We present protease specificity profiling based on quantitative proteomics in combination with proteome-derived peptide libraries. Peptide libraries are generated by endoproteolytic digestion of proteomes without chemical modification of primary amines before exposure to a protease under investigation. After incubation with a test protease, treated and control libraries are differentially isotope-labeled using cost-effective reductive dimethylation. Upon analysis by liquid chromatography-tandem mass spectrometry, cleavage products of the test protease appear as semi-specific peptides that are enriched for the corresponding isotope label. We validate our workflow with two proteases with well-characterized specificity profiles: trypsin and caspase-3. We provide the first specificity profile of a protease encoded by a human endogenous retrovirus and for chlamydial protease-like activity factor (CPAF). For CPAF, we also highlight the structural basis of negative subsite cooperativity between subsites S1 and S2. For A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) -4, -5, and -15, we show a canonical preference profile, including glutamate in P1 and glycine in P3. In total, we report nearly 4000 cleavage sites for seven proteases. Our protocol is fast, avoids enrichment or synthesis steps, and enables probing for lysine selectivity as well as subsite cooperativity. Due to its simplicity, we anticipate usability by most proteomic laboratories. Molecular & Cellular
Identifying antibody epitopes on membrane proteins can help differentiate antibody binding mechanisms, identify immunodominant structures, distinguish and classify antibodies, and identify cancer state-specific antigens, providing valuable information for therapeutic and diagnostic applications. However, membrane proteins represent challenging targets for epitope mapping studies using conventional methodologies such as X-ray crystallography or peptide scanning. We have developed a technology, ‘Shotgun Mutagenesis Epitope Mapping’ for rapidly mapping antibody epitopes on structurally-intact proteins expressed in mammalian cells. Here, we applied Shotgun Mutagenesis to create a comprehensive mutation library for CXCR4, a GPCR associated with tumor metastasis. The CXCR4 mutation library, which comprised >700 clones with 2.7x coverage of each of the 352 residues, was screened in triplicate with 5 different anti-CXCR4 MAbs to identify their epitopes. Our analysis revealed that all CXCR4 MAb epitopes were conformational and showed a requirement for charged residues located on the second extracellular loop. CXCR4 MAb epitopes were mapped onto the crystal structure to visualize and identify common structural features. Using epitope information, we generated point mutations in CXCR4 at positions of critical contact residues, incorporated them into virus-like particles (‘Lipoparticles’) and measured their binding kinetics. These approaches helped define the electrostatic contribution of individual amino acid residues on CXCR4 to the association rate, dissociation rate, and overall binding interaction with CXCR4 MAbs. Taking the same approach, we generated a comprehensive mutation library for the biomarker Claudin-4, a tight junction protein upregulated in epithelial cancers. The Claudin-4 mutation library, which comprised >400 clones with complete coverage of each of the 209 residues, was screened with a panel of 7 different anti-Claudin-4 antibodies to map their epitopes. We identified several distinct conformational epitopes that all map exclusively to the large first extracellular loop, identifying this domain as an immunodominant region of claudin-4. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4742. doi:1538-7445.AM2012-4742
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