Dataset Information

Tag-Free Identification of Protease Specificity Using Proteome-Derived Peptide Libraries

ABSTRACT: Proteome-derived peptide libraries are a powerful tool for the investigation of protease specificity, conventionally involving the biotinylation of cleavage products to enable for their enrichment. Here we present a modified strategy for protease specificity profiling using proteome-derived peptide libraries in a tag-free manner. In comparison to the original method the new protocol is faster, avoids cost- and time-intensive enrichment steps, and enables probing for lysine selectivity. In the new workflow, peptide libraries are generated by endoproteolytic digestion of proteomes without chemical modification of primary amines prior to exposure to a protease under investigation. After incubation with the test protease, treated and control library are differentially labeled using stable isotopes by means of reductive dimethylation. Upon analysis by liquid chromatography mass spectrometry, cleavage products of the test protease appear as peptides enriched for the respective isotopic label. With “PICS 2.0” we identified more than 2800 cleavage sites in five specificity experiments for four proteases, including trypsin, caspase-3, and chlamydial protease-like activity factor (CPAF). Hence, this method is a valuable addition to the different strategies currently employed for specificity profiling of proteases

INSTRUMENT(S): Q Exactive

ORGANISM(S): Escherichia Coli

SUBMITTER: Oliver Schilling

LAB HEAD: Oliver Schilling

PROVIDER: PXD002785 | Pride | 2016-05-02

REPOSITORIES: Pride

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Publications

Identification of Protease Specificity by Combining Proteome-Derived Peptide Libraries and Quantitative Proteomics.

Biniossek Martin L ML Niemer Melanie M Maksimchuk Ken K Mayer Bettina B Fuchs Julian J Huesgen Pitter F PF McCafferty Dewey G DG Turk Boris B Fritz Guenther G Mayer Jens J Haecker Georg G Mach Lukas L Schilling Oliver O

Molecular & cellular proteomics : MCP 20160427 7

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 ...[more]

PMID: 27122596

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Project description:Butyrophilin 1A1 (BTN1A1) was first identified as a major integral protein of both the apical surface of mammary epithelial cells during lactation and the surface membrane surrounding lipid droplets in milk. Current evidence supports a role for BTN1A1 in the secretion of milk lipids as a membrane receptor, which forms a secretion complex with the redox enzyme, xanthine oxidoreductase (XDH). Capping of cytoplasmic lipid droplets with BTN1A1 and XDH serves to envelop the droplets with cellular membranes at the cell apex as an initial step in their subsequent release from the cell. The first evidence that BTN1A1 functions in this process was the generation of Btn1a1-/- mice, in which lipid secretion was disrupted and large unstable droplets were released into alveolar spaces with fragmented surface membranes. We have revisited this mutant mouse line using RNAseq and proteomic analysis to fully assess the consequences of ablating the Btn1a1 gene on the expression of other genes and proteins. Disruption of Btn1a1 expression led to a large build-up of Xdh (6-fold over wild-type levels) in the cytoplasm, induction of acute phase response genes and Lif-activation of STAT3 phosphorylation. At peak lactation, approx. 10% of the cells were dying, as assessed by TUNEL-analysis of nuclear DNA. Cell death appeared to proceed through expression of caspase 8 and activated caspase 3 and not via lysosomal lysis, which is the principal route for elimination of cells during mammary involution in wild-type mice. Other potential death pathways include autophagy and Slc5a8-mediated inactivation of survivin (Birc5). Milk secretion was prolonged by renewal of the secretory epithelium, as evidenced by the expression of cyclins, Fos/Jun and upregulation of Ki67 in approx. 10% of secretory cell nuclei. Thus a 21-day lactation cycle can be maintained, despite significant cell death by compensatory cell division, provided a milking stimulus is maintained. These data highlight the importance of BTN1A1 expression for the maintenance of terminally differentiated mammary epithelial cells and optimal milk production throughout lactation. From a practical standpoint, the complimentary RNAseq and proteomics data should provide a useful reference database for gene and protein expression in C57/Bl6 mice at peak lactation.

Dataset Information

Tag-Free Identification of Protease Specificity Using Proteome-Derived Peptide Libraries

Publications

Identification of Protease Specificity by Combining Proteome-Derived Peptide Libraries and Quantitative Proteomics.

Similar Datasets

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