Phosphatidylinositol 3,4,5-trisphosphate activity probes for the labeling and proteomic characterization of protein binding partners

Abstract

Phosphatidylinositol polyphosphate lipids, such as phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P₃], regulate critical biological processes, many of which are aberrant in disease. These lipids often act as site-specific ligands in interactions that enforce membrane association of protein binding partners. Herein, we describe the development of bifunctional activity probes corresponding to the headgroup of PI(3,4,5)P₃ that are effective for identifying and characterizing protein binding partners from complex samples, namely cancer cell extracts. These probes contain both a photoaffinity tag for covalent labeling of target proteins and a secondary handle for subsequent detection or manipulation of labeled proteins. Probes bearing different secondary tags were exploited, either by direct attachment of a fluorescent dye for optical detection or by using an alkyne that can be derivatized after protein labeling via click chemistry. First, we describe the design and modular synthetic strategy used to generate multiple probes with different reporter tags of use for characterizing probe-labeled proteins. Next, we report initial labeling studies using purified protein, the PH domain of Akt, in which probes were found to label this target, as judged by in-gel detection. Furthermore, protein labeling was abrogated by controls including competition with an unlabeled PI(3,4,5)P₃ headgroup analogue as well as through protein denaturation, indicating specific labeling. In addition, probes featuring linkers of different lengths between the PI(3,4,5)P₃ headgroup and photoaffinity tag led to variations in protein labeling, indicating that a shorter linker was more effective in this case. Finally, proteomic labeling studies were performed using cell extracts; labeled proteins were observed by in-gel detection and characterized using postlabeling with biotin, affinity chromatography, and identification via tandem mass spectrometry. These studies yielded a total of 265 proteins, including both known and novel candidate PI(3,4,5)P₃-binding proteins.

Figure 1

Design of bifunctional PIPn activity-based probes. Probes consist of the binding moiety (PIPn headgroup), linked to a Y-shaped lysine linker containing both a photoaffinity group (benzophone) and a secondary tag that consisted of either fluorescein (probe 1) or an alkyne as a bioorthogonal tag (probes 2–3).

Figure 2

Gel images of labeling studies using purified Akt-PH (fluorescent gel image shown in grey scale). Studies indicated successful labeling of Akt-PH protein by both fluorescein-probe 1 (lane 2) and alkyne-probe 2 (lane 4, after click chemistry post-derivatization) during studies. Additionally, control studies involving no photo-cross-linking (lane 1), or heat denaturation of the protein prior to probe incubation (lanes 3 and 5) yielded no fluorescence, indicating the absence of non-specific labeling. Finally, Coomassie Blue stains indicate that the protein is still present despite the abrogation of probe labeling. Please also see color fluorescence gel scans in Figure S1 of the supplementary information.

Figure 3

Competition studies involving Akt-PH labeling (fluorescent gel image shown in grey scale). In lanes 1–4, pre-incubation of Akt-PH with excess inhibitor 4b suppressed labeling by probe 2 as visualized by a reduction in fluorescence intensity of the AKt-PH band. In lane 5, the bifunctional lysine lacking the PIP headgroup did not label the Akt protein.

Figure 4

Akt-PH labeling studies with probes 2 and 3 indicate that the shorter linker (probe 2) results in significantly enhanced protein labeling (fluorescent gel image shown in grey scale).

Figure 5

Illustration of the mechanisms of protein labeling and subsequent analysis using alkynyl-PI(3,4,5)P3 activity probes 2 and 3. Following the incubation of the probes with cell extracts, protein−probe binding events were captured through irradiation of the benzophenone tag. Next, labeled proteins were selectively derivatized via click chemistry using the secondary functional tag of the probe to introduce rhodamine (11) for in-gel fluorescence analysis or biotin (12) for protein enrichment via affinity chromatography and subsequent protein identification using liquid chromatography-tandem mass spectrometry.

Figure 6

Labeling studies to ascertain effective probe concentrations (fluorescent gel image shown in grey scale). Probe concentrations were screened using MDA-MB-435 cancer cell extracts (soluble fraction) to identify concentrations that were effective for studies. Concentrations ranging from 200 nM to 1nM were used to label the same amount of the cell extracts with heat-denatured controls performed for each protein concentration. From fluorescence gel imaging results, probe concentrations of approximately 50 µM (lanes 5 and 6) were selected for other labeling studies. Please also see color fluorescence gel scans in Figure S2 of the supplementary information.

Figure 7

Labeling of MDA-MB-435 soluble proteome using various conditions (fluorescent gel image shown in grey scale). Studies using fluorescein-probe 1 resulted in significantly stronger labeling in the heat denatured control (lane 2) compared to the normal study (lane 1), indicating that the presence of the fluorophore during labeling is problematic. Studies using probe 2 and post-labeling led to diminished labeling in heat-the denatured control (lane 5, when compared to lane 4). No probe (lane 6) and no light (lane 8) controls negated labeling, and click chemistry ligands TBTA (lane 7) and THPTA (lane 9) yielded similar results. Please also see color fluorescence gel scans in Figure S3 of the supplementary information.

Figure 8

Analysis of the effect of probe linker length using MDA-MB-435 cancer cell extracts (fluorescent gel image). Labeling studies with probe 2 (shorter linker) yielded significantly enhanced labeling compared to probe 3. Control compound 8a lacking the PIPn headgroup yielded minimal protein labeling of an orthogonal subset of proteins.

Scheme 1

General synthetic route to PIPn activity probes using headgroup aminoconjugates of type 4.

Scheme 2

Synthesis of bifunctional lysine moieties bearing different secondary tags to generate PIPn activity probes 1−3.