title: Global analysis of cellular proteolysis by selective enzymatic labeling of protein N-termini.
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.
authors: Department of Pharmaceutical Chemistry, University of California, San Francisco, California, USA; Department of Laboratory Medicine, University of California, San Francisco, California, USA.
authors: Arun P
authors: AP
authors: Wiita
authors: Department of Pharmaceutical Chemistry, University of California, San Francisco, California, USA.
authors: Julia E
authors: JE
authors: Seaman
authors: Department of Pharmaceutical Chemistry, University of California, San Francisco, California, USA; Department of Cellular and Molecular Pharmacology, University of California, San Francisco, California, USA. Electronic address: jim.wells@ucsf.edu.
authors: James A
authors: JA
authors: Wells
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D010447:Peptide Hydrolases / Q000235:genetics / Q000378:metabolism*; D010453:Peptide Synthases / Q000235:genetics / Q000378:metabolism*; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D059748:Proteolysis; D040901:Proteomics / Q000379:methods*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013194:Staining and Labeling / Q000379:methods; D013379:Substrate Specificity; D013381:Subtilisins / Q000235:genetics / Q000378:metabolism*
chemical_list: D011506:Proteins; D011994:Recombinant Proteins; D010447:Peptide Hydrolases; D013381:Subtilisins; D010453:Peptide Synthases; C117282:subtiligase
keywords: Apoptosis; Caspase; Degradomics; Mass spectrometry; Proteolysis; Proteomics; Selected reaction monitoring; Subtiligase