What can you discover using in vivo crosslinking techniques?

The Ideal:

  • To "freeze" protein interactions in place and time so that they can be characterized temporally and spatially relative to treatment conditions.
  • To stabilize transient complexes and weak interactions for analysis by immunoprecipitation (IP), Co-IP, ChIP, EMSA, Western blot, IF, IHC and other methods.

The Myths:

  • Crosslinking experiments are difficult and not very reproducible.
  • Crosslinking creates too many aggregates and other artifacts that make data interpretation difficult and extremely unreliable.
  • Only a few traditional crosslinkers are compatible with in vivo experiments, and no new crosslinker innovations have been made in recent years.

The Facts:

  • With the appropriate controls, crosslinking experiments enable unambiguous identification of protein interactions when used together with a variety of techniques, including immunoprecipitation, Western blotting and mass spectrometry (MS).
  • Several classes and sizes of in vivo crosslinkers are available to target cell surface or intracellular proteins and to accommodate different downstream analysis methods.
  • New types of crosslinking reagents have been developed that complement and expand the range of information that can be acquired about protein interactions.

Popular crosslinkers for in vivo crosslinking analysis

Homobifunctional, amine-reactive, NHS-ester crosslinkers

The NHS-ester chemistry is widely used and referenced in the literature. NHS-esters reactions are fast and amine-specific. Because lysine residues are abundant and easily accessible of on the hydrophilic surface of most proteins, they usually crosslink with high efficiency. Using homobifunctional linkers is a "shotgun" approach to capturing interaction complexes; the reagents will crosslink any and all interacting molecules whose respective lysine residues come within the spacer length of the crosslinker. All that is needed to detect a particular interaction complex after crosslinking and (usually) cell lysis is a specific antibody or other probe for one of the target molecules in the complex.

  • Cell surface protein crosslinking
    • BS3 (= Sulfo-DSS), 11.4 Å spacer (Ref 8)
    • DTSSP, 12.0 Å spacer, cleavable
    • Sulfo-EGS, 16.1 Å spacer, cleavable
  • Intracellular protein crosslinking
    • DSG, 7.7 Å spacer (Ref 1, 4)
    • DSP, 12.0 Å spacer, cleavable (Ref 5, 7)
    • DSS, 11.4 Å spacer (Ref 4)
    • EGS, 16.1 Å spacer, cleavable (Ref 2, 6)

Comparison of several in vivo crosslinking methods.

HeLa cells treated with 1% Formaldehyde (HCHO) or 1mM homobifunctional NHS-ester crosslinker (Thermo Scientific DSG and DSS) in PBS for 10 minutes before quenching. A fourth set of HeLa cells were treated and crosslinked for 10 minutes with 4mM Photo-Leucine, 2mM Photo-Methionine (Photo-AA) according to the procedure (see subsequent section). Formaldehyde-treated and NHS-ester-treated cells were quenched with 100mM glycine pH 3 and 500mM Tris pH 8.0, respectively for an additional 15 minutes. One million cells from each condition were then lysed and 10µg of each sample was heated at 65°C for 10 minutes in reducing sample buffer containing 50mM DTT and analyzed by SDS-PAGE and Western blotting with Stat3 specific antibodies (Cell Signaling). Gapdh (Santa Cruz) and beta-actin (US Biologicals) were blotted as loading controls.

Heterobifunctional, NHS-ester/diazirine crosslinkers

The new diazirine photo-reactive linkers combine robust NHS-ester chemistry with a light-activatable reactive group that is able to crosslink with molecules where no amine residue is available or accessible (even DNA, polysaccharides and other molecules). Diazirines are more efficient than traditional photo-reactive moieties (aryl-azides) and give higher yields. These heterobifunctional linkers enable “two-step” reactions in which "bait" proteins can be labeled, added to a cell and light-activated to crosslink at the desired time (e.g., upon cell stimulation when the interaction of interested is presumed to occur).

  • Cell surface protein crosslinking
    • Sulfo-SDA, 3.9 Å spacer
    • Sulfo-LC-SDA, 12.5 Å spacer
    • Sulfo-SDAD, 13.5 Å spacer, cleavable
  • Intracellular protein crosslinking
    • SDA, 3.9 Å spacer
    • LC-SDA, 12.5 Å spacer
    • SDAD, 13.5 Å spacer, cleavable

Intracellular crosslinking of EEA1 protein complex using NHS-ester diazirine crosslinkers.

HeLa cells (2 x 10^6) were labeled for 10 minutes with 1mM SDA, Sulfo-SDA or SDAD in PBS. Non-reacted NHS-esters were quenched with Tris•HCl, pH 8.0 at a final concentration of 100mM for 5 minutes and then rinsed with PBS. NHS-diazirine labeled cells and a mock-treated control (-) were UV irradiated using a Stratalinker 2400 at 365nm for 15 minutes at a distance of 4cm in PBS. After UV treatment, cells were lysed with Thermo Scientific M-PER Mammalian Protein Extraction Reagent (Part No. 78501) and analyzed for total protein concentration using the Thermo Scientific Pierce BCA Protein Assay (Part No. 23225). Reducing sample buffer was added to 10 µg of each sample with the exception of a duplicate SDAD treated sample (-DTT) and separated by SDS-PAGE. Results were analyzed by Western blot using anti-EEA1 and GAPDH antibodies.

Photoreactive amino acids

The two Photo-amino acids Photo-Leucine and Photo-Methionine are used in metabolic labeling approaches as they are incorporated into proteins by the protein synthesis machinery (Ref 3, 4). They do not alter the cell’s metabolism and are essentially non-invasive. They are relatively easy to use (no need for artificial tRNA) and non-toxic. As zero-length linkers, they are very specific, do not result in large artificial aggregates and provide excellent recovery yields (see "Stat3" figure above). They are available for two hydrophobic amino acids that cannot be targeted by traditional chemical crosslinkers, making them an ideal complement and especially interesting for studies of hydrophobic interactions. They are equally well suited for cell-surface and intracellular proteins.


Protocol summary for in vivo labeling and crosslinking using photoreactive amino acids as crosslinkers.

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