ReCLIP (reversible cross-link immuno-precipitation): an efficient method for interrogation of labile protein complexes

Abstract

The difficulty of maintaining intact protein complexes while minimizing non-specific background remains a significant limitation in proteomic studies. Labile interactions, such as the interaction between p120-catenin and the E-cadherin complex, are particularly challenging. Using the cadherin complex as a model-system, we have developed a procedure for efficient recovery of otherwise labile protein-protein interactions. We have named the procedure "ReCLIP" (Reversible Cross-Link Immuno-Precipitation) to reflect the primary elements of the method. Using cell-permeable, thiol-cleavable crosslinkers, normally labile interactions (i.e. p120 and E-cadherin) are stabilized in situ prior to isolation. After immunoprecipitation, crosslinked binding partners are selectively released and all other components of the procedure (i.e. beads, antibody, and p120 itself) are discarded. The end result is extremely efficient recovery with exceptionally low background. ReCLIP therefore appears to provide an excellent alternative to currently available affinity-purification approaches, particularly for studies of labile complexes.

Figure 1. Titration of intracellular cross-linking of p120 and E-cadherin.

Western blot analysis of E-cadherin (top panels) and p120 (bottom panels) in whole cell lysates of A431 cells treated with the indicated concentrations of DSP (a, b) or DTME (c, d) between 0.01 mM and 1.0 mM. Samples were prepared under non-reducing (a, c) and reducing conditions (b, d) as indicated. Arrowheads indicate monomeric E-cadherin and p120, large cross-linked species are indicated with arrows, smaller crosslinked E-cadherin species are indicated with a diamond (⧫), and a possible cysteine-induced E-cadherin dimer is indicated with an asterisk (*). The chemical structures of DSP (e) and DTME (f) are shown, images were constructed with the DrawIt application in KnowItAll Informatics System v. 4.1 (Bio-Rad).

Figure 2. In-cell cross-linking preserves the interaction of p120 and E-cadherin and is specific for interacting proteins.

(a) Western blot analysis of p120, E-cadherin, FAK, and p42/44 MAPK in p120 immunoprecipitates and lysates from A431 cells lysed in 1% digitonin or RIPA buffer following treatment with DMSO vehicle or 0.5 mM cross-linker as indicated. (b) Cadherin-negative A431-D cells, and A431-D cells stably expressing wild type (WT E-cad) or p120-uncoupled (764 E-cad) E-cadherin were prepared and analyzed as in A. (c) Western blot analysis of p120, E-cadherin, β-catenin, and α-catenin in p120, control IgG immunoprecipitates, and lysates from A431 cells treated as in panel a.

Figure 3. Elution of binding partners from p120.

(a) A schematic of the elution strategy. Following immunoprecipitation and washing of cross-linked complexes on p120 mAb beads, binding partners are released by incubation with DTT in RIPA buffer, cleaving the cross-links and releasing interacting proteins from p120. (b) A representative western blot demonstrating depletion of p120 from A431 cell lysates following immunoprecipitation with p120 mAb beads of control IgG beads. Tubulin is shown as a loading control. (c) Elution of known binding partners, but not p120, from p120 mAb beads. Whole cell lysate is shown as a control, and 10% of the DTT eluate was analyzed for E-cadherin, β-catenin, α-catenin, and p120 by Western blot.

Figure 4. Cross-linkers can be combined to enhance complex recovery.

(a) Average number of distinct peptides identified in 2 LC-MS/MS runs for E-cadherin, α-catenin, β-catenin, and plakoglobin from A431 cells treated with DSP, DTME, or both compounds simultaneously (DSP + DTME). Error bars represent standard error of the mean. Background levels were similar across all conditions. (b) Western blot analysis of E-cadherin levels in lysates (Lysate), p120 eluates (p120), and control IgG eluates (Control) from A431 cells treated with the indicated cross-linkers. (c) Silver stain analysis of total protein recovery from p120 and control IgG eluates from each condition (DSP, DTME, or DSP + DTME). (d–f) Average distinct peptide recovery of 15 additional putative p120 binding partners under each cross-linking condition. Proteins were grouped based on whether more peptides were detected using the combination of DSP and DTME (d), DSP alone (e) or DTME alone (f).