleavage event to affect other molecular networks and cellular processes in physiological and pathological conditions. This review is aimed at researchers with a keen interest in state of the art systems level positional proteomic approaches that: (i) enable the study of complex protease-protease, protease-inhibitor and protease-substrate crosstalk and networks; (ii) allow the identification of proteolytic signatures as candidate disease biomarkers; and (iii) are expected to fill the Human Proteome Project missing proteins gap. We predict that these methodologies will be an integral part of emerging precision medicine initiatives that aim to customize healthcare, converting reactive medicine into a personalized and proactive approach, improving clinical care and maximizing patient health and wellbeing, while decreasing health costs by eliminating ineffective therapies, trial-and-error prescribing, and adverse drug effects. Such initiatives require quantitative and functional proteome profiling and dynamic disease biomarkers in addition to current pharmacogenomics approaches. With proteases at the pathogenic center of many diseases, high-throughput protein termini identification techniques such as TAILS (Terminal Amine Isotopic Labeling of Substrates) and COFRADIC (COmbined FRActional DIagonal Chromatography) will be fundamental for individual and comprehensive assessment of health and disease.
    Full-text · Article · Nov 2015
    • "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 a-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. 13.1B ). "
    [Show abstract] [Hide abstract] ABSTRACT: Proteolysis is a critical modification leading to alteration of protein function with important outcomes in many biological processes. However, for the majority of proteases, we have an incomplete understanding of both cellular substrates and downstream effects. Here, we describe detailed protocols and applications for using the rationally engineered peptide ligase, subtiligase, to specifically label and capture protein N-termini generated by proteases either induced or added to complex biological samples. This method allows identification of the protein targets as well as their precise cleavage locations. This approach has revealed >8000 proteolytic sites in healthy and apoptotic cells including >1700 caspase cleavages. One can further determine substrate preferences through rate analysis with quantitative mass spectrometry, physiological substrate specificities, and even infer the identity of proteases operating in the cell. In this chapter, we also describe how this experimental method can be generalized to investigate proteolysis in any biological sample.
    Full-text · Article · Jun 2014
    • "We have never observed transfer onto peptide or protein side chains, suggesting an enzymatic specificity thus far unachievable via small molecule approaches. 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. "
    [Show abstract] [Hide abstract] ABSTRACT: Proteolysis is a key regulatory post-translational modification in diverse cellular processes including programed cell death, immune function, and development. Tracking proteolytic events has become a focus of researchers assessing the downstream consequences of protease activation. In this review we summarize unbiased methods for identifying protease substrates and tracking the extent of cleavage, a field termed 'degradomics'. These include one-dimensional and two-dimensional gel-based methods for identifying protease substrates, N-terminal peptide identification methods for simultaneously identifying substrates and cleavage sites, and approaches for the quantitation of cleavage events during endogenous proteolysis. Individual methods have identified more than 300 caspase-cleaved targets during apoptosis suggesting broad future applications for these technologies.
    Full-text · Article · Oct 2009
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