The cell permeable crosslinker DSP [dithiobis-(succinimidyl propionate)] stabilizes transient and labile interactions in vivo, which allows their isolation using stringent protein complex purification techniques. Here we present a technique for crosslinking cells grown in culture followed by isolation of protein complexes by immunoprecipitation.
The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude stringent methods for the isolation and identification of protein networks around a protein of interest. The use of chemical crosslinkers allows the selective stabilization of labile interactions, thus bypassing biochemical limitations for purification. Here we present a protocol amenable for cells in culture that uses a homobifunctional crosslinker with a spacer arm of 12 Å, dithiobis-(succinimidyl proprionate) (DSP). DSP is cleaved by reduction of a disulphide bond present in the molecule. Cross-linking combined with immunoaffinity chromatography of proteins of interest with magnetic beads allows the isolation of protein complexes that otherwise would not withstand purification. This protocol is compatible with regular western blot techniques and it can be scaled up for protein identification by mass spectrometry1.
Stephanie A. Zlatic and Pearl V. Ryder contributed equally to this work.
1. Preparing for Crosslinking
2. Preparing the Crosslinking Solution
3. Prepare Cells for Crosslinking
4. Inactivation of DSP Reaction
5. Cell Lysis
6. Preparing Immuno-magnetic Precipitation Beads
Note: This step is usually started directly after beginning the 2 hour crosslinking incubation.
7. Incubate Crosslinked Lysate with Immuno-magnetic Beads
8. Wash Unbound Lysate from Beads
9. Denature Sample and Collect from Beads
10. Elution of Crosslinked Complexes with Antigenic Peptides (Immunoaffinity Chromatography).
Figure 1. Isolation of AP-3 interacting protein complexes and membrane proteins. HEK293 cells (A) or PC12 cells (B) were treated either in the presence of vehicle control (DMSO, odd lanes Fig 1A) or DSP (even lanes Fig. 1A or all lanes in Fig 1B). Clarified extracts were incubated either with beads alone (Fig. 1A, Lanes 3-4), transferrin receptor antibodies (Fig. 1A, Lanes 5-6), or AP-3 δ antibodies (Fig. 1A, Lanes 7-10; Fig. 1B, lanes 1-10). Immune complexes were eluted with SDS-PAGE sample buffer (Fig. 1A), Buffer A alone or (Fig. 1B, lanes 1-2), or Buffer A supplemented with increasing concentrations of the δ antigenic peptide corresponding to the amino acids 680 710 of human δ-adaptin (NCBI:AAD03777; gi:1923266)(Fig. 1B, lanes 3-10). This peptide binds the δ antibody. After peptide elution, supernatants (S) and beads (B) were analyzed by immunoblot (Fig. 1B). AP-3, which is detected with antibodies against the δ, β3, and σ3 subunits, coprecipitates with the following soluble factors: clathrin heavy chain (CHC), the BLOC-1 subunit pallidin, and the HOPS complex subunit vps33b; as well as the membrane proteins phosphatidylinositol-4-kinase type II alpha (PI4KIIα) and the zinc transporter 3 (ZnT3). Note the absence of IgG mouse heavy chains in the peptide eluted supernatant in Fig. 1B.
DSP, a membrane-permeable, chemically reducible crosslinker with a spacer arm of 12 Å is used to stabilize transient protein interactions 1,2,3,4. Here we exemplified this strategy with the adaptor complex AP-3 a soluble protein complex that recognizes and sorts membrane proteins into vesicles from endosomes 5. AP-3 selectively binds to the zinc transporter ZnT3 and the lipid kinase phosphatidylinositol-4-kinase type II alpha but not transferrin receptor 1,4,6. We expanded these observations to the identification of an AP-3 protein interaction network of membrane proteins and cytosolic factors by mass spectrometry 1. Low background immuno-magnetic precipitation follows crosslinking to identify interacting proteins. Moreover a published catalogue of proteins that bind non-selectively to magnetic beads allows for a first pass elimination of non-specific protein isolation and identification by mass spectrometry 7. DSP utilizes amine-reactive ester groups to link primary amines such as lysines or the amino acid terminus of proteins. Upon denaturing conditions, the DSP molecule is cleaved in half, leaving short chemical groups on the linked amino acids. For some antigens there is a decrease in immunoreactivity signals by immunoblot when comparing equal protein loads between DMSO control and DSP treated cells (Figure1A). It is possible that this decrease results from decreased antibody affinity at the DSP modified lysines. Aside from these rare cases, the use of DSP chemical crosslinking enhances the ability to detect closely interacting membrane-associated proteins.
Proteins or proteins complexes peripherally associated with the cytosolic leaflet of membranes, such as AP-3 are localized to membranes at normal incubation temperatures of 37°C. However, at room temperatures of 15-20°C the AP-3 complex slowly redistributes to the cytosol. Thus, in order to capture interactions between AP-3 and membrane-associated proteins, the internal temperature of these cells must be rapidly cooled to 4°C. The best way to accomplish this is to 1) have all buffers incubating in an ice bath prior to removing cells from the 37°C incubator, 2) to immediately remove all warm media from the plate of cells and replace with ice cold buffer, and 3) to keep the cells suspended on an ice bath for the entirety of the crosslinking experiment.
DSP is not soluble in water and thus warming the PBS/Ca/Mg solution to 37°C before addition of DSP/DMSO solution is advisable. However, since the cells need to maintain a temperature of 4°C, the DSP crosslinking solution should be placed in an ice bath once the DSP is completely solubilized. If the DSP is not completely solubilized large quantities of precipitate will form as the solution cools (a small amount of precipitation is normal). If a large amount of precipitation occurs, try heating the solution back to 37°C for complete solubilization and recool. If the DSP repeatedly falls out of solution, you may need to prepare fresh crosslinking solution. The rapid removal of DSP from solution due to incomplete solubilization should not be confused with the slow formation of a crystalline layer that appears in the wells of DSP treated cells. The presence of this DSP crystalline layer does not interfere with the cross-linking reaction in cells.
This work was supported by grants from the National Institutes of Health to V.F. (NS42599 and GM077569).
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
Phosphate Buffered Saline | Invitrogen | P4417 | Dissolve 1 tablet in 200 mL water; add MgCl2 to a final concentration of 1 mM and CaCl2 to a final concentration of 0.1 mM | |
Dithiobis (succinimidyl propionate) (DSP) | Thermo Scientific | 22585 | Moisture sensitive, store in air tight container 4°C | |
Dimethyl sulphoxide (DMSO) Hybri-Max | Sigma | D2650 | ||
Triton X-100, SigmaUltra | Sigma | T9284 | ||
Dynabeads, Sheep anti-Mouse IgG | Invitrogen | 110.31 | Beads are also available as sheep anti-rabbit | |
Dyna-Mag-2 magnet | Invitrogen | 123-21D |