Characterization of Proteins by Size-Exclusion Chromatography Coupled to Multi-Angle Light Scattering (SEC-MALS)

SEC-MALS is a quantitative method for determining the molecular weight, size and conformation of macromolecules in solution. It can identify oligomers and complexes, and characterize difficult samples, like glycoproteins and membrane proteins. SEC-MALS is an absolute method.

It relies on fundamental physics, and does not depend on collaboration against molecular standards, the molecule's conformation, or how we interact with the separation medium. SEC-MALS can be applied to many systems that are separated by sec. From peptides and small polymers to proteins, nucleocapsids, polysaccharides, synthetic polymers, virus-like particles, and small viruses.

It is important to verify that your system and buffers miss the vendor's recommendations for low particulate content. Consult with the vendor's technical support representative to be sure. Begin this procedure with preparation of the SEC-MALS system as described in the text protocol.

Using each PLC-grade reagents, prepare one liter of phosphate-buffered saline with 50 to 100 millimolar sodium chloride. Filter the buffer to 0.1 micron using a bottled up polyether cell foam filter, or similar. Filter the first 50 to 100 milliliters of buffer to a waste bottle in order to eliminate the particulates from the dry filters.

Then, filter the remainder to a clean, sterile bottle that has been previously washed with filtered, deionized water and kept to prevent dust from entering. Flush the column overnight at a flow rate of 0.5 milliliters per minute to equilibrate the column in the buffer and to remove particulates. Use the FPLC's continuous flow mode and ensure that the flow does not stop until all SEC-MALS runs are complete.

Place the DRI flow cell in purge mode during the overnight flush. When beginning the flush, gradually ramp the flow rate to prevent the column shedding effect, caused by a sudden change of pressure in the column. Turn the purge off before beginning sample runs.

Check system cleanliness by lightly tapping the tubing downstream of the column to release accumulated particles. Observe the signal in the 90 degree detector on the front panel display of the MALS instrument. Verify that the peak-to-peak noise is no more than 50-100 microvolts.

Also verify that the refractive index, or RI signal, is stable to less than 1 times ten to the minus seven refractive index units. Perform a blank injection to verify that the injector is clean of particles. A blank is simply the running buffer prepared in a fresh, sterile vial.

If the particle peak is no more than one milliliter in volume, and no more than five millivolts above baseline, then the system is ready for samples. Otherwise, perform additional blank injections until clean, or perform maintenance to clean the injector. Now, prepare at least 200 microliters of BSA at one to two milligrams per milliliter in the SCC buffer.

Filter the protein to 02 microns, using a syringe tip filter. Discard the first few drops of filtrate, in order to eliminate particles from the dry filters. Alternatively, centrifuge the sample at 10, 000 times G for fifteen minutes to enable precipitation of non-soluble aggregates and other large particles.

Then, inject 100 microliters of the BSA solution into the loop. Open new experiment from method in the MALS software menu. Then, select the online method from the light scattering system methods folder.

If a DLS detector is present, select the online method from the light scattering with QELS folder. Set parameters of the sample and mobile phase in the configuration section. In the generic pump view, set the flow rate to that used in the FPLC.

Then, navigate to the generic pump view, solvent branch, name field, and select PBS. In the injector view, sample branch, enter the name as BSA. Set dn/dc to 0.185, set A2 to 0, and set the UV Extinction Coefficient to 0.667 milliliters per milligram centimeter.

In the procedures section, basic collection view, select the checkbox trigger on auto-inject. Set the duration of the run to 70 minutes, so that data are collected for the entire allusion, until the total permeation volume of the SEC column is reached. Start the experiment in the MALS software by clicking the run button.

It will start reading the data after receiving the pulse signal from the FPLC instrument via the MALS detector. Zero the DRI signal by clicking the auto-zero button on the instrument's front panel. Working in the FPLC software, navigate to manual, execute manual instructions, set mark, and insert the name of the protein and the run.

Switch the injection valve from manual load to inject, under flow path, injection valve. Include a pulse signal by inserting a 0.5 second pulse under IO box pulse digital out. This will trigger data collection in the MALS software.

Now, inject the sample into the loop. Click execute in the FPLC software to start the experiment run. Perform analysis step-by-step, under the procedures section in the MALS software.

Verify that peaks appear at approximately the same allusion volume in UV, MALS, and RI by checking the basic collection view. In the baseline view, define the baseline for all signals. In the peaks view, define the peaks to be analyzed by clicking and dragging the mouse.

Select the central 50%of each peak. First select the monomer peak, and then the dimer peak. Under each peak, verify the correct values of dn/dc and extinction coefficient at 280 nanometers for BSA.

In the procedures, alignment view, select the central region of the peaks by clicking and dragging the mouse. Click align signals, and then okay. In the procedures, band broadening view, choose the central 50%of the monomer peak.

Make sure the refractive index detector is specified as the reference instrument. Then click perform fit, and apply to match the UV and light scattering signals to the refractive index signal. Zoom into the peaks to verify that they overlap very closely within the central 50 to 70%and then click okay.

In the procedures, normalization view, select peak one. Enter 3.0 nanometers as the RG value, click normalize, and then click okay. View the graph of the results in the EASI graph view.

Select molar mass from the display drop down at the top of the window. Use control, click, and drag to zoom into the peak region. To view the final tabulated weight average molar mass results for the monomer and dimer peaks, navigate to peak results, molar mass moments, MW, and select to the results, report summary.

Purity is reported under peak results, mass fraction. From the file menu, select save as method, and save the analyzed BSA data as a standard method for future measurements of all types of proteins. The normalization and band broadening parameters determined for BSA will be carried over in the analysis.

Shown here are SEC-MALS analyses of BSA, using a 200 Angstrom pore size exclusion column. Chromatogram traces are normalized to the monomer peak, and offset for clarity. Common artifacts that may be ignored are pointed out, including a particle peak near the beginning of the light scattering signal, as well as salt and dissolved air peaks near the total permeation volume in the refractive index signal.

The chromatogram exhibits excellent monomer-dimer-trimer separation, and the light scattering signal exhibits high signal to noise. The monomer and dimer weight average molar mass values exhibit high homogeneity. Here are examples of low quality SEC-MALS analyses.

In this example, inadequate separation on a 75 Angstrom pore size exclusion column results in a particle peak between eight to nine minutes, that is not well separated from the proteins. Here, there is an inadequate signal to noise ratio, and extensive particles adjacent to the proteins are apparent in the light scattering signal. The keys to good SEC-MALS results are selecting an appropriate column, ensuring that the system is calibrated with low light scattering noise, and pre-filtering or centrifuging the samples to remove particles.

Following SEC-MALS, protein samples can be further analyzed for binding affinity, bioliza, surface plasmon resonance, isothermal titration calorimetry, biolerid phorometry, microscale thermophoresis, or composition gradient, multi-angle light scattering. The protein structure may be determined by x-ray crystallography, cryo-electron miscroscopy, or NMR.