The central goal for the development of the csBN-MS technique was getting comprehensive access to the organization and assembly of protein complexes that underlie signal transduction at and across the plasma membrane of various types of tissue and organs. csBN-MS currently provides the highest resolution versatility for analysis of native protein complex and their subunit composition, in particular with respect to membrane proteins. Although the csBN-MS technique was developed to analyze complexed mixtures of membrane protein assemblies in rodent brain it can easily be adapted to the analysis of any type of biological sample.
One of the key steps of our method is the embedding of the gel piece and its correct alignment to the cutting plane before slicing. Be careful not to tear or to compress the gel and be sure that it is not tilted to the cutting plane since this would reduce the resolution of the analysis. Our csBN-MS technique has several practical steps that are crucial for good results.
It is easier to show how to perform those steps in detail than by just describing them. To begin this procedure use a stirring two-chamber gradient mixer driven by a pump to cast a linear or a hyperbolic pore gradient gel. Prepare solutions for the front mixing chamber and reservoir chamber as outlined in the text protocol.
Start the stirrer and add 30 microliters of APS and 2.5 microliters of TEMED to the solution in the front chamber. Then, start the pump and open the front valve. After approximately one minute, add 90 microliters of APS and five microliters of TEMED to the reservoir chamber and open the chamber connection.
Allow the gel to polymerize slowly and thoroughly for at least 24 hours at room temperature to generate a homogenous pore size gradient. If kept moist the polymerized gel can be stored upright at four degrees Celsius for up to one week. Next, prepare the loading slots by inserting the appropriate spaces between the glass plates for separating between 0.5 and 2.0 milligrams of protein.
The slots should be at least three centimeters wide. For running buffers, prepare a standing cathode buffer consisting of 50 millimolar tricine, 50 millimolar Bis-Tris, and 0.01%Coomassie G-250. Prepare a standard anode buffer consisting of 50 millimolar Bis-Tris.
Solubilize approximately 2.5 milligrams of membrane and two milliliters of solubilization buffer containing 1%non-denaturing detergent on ice for 30 minutes. Then ultracentrifuge at 130, 000 times G for 11 minutes. Concentrate the solubilisate on a short 50%20%sucrose step gradient by ultracentrifugation at 400, 000 times G for one hour.
After this, harvest the sucrose gradient from the bottom of the tube, add 0.05%Coomassie G-250 to the solubilisate, and load the sample on the gel. Run a preparative BN-PAGE at 10 degrees Celsius overnight using a three-step voltage protocol, as outlined in the text protocol. After the gel has run scan the gels for documentation purposes while keeping it between the glass plates.
Inspect the quality of the gel separation. Next, dissemble the plates and excise the lane sections of interest. Fix the lanes twice with 30%ethanol and 15%acetic acid for at least 30 minutes.
Transfer the sample of embedding medium and allow it to soak and equilibrate for at least two hours while keeping the gel slab in slow motion on an orbital shaker. Then, cut the fixed gel lanes into sections exactly parallel to the protein migration front, or band pattern. Place each section on a plastic film support with equal dimensions for easier handling.
Transfer the lanes into an open tube with stoppers that are closed on the bottom and centrally perforated on the top, both precisely aligned with the upper and lower ends of the gel section. Dip the cylinder into the liquid nitrogen briefly to rapidly initiate the solidification. The transparent embedding medium solidifies within seconds and becomes white in color.
Fill the cavity with embedding medium, briefly dip the cylinder into liquid nitrogen, and then let it freeze thoroughly at minus 20 degrees Celsius for several hours. After disassembly, remove the plastic film and transfer the block with the embedded gel section to a cooled metal cylinder. The cylinder is larger in diameter and sealed on the outside with embedding medium.
It is placed on a flat support. Fill the cylinder with embedding medium and freeze thoroughly as mentioned before. Repeat this procedure with the other side of the cylinder to obtain a solid block with a coplanar bottom surface.
Then, remove the block from the cylinder and use embedding medium to glue it to a pre-cooled metal holder. Insert the metal holder into the cryoslicing machine. The holder has to be carefully aligned with respect to the slicing plane.
Allow the block to equilibrate to the optimum temperature for the slicing process. After this harvest the gel slices one after another with a final desired thickness of 0.25 millimeter step size and transfer them individually to reaction tubes with low protein binding properties. Then, the slices are subjected to tryptic digest and mass spectrometric analysis.
This protocol extends the application of high resolution complexome profiling to non-mitochondrial membranes expressing low-abundant proteins. Complex separation shows strongly stained protein bands with very little migration artifacts. Deviations of peptide signals in mass and retention time indicate a very low rate for false positive peak volume assignment.
Run-to-run variations are easily eliminated by rescaling of the peak volume data sets. The resulting peptide intensity information is then used to reconstruct 2545 protein profiles of relative abundance. The relevance of gel sampling step size for the resolution of protein complexes was assessed by joining datasets of adjacent slices.
At 0.25 millimeters, size separation of TPC1 associated complex populations is nicely in agreement with the results from western blot analysis. Joining of more than two slices abolishes discrimination of TPC1 complex subpopulations. Finally, the analysis provides information on well-characterized complexes and demonstrates the existence of novel subunits and complex assemblies.
For ferritin, the subunit profiles adjusted for their relative abundance suggest the existence of at least three complex isoforms with distinct heavy/light chain stoichiometries. In contrast, nicalin-nomo1 complexes, the gamma-secretase core complex, and the GPI-transamidase machinery show fixed abundance ratios of their core subunits over the entire size range and independent from association with additional proteins. The key parameter of csBN-MS analysis is the effective resolution of complexes, which is determined by sample biochemistry, BN gel quality, proper alignment during embedding and slicing, and quality of the acquired MS data.
Combing csBN-MS with isotope labeling of proteins and samples will allow for direct comparison of complexomes at different pathophysiological, biological, or developmental states. csBN-MS performed on membranes from the rodent brain revealed the enormous diversity of receptors, ion channels, and transporters with respect to their subunit composition and their function, and it will be instrumental for analyzing the 3D structure in native preparations.
