The overall goal of the capture compound mass spectrometry experiment is to capture and identify cyclic die GMP binding proteins. This is achieved by fractionating, a cell culture by French press and removing the free cyclic die gmp. Using a PD 10 column as a second step, the capture compound is added and activated by UV light to covalently cross-link the cyclic die GMP binding proteins bound to the capture compound in either the absence or presence of an excess of free cyclic die GMP.
Next, the capture compound is bound to streptavidin coated magnetic beads in order to intensively wash the captured proteins. Finally, the captured proteins are digested and prepared for mass spectrometry to identify the generated peptides Results are then analyzed in silico. The main advantage of CCMS of our existing methods like co precipitation, is that the capture compound improves the specific capture of direct partners and allows harsh washing conditions thanks to the covalent cost linking.
Now this method can provide insights into bacterial cyclic di GMP signaling. It also can be applied to other systems such as nucleotide signaling in general and virtually any molecule fueled to the capture compound. Prior to starting this procedure, prepare and lyse p aerogen cells through a French pressure cells ultra ultracentrifuge the cell lysate at 100, 000 times G for one hour at four degrees Celsius.
After saving the supernatant, wash the pellet with one milliliter of one x lysis buffer by pipetting up and down following ultracentrifugation using the same conditions as before. Flash freeze the pellet in liquid nitrogen, then store the frozen sample at minus 20 degrees Celsius until used for the capture of membrane proteins. Following this wash a PD 10 desalting column with 10 milliliters of cold lysis buffer.
Pour the supra natant onto the PD 10 in order to remove nucleotides, then elute with four milliliters of cold lysis buffer in 500 microliter increments. Use the Bradford assay to determine the most concentrated fractions. Then pool the selected fractions in a 1.5 milliliter tube Resus.
Suspend the previously stored pellet in 500 to 1000 microliters of one x capture buffer without detergent. Add a 1%weight per volume solution of end doil Beta D multi purin aside to the sample. Next, incubate the sample at four degrees Celsius for at least two hours on a rotating wheel.
When finished, ultracentrifuge the sample at 100, 000 times G for one hour at four degrees Celsius. Following ultracentrifugation, transfer the supernatant to a new 1.5 milliliter tube. At this point, mix 300 micrograms of protein with one millimolar of G-D-P-G-T-P-A-T-P-C-T-P, and 20 microliters of five x capture buffer.
Adjust the reaction volume to 100 microliters with water. After incubating the sample at four degrees Celsius on a rotating wheel, add 10 micromolar of cyclic die GMP capture compound. Then incubate the sample at four degrees Celsius for at least two hours for the soluble fraction or overnight for the membrane fraction on a rotating wheel in the dark.
After a short spin and lid removal cross-link by activation of the reactive moiety of the cyclic die GMP capture compound with UV light for four minutes using a cap row box. Following this add 25 microliters of five x wash buffer and 50 microliters of well resuspended strep AVID in magnetic beads. Gently homogenize the sample.
Then incubate the sample at four degrees Celsius for one hour on a rotating wheel. After capturing the beads in the lid with a magnet, wash the soluble fractions six times in 200 microliters of one X washing buffer after washing once in 200 microliters of HPLC grade water wash six times in 200 microliters of 80%aceto nitrile, and two times in 200 microliters of HPLC grade water for the membrane fraction wash five times in 200 microliters of one X washing buffer and decreasing concentrations of end doil beta D multi puric aside. Next, resuspend the beads in 20 microliters of 100 millimolar ammonium bicarbonate for the soluble fraction and 100 millimolar ammonium bicarbonate plus eight molar urea.
For the membrane fraction, transfer each suspension into 1.5 milliliter tubes. After incubating the membrane fraction, add 0.5 microliters of 200 millimolar tris. Two carboxy ethyl phosphine to both fractions and incubate at 60 degrees Celsius for one hour with shaking at 500 RPM.
Once the samples have cooled to 25 degrees Celsius, add 0.5 microliters of freshly prepared 400 millimolar I oto acetamide following incubation in the dark. Add 0.5 microliters of 0.5 molar and acetylcysteine to each fraction. Then incubate the samples at 25 degrees Celsius for 10 minutes with shaking at 500 RPM.
At this point, add one microliter of LY C to the membrane fraction following overnight incubation at 37 degrees Celsius. Add two micrograms of trypsin to each sample and wrap them in paraform to prevent drying. After incubating and spinning down the samples, transfer the supernatant into a new 1.5 milliliter tube with the help of the magnet to avoid collecting the beads.
Next, add five microliters of 5%trichloroacetic acid and one microliter of two molar hydrochloric acid to the soluble fraction, add 15 microliters of 5%trichloroacetic acid and five microliters of two molar hydrochloric acid to the membrane fraction condition. C 18 micro spin columns with 150 microliters of aceto nitrile by spinning 20 seconds at 2, 400 RPM. Once the columns have been equilibrated twice with 150 microliters of 0.1%tri fluoro acetic acid, load the sample and spin for two minutes.
At 2000 RPM, reload the flow through onto the columns. After repeating the spinning and washing the columns with 0.1 tri fluoro acetic acid and aceto nitrile, take a new tube and elute twice with 150 microliters of 0.1%tri fluoro acetic acid, and 50%aceto nitrile. Once the peptides have been dried in a speed vac Resus resuspend them in 40 microliters of 98%water, 2%acetyl nitrile and 0.15%formic acid.
Once the samples have been sonicated for 20 seconds, spin them down for five seconds at 12, 000 RPM After vortexing and spinning, transfer the samples to HPLC vials for lc MS MS analysis. The initial hit list from a P aerogen log phase culture comprises 768 soluble fraction proteins and 433 membrane fraction proteins After removing non-specifically captured proteins, the list was decreased to 76 soluble fracture proteins and 133 membrane fraction proteins. This included 13 soluble and 21 membrane proteins from P aerogen that are known or predicted to bind cyclic dye.GMP.
The other 63 soluble and 112 membrane proteins are new putative cyclic die. GMP binding proteins, which do not contain one of the known cyclic die GMP binding domains. CCMS with cell extracts harvested from different growth conditions and with various intracellular cyclic die.
GMP concentrations were also used. Overall, 74%of the known or predicted p aerogen, PO one cyclic die, GMP signaling components were captured. Given that at least nine of these genes were shown to be transcribed under specific conditions and that some may not bind cyclic die, GMP at all, this degree of coverage might be close to saturation.
This coupled with the observation that most of these components were captured with high specificity, strongly argues that this technique is effective and powerful. While attempting this procedure, it's important to remember that the selection of potent candidates will be achieved by comparing the capture specificity of known psychic DGP Effectors. So following this procedure, methods like Drac, ubi, cross linking, differential scanning, fluorometry micros or isothermal telemetry can be performed in order to validate a specific binding of the candidates to cycling that GMP.
