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Functional proteomics using chromophore‐ assisted laser inactivation
Author(s) -
Rubenwolf Susanne,
Niewöhner Jens,
Meyer Elisabeth,
PetitFrère Corinne,
Rudert Fritz,
Hoffmann Philipp R.,
Ilag Leodevico L.
Publication year - 2002
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/1615-9861(200203)2:3<241::aid-prot241>3.0.co;2-7
Subject(s) - phosphorylation , proteomics , glycosylation , chemistry , cell signaling , small molecule , chromophore , function (biology) , biochemistry , signal transduction , protein function , proteome , target protein , biophysics , microbiology and biotechnology , biology , organic chemistry , gene
Proteins are the molecules that fulfil most cellular functions and represent over 90% of drug targets in the market. Chromophore‐assisted laser inactivation (CALI) provides a timely and locally restricted protein inactivation and has proven to specifically destroy protein function using dye‐coupled ligands and laser irradiation. CALI involves the generation of short‐lived radicals thus limiting the radius of covalent modifications to spatially restricted sites on the target molecule. A transient functional inactivation occurs if the radicals modify amino acids of the target protein that are responsible for function. Here we show specific inactivation of several protein targets, that are members of relevant signal transduction pathways. For each of these targets, simple and high throughput screening‐scaleable assays have been developed, making it possible to quantify the observed inactivation. Activities of target proteins have been addressed in cell‐free as well as cell‐based assays employing human primary and tumor‐derived cell lines. In all cases, at least 50% inactivation was achieved. The data presented here demonstrate that CALI is a highly versatile tool for validating disease relevant targets at the protein level. This approach also takes into account post‐translational modifications like phosphorylation, glycosylation or acylation, thereby enlarging its applicability for many different types of targets.