Functional proteomics using chromophore- assisted laser inactivation

Abstract

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.

Functional Proteomic Screen Identifies a Modulating Role for CD44 in Death Receptor–Mediated Apoptosis

“…It has previously been shown that FALI of Fas or caspase-3 can block apoptosis as measured by a viability assay (22). In this study, we sought to adapt FALI to a miniaturized format that would lend itself readily to high-throughput functional proteomic analysis (Fig.…”

“…Furthermore, by not loading antibody into cells, it is possible to exclusively examine the function of the extracellular compartment of a targeted protein (19,22).…”

Disease proteomics

“…One advantage of these approaches is the ability to inactivate a specific site in a protein in a time-dependent and localization-restricted manner, as with chromophore-assisted laser inactivation (CALI). Such inactivation allows assessment of the disease relevance of the site and provides a means to revert a cellular phenotype to a more normal state 49 (Fig. 4).…”

Chromophore‐Assisted Light Inactivation: A Twenty‐Year Retrospective

“…The commonly used fluorophore fluorescein was highly efficient in loss of b-galactosidase activity in CALI while green fluorescent protein (GFP) was about the same as Malachite green. Fluorophore-assisted light inactivation (FALI) was developed [10], which took advantage of the greater efficiency of fluorescein to permit use of non-laser light sources (such as a slide projector) to simultaneously irradiate many samples in multi-well plates [11], thus providing the potential for high-throughput CALI [12,13].…”