Combining Chemical Cross-linking and Mass Spectrometry of Intact Protein Complexes to Study the Architecture of Multi-subunit Protein Assemblies

The overall goal of combining cross-linking with native mass spectrometry is to obtain complementary structural information on multi-subunit protein assemblies which helps elucidate their three-dimensional arrangement. Chemical cross-linking and native mass spectrometry help to answer key questions in structural biology such as composition, connectivity and stoichiometry. The main advantage of combining several techniques is that complementary information is obtained which is not available with only one technique.

Our workflow can be applied to any other protein complex. It can be purified from salts or tissue or that can be reconstituted from purified proteins. First, apply 7.5 microliters of four-fold simple buffer and three microliters of 10-fold reducing agent to the desired protein complex.

Spin down the sample for one minute at 16, 200 times the gravity. After centrifugation, heat the sample for 10 minutes at 70 degrees Celsius. Prepare a four to 12%gradient gel for electrophoresis and place the gel into the electrophoresis chamber.

Dilute previously prepared running buffer 20 times with water and fill the electrophoresis chamber. Then, add 0.5 milliliters of antioxygen to the inner chamber. Load a suitable protein marker in the first cavity of the gel, and the protein samples into the remaining cavities.

Then, separate the proteins for the appropriate amount of time at 200 volts. To stain the protein bands, transfer the gel into a gel-staining box and cover the gel with a water-base Coomassie standing solution. Incubate the gel overnight at room temperature on a horizontal gel shaker.

On the following day, de-stain the gel by replacing the staining solution with water. Repeat the previous step approximately three to five times until the gel background appears clear. Now, cut the protein bands that are visualized by blue Coomassie stain from the gels using a scalpel.

Carefully cut the protein bands into small pieces of approximately one-by-one milliliter. Then, wash the protein bands with water and acetonitrile. Reduce the disulfide bonds with DTT, and then alkylate the cysteine residues with iodoacetamide.

Following this, digest the proteins with trypsin as described in the text protocol. To extract the peptides, incubate the gel pieces with ammonium bicarbonate and acetonitrile and collect the peptide-containing supernatant. Following this, incubate the gel pieces with 5%formic acid and acetonitrile and collect the peptide-containing supernatant.

After collecting the peptide-containing supernatant, combine both supernatants and dry the extracted peptides by evaporating the solvents in a vacuum centrifuge. Once the peptides are dry, mix them with 20 microliters of 2%acetonitrile and 0.1%formic acid solution. Dissolve the peptides with a sonication bath for two to three minutes.

Then, spin down the sample in a centrifuge at 16, 200 times the gravity for 30 minutes. After transferring the sample into a autosampler vial, inject five microliters into a nano LC-MS/MS system and load the peptide mixture onto a reversed-phase C18 pre-column to desalt and concentrate the peptides online. Remove the storage buffer from a size-exclusion spin column by centrifugation at 1, 000 times gravity and four degrees Celsius for one minute.

After discarding the flow-through, wash the column three times by adding 500 microliters of 200 millimolar ammonium acetate followed by centrifugation. Following centrifugation, load 20 microliters of the protein sample onto the column and centrifuge at 1, 000 times the gravity and four degrees Celsius for four minutes. Now, place a previously prepared gold-coated capillary in a capillary holder.

Open the tip of a needle and fill the capillary with one to four microliters of protein sample. Connect the capillary holder with the nano electrospray source of the mass spectrometer and position the tip of the capillary at 0.5 to 1.5 centimeters to the cone orifice. Use 80 to 150 liters per hour nanoflow gas to initiate the spray and adjust the gas flow to maintain a stable spray.

Adjust the parameters and the tune page of the Q-TOF instrument. Then, start the acquisition by clicking the Acquire button and combine as many scans as possible to obtain a good mass spectrum. Dissolve 1.43 milligrams of BS3 and 100 microliters of water to prepare a 25-millimolar stock solution.

Next, add BS3 to the protein complex. Use varying amounts of BS3 to identify the optimal cross-linker concentration. Incubate the reaction mixtures at 25 degrees Celsius for one hour in a thermomixer.

Now, perform SDS page of the cross-linked proteins with a four to 12%gradient gel. Cut the gel bands and digest the protein by in-gel digestion as previously described. After obtaining the peptides from in-gel digestion, perform liquid chromatography coupled mass spectrometry as previously described.

All protein subunits of the heterododecamer RvB1/B2 and heterooctameric CPS complexes were identified with high confidence. The mass spectrum of RvB1/RvB2 reveals two species that correspond to the hexameric and dodecameric double rings. A crystal structure for the RvB1/B2 complex shows the arrangement of the double ring.

Addition of BS3 to the RvB1/RvB2 complex cause covalent linkage of the proteins resulting in protein bands at a higher molecular weight. And increasing amounts of BS3 yield higher amounts of cross-linked species, while non-cross-linked protein subunits are reduced. The cross-linked dipeptide spectrum shows Y ion series of both peptides confirming this protein interaction.

The results from BS3 cross-linking are visualized in an interaction network showing intramolecular interactions as well as interactions between different subunits. The native mass spectrum of CPS shows the heterodimer, heterotetramer and heterooctamer. Tandem mass spectrometry of the tetramic and octameric CPS reveals dissociation of the small CPS subunit.

Chemical cross-linking of CPS with BS3 cross-linker shows several intramolecular cross-links between two copies of the large CPS subunit. The crystal structure reveals that the interaction surface between the tetramic core and the peripheral small subunits is very small which might explain the absence of intersubunit interactions. Once mastered, our workflow can be done one week if performed properly.

As the protein assembly's too complex, additional time is required for data analysis. Following this procedure, computational modeling can subsequently be performed to obtain three-dimensional models of the protein complexes. After watching this video, you should have a good understanding of how to perform chemical cross-linking and native mass spectrometry experiments until these two complementary techniques help elucidate the structures of protein assemblies.