Profiling protein function with small molecule microarrays

Abstract

The regulation of protein function through posttranslational modification, local environment, and protein-protein interaction is critical to cellular function. The ability to analyze on a genome-wide scale protein functional activity rather than changes in protein abundance or structure would provide important new insights into complex biological processes. Herein, we report the application of a spatially addressable small molecule microarray to an activity-based profile of proteases in crude cell lysates. The potential of this small molecule-based profiling technology is demonstrated by the detection of caspase activation upon induction of apoptosis, characterization of the activated caspase, and inhibition of the caspase-executed apoptotic phenotype using the small molecule inhibitor identified in the microarray-based profile.

Fig 1.

Screening of PNA-encoded libraries. Individual small molecules (red shapes) are tethered to a unique PNA sequence (blue bars), which encodes both their synthetic history and their location upon hybridization to an oligonucleotide microarray. The PNA are capped with fluorescein (yellow oval) for fluorescence detection. The library of PNA-encoded small molecules is incubated with a protein mixture of interest, passed through a size-exclusion filter to separate the small molecule-PNA adducts bound to a macromolecule from the unbound ones, and the high molecular weight fraction is hybridized to the oligonucleotide microarray.

Fig 2.

Chemical structure of selected protease inhibitors 1–7.

Fig 3.

Quantification of protease activity. (A) Probes 1–7 (20 μl at 1.0 μM) were incubated with various concentrations of caspase-3 (10–500 nM), passed through a size-exclusion filter, and hybridized to a GenFlex (Affymetrix) microarray (seen in a false color scale). The probes at the top of the image are control probes. The intensity has been standardized in the five images for qualitative viewing purposes. (B) Correlation of protease activity and observed probe intensity. Plot of the fluorescence intensity (x axis) vs. caspase-3 concentration (y axis) with an SE of 10%.

Fig 4.

Crude cell lysate profiling. (A) Direct hybridization of compounds 1–7; (B) Incubation of compounds 1–7 with granzyme B, size exclusion, hybridization. (C) Incubation of compounds 1–7 with purified caspase-3, size exclusion, hybridization. (D) Incubation of compounds 1–7 with Jurkat crude cell lysate, size exclusion, hybridization. (E) Incubation of compounds 1–7 with crude cell lysate from Jurkat cells pretreated with granzyme B, size exclusion, hybridization.

Fig 5.

MS/MS spectra of (A) triply charged and (B) doubly charged SGTDVDAANLRETFR peptides derived from human caspase-3. Prominent y₉²⁺ and y₁₁²⁺ ions in the MS/MS spectrum of the doubly charged precursor in B are consistent with facile cleavage C-terminal to aspartic acid residues reported in the literature (24). *, loss of the elements of water; #, loss of ammonia.

Fig 6.

Inhibition of the apoptosis phenotype. (A) Inhibition of downstream caspase-3 mediated autoprocessing and cleavage of DFF-45 upon incubation of granzyme B-activated Jurkat lysates with inhibitor 6c. The blots were probed with anti-caspase 3 and anti-DFF45 and then visualized. (B) Inhibition of Fas-mediated apoptosis by caspase inhibitor [Z-D(OMe)-E(OMe)-V-D(OMe)-FMK]. Cells were stained with both Annexin-V-EGFP and propidium iodide.