Protocols and SOPs
The Overall Lab goes Wikipedia
Terminal amine isotopic labeling of substrates (TAILS)
Degradomics (by Parker Jobin)
Identifying the proteases and their inhibitors in vivo is an essential step in understanding their clinical relevance. For such analyses, we annotated the human and mouse protease genomes . The CLIP-CHIP™ is a complete protease and protease inhibitor DNA microarray chip for human and mouse, and includes 70-mer oligonucleotide probes for all 1561 human and murine proteases, inactive homologues and inhibitors [2,3]. [read more …]
TAILS is a novel multiplex quantitative proteomics platform for the determination of N-terminomes and the system-wide identification of protease substrates and cleavage sites in complex biological samples. In this method, all natural and cleaved N-termini and lysines in a proteome (exposed or not exposed to a protease) are blocked by differential isotopic labeling. Pooled samples are trypsinized and then, to reduce sample complexity, incubated with an aldehyde-derivatized amine-reactive polymer that removes all unlabeled peptides. [read more …]
TopFIND with TopFINDer and PathFINDer
TopFIND is the Termini oriented protein Function Inferred Database (TopFIND) is an integrated knowledgebase focused on protein termini, their formation by proteases and functional implications. It contains information about the processing state of proteins and functional implications thereof derived from research literature, contributions by the scientific community and biological databases. Two software tools for the analysis of protein termini and protease cleavage are available through TopFIND:
- TopFINDer (TopFIND ExploreR) automatically retrieves data from TopFINDer for large lists of N- or C-termini.
- PathFINDer identifies indirect connections between a protease and list of substrates or termini thus supporting the evaluation of complex proteolytic processes in vivo.
Protease specificity relies largely on the active site-mediated recognition of amino acid sequences that encompass the scissile peptide bond. Mapping of active site specificity is essential to characterise substrate preferences as well as for assay development and inhibitor studies. We have developed PICS (Proteomic Identiﬁcation of protease Cleavage Sites), a technique that employs proteome-derived peptide libraries as proteolytic substrate screens for the characterisation of protease active site specificity (Schilling & Overall 2008). PICS simultaneously profiles prime and non-prime specificity and allows to investigate subsite cooperativity by identifying 10s – 100s of individual cleavage products. [read more …]