Analysis of Protein Complex Formation at Micromolar Concentrations by Coupling Microfluidics with Mass Photometry

This protocol demonstrates a completely new way to analyze low affinity protein complexes. It allows researchers to detect and characterize certain protein interactions even when they are weak or transient, which has generally been very difficult or even impossible before. Protein interactions are crucial for cell function, but detecting and measuring them can be challenging.

Mass photometry is a great tool for quantifying protein complex formation, but it has limitations with dilute samples. We're solving this problem with mass fluidics. The research gap we are addressing is how scientists can reliably characterize low affinity interactions between proteins in solution.

The advantages of our approach are that it allows you to look for weak protein interactions in solution without the need for labels or immobilization. So researchers can observe their proteins of interest and how they interact without worrying that they're disrupting the system they're looking at. To begin, turn on the mass photometer and allow it to operate for one hour before initiating any measurements.

Switch on the power and engage the isolation button to activate the anti-vibration table. Subsequently, turn on the air compressor to activate the microfluidics box. Then initiate the data acquisition software followed by the microfluidics control software.

To load the sample and calibrant, position the calibrant centrifuge tube at slot number four and the sample centrifuge tube at slot number five within the microfluidics box. Place cleaning solutions, CS2, CS2, and CS3 at positions one, two, and three respectively in the microfluidics box. Attach the cap that connects the buffer line to the 200 milliliter buffer bottle of pH 7.4.

Then set the microfluidics chip on a preparation plate. Connect the other tube end of the S-flow unit sensor to the sample inlet of the first channel on the microfluidics chip, completing the sample line. Insert the other end of the L-flow unit sensor into the buffer inlet of the first channel on the microfluidics chip, finalizing the buffer line.

For outflow collection, attach a tube to the outlet of the first channel on the microfluidics chip and position the other end into a waste receptacle flask or beaker. Apply a small amount of microscope immersion oil onto the objective lens of the mass photometer. Position the microfluidics chip on the mass photometer's holder ensuring the sample inlet faces upwards, and secure it using the stage clamps.

Use the data acquisition software to adjust the stage, aligning the observation area of channel one with the photometer's objective. Then close the lid of the mass photometer and activate the Droplet-dilution find focus feature in the data acquisition software. Inspect the white focus ring in the bottom left corner of the software interface.

If gaps are present indicating air bubbles in the immersion oil, increase the stage speed to the maximum and gently shift the stage side to side to eliminate them. Press the record button to begin a one-minute measurement ensuring no impurities are present during the measurement. Remove the cap from the 200 milliliter buffer bottle with pH 7.4 and attach it to the 200 milliliter buffer bottle with pH 5.0.

Place the microfluidics chip onto a preparation plate as demonstrated earlier, and use the second channel on the microfluidics chip instead of the first for outflow collection. In the microfluidics control software, change the M-switch to position five, set the sample line flow rate to eight microliters per minute, and ensure the pressure in the sample line remains below 350 millibars. When calculating flow rates, match the full difference in flow rate to the desired sample dilution factor.

After concluding the experiment, follow the cleaning protocol to maintain the cleanliness of the lines. To begin, position the microfluidics chip on the mass photometer's holder and measure the mass photometry sample. After completing data acquisition, open the data analysis software.

To load a file for analysis, click on the plus icon on the top left and select the mp calibrant file. The software will start analyzing the file, and the time taken may vary based on file size and measurement count. To calibrate mass, press the Create Mass Calibration button located on the bottom right.

A dialogue box displaying a table with a contrast values of the fitted peaks will appear. Modify the table values to match the known mass values for the fitted peaks, then click Save. The new calibration file will be displayed in the mass calibration panel at the bottom left of the data analysis software.

To add a sample file, click the plus icon on the top left and select the mp sample file. For creating a mass histogram, navigate to the Analysis tab, select Histogram Mode, and choose the Mass Plot option. Adjust the bin width, mass limits, and other necessary parameters as required.

Further customize the plot in the Figures tab as desired. After customization, export figures or save the entire workspace as a DMP file. The mass photometry measurements with manual dilution resulted in a mass histogram where the two largest peaks corresponded to unbound FcRn monomers and immunoglobulin G antibody monomers.

Also, two additional peaks were clearly observed at 196 and 251 kilodaltons corresponding to immunoglobulin G FcRn complexes with one to one and one to two stoichiometries.