This method provides solutions for today's demanding biological questions. From an in-cell FPOP experiment, we can study protein-ligand interactions, protein-protein interaction sites, and regions of conformational change. The advantage of this technology is the use of an automated six-well plate-based IC-FPOP platform that makes it possible to grow cells and perform in-cell FPOP studies right at the laser.
This method can be used in pharmaceutical and biotech industries, as well as major academic and clinical research institutes, especially by biological investigators in universities studying protein dynamics. To begin, unscrew the platform incubator from the positioning stage and disconnect the temperature, gas, and humidifier lines. Spray the incubator with 70%ethanol and place it in the cell culture hood.
Remove the six-well plate from the platform incubator, secure the plate's original lid, and confirm cell confluency using a microscope, then place the six-well plate with confluent cells back in the platform incubator inside the cell culture hood. Insert three pre-cut Tygon tubes in each well via the embedded ports by flushing the tubes to the well walls and secure them with custom 3D printed rings. Prepare an integration software script for pump withdrawal.
Use one peristaltic pump to completely remove cell media from all six wells. Prime solvents in each channel of the other three peristaltic pumps, then infuse hydrogen peroxide and Quench buffer in their respective alternating tubes until the reagents reach the incubator ports. Confirm that the laser beam has been angled correctly and reaches the incubator uninhibited.
Check laser energy using an external sensor. Prepare the integration software script for pump infusion after confirming beam alignment. Infuse 200 millimolar hydrogen peroxide at 35 milliliters per minute into the first well.
Press the start button on the laser software at the five-second mark to trigger the pulse at the 11-second mark on the timer. Infuse 125 millimolar Quench solution at 35 milliliters per minute into the first well immediately after the laser pulse. Manually move the positioning stage to align the next well with the laser beam and repeat the process for all the wells.
In a cell culture hood, use a cell scraper to transfer the cells from each well into individual 15 milliliter conical tubes. Centrifuge cells at 1, 200 times G for five minutes. Discard the supernatant and resuspend the cells in 100 microliters of RIPA lysis buffer.
Transfer cells to individual 1.2 milliliter polypropylene tubes. Flash freeze all samples in liquid nitrogen and place them in a minus 80 degree freezer until use. To localize FPOP modifications, analyze the digested cell lysate using liquid chromatography tandem mass spectrometry analysis, then calculate the extent of modification.
To confirm that the platform incubator conditions are sufficient for cell culture at the laser platform, GCaMP2 was transiently transfected into HEK293T and transfection efficiency for both plates was assessed via fluorescence imaging. A luciferase assay was performed to quantitate transfection efficiency. FPOP modifications in HEK293T cells labeled in the flow system were compared to those labeled in the platform incubator.
The platform incubator outperformed the flow system both in the number of proteins modified and the total FPOP coverage in those proteins. In the flow system, two modified peptides were detected, providing limited structural information. However, five modified peptides spanning the actin sequence were detected in the platform incubator.
Tandem MS spectra of actin with modified proline in both systems and unmodified actin peptide are shown here. The FPOP modified residues in the platform incubator samples contained 12 modified residues, three modified residues in the flow system, and one overlapping modified residue. LC-MS MS analysis revealed that 792 proteins were modified by in vivo FPOP in the platform incubator compared to the 545 proteins modified with the flow system.
It is imperative to keep cells under sterile conditions. Always bring the platform incubator into the sterile cell culture hood to insert all necessary six-well plates, tubings, and rings. This ensures the integrity of the experiment.
