| Autor: |
Kuljanin M; Department of Cell Biology, Harvard Medical School, Boston, MA, USA.; Division of Radiation and Genome Stability, Department of Radiation Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA., Mitchell DC; Department of Cell Biology, Harvard Medical School, Boston, MA, USA., Schweppe DK; Department of Cell Biology, Harvard Medical School, Boston, MA, USA.; Department of Genome Sciences, University of Washington, Seattle, WA, USA., Gikandi AS; Division of Radiation and Genome Stability, Department of Radiation Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA., Nusinow DP; Department of Cell Biology, Harvard Medical School, Boston, MA, USA., Bulloch NJ; Department of Cell Biology, Harvard Medical School, Boston, MA, USA., Vinogradova EV; Department of Chemistry, The Scripps Research Institute, La Jolla, CA, USA., Wilson DL; Department of Chemistry, Stanford Cancer Institute and ChEM-H Institute, Stanford University, Stanford, CA, USA., Kool ET; Department of Chemistry, Stanford Cancer Institute and ChEM-H Institute, Stanford University, Stanford, CA, USA., Mancias JD; Division of Radiation and Genome Stability, Department of Radiation Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA., Cravatt BF; Department of Chemistry, The Scripps Research Institute, La Jolla, CA, USA., Gygi SP; Department of Cell Biology, Harvard Medical School, Boston, MA, USA. steven_gygi@hms.harvard.edu. |
| Abstrakt: |
Current methods used for measuring amino acid side-chain reactivity lack the throughput needed to screen large chemical libraries for interactions across the proteome. Here we redesigned the workflow for activity-based protein profiling of reactive cysteine residues by using a smaller desthiobiotin-based probe, sample multiplexing, reduced protein starting amounts and software to boost data acquisition in real time on the mass spectrometer. Our method, streamlined cysteine activity-based protein profiling (SLC-ABPP), achieved a 42-fold improvement in sample throughput, corresponding to profiling library members at a depth of >8,000 reactive cysteine sites at 18 min per compound. We applied it to identify proteome-wide targets of covalent inhibitors to mutant Kirsten rat sarcoma (KRAS) G12C and Bruton's tyrosine kinase (BTK). In addition, we created a resource of cysteine reactivity to 285 electrophiles in three human cell lines, which includes >20,000 cysteines from >6,000 proteins per line. The goal of proteome-wide profiling of cysteine reactivity across thousand-member libraries under several cellular contexts is now within reach. |
Full text from SpringerLink