The overall goal of this procedure is to enrich low abundance cell surface membrane proteins. This is accomplished by first denaturing and digesting membrane proteins. The second step is to oxidize the glycans and capture the glycosylated peptides on a resin surface.
The non glycopeptides are then washed off the resin. The final step is release of the N-Linked Glycopeptides off the resin for lc MS analysis. Ultimately, the identified peptide sequence can be mapped back to proteins for identification and quantification purposes.
Today I'm going to demonstrate you how the Geico peptide capture ral works. The main advantage of this technique overdose, ex existent ones, such as glycoprotein capture, is that the membrane protein will first be digested and the capture of the glycopeptide can be performed in one vessel. The application of this technique can extend to the discovery of the disease biomarker.
Since gator protein can be shed in the blood and other body fluid and the circulating glycoprotein in blood, which are specific to the pathological process, can be used as prognosis and diagnosis Biomarkers. First, prepare four milliliters of hypotonic buffer with one to 100 protease inhibitor cocktail. Add one milliliter of hypotonic buffer onto a cell pellet containing approximately 10 to the eighth cells, and incubate the mixture for 15 to 30 minutes On ice, transfer the cells into a two milliliter vial and lice the cells by passing the sample five to 10 times through a syringe needle.
Then use a hemo cytometer and trian blue staining. To check the efficiency of lysis, combine the one milliliter lysate with three milliliters of leftover hypotonic buffer. Spin the tube at 3000 times G at four degrees Celsius for 15 minutes.
Transfer the super supernatant to an ultracentrifuge tube and store the pellet in a negative 80 degrees Celsius freezer. Spin the ultracentrifuge tube at 100, 000 times G at four degrees Celsius for two hours. Next, dissolve the microsomal fraction in 200 microliters of denaturation buffer, and then boil the solution at 100 degrees Celsius for 10 minutes.
After cooling the heated solution to room temperature, add ultra high purity urea powder to give an eight molar final concentration and incubate the sample at 37 degrees Celsius for 30 minutes. After incubation, add 500 millimolar. I oto Acetamide stock solution to the sample to give a final concentration of 15 millimolar.
Then incubate the solution in the dark for 30 minutes at room temperature to alkylate the free thiols in the sample. Following this, add one molar DTT stock solution to the sample to give a final concentration of 10 millimolar and incubate the solution for another 10 minutes at room temperature. To quench the excessive I oto Acetamide dilute the obtained solution 10 times with 40 millimolar tris buffer at PH eight, and subsequently add trypsin to the sample at a one to 20 ratio of trypsin to total protein quantity.
Obtain the quantity of total protein using the Bradford assay. Maintain the digestion reaction in a 37 degrees Celsius oven overnight to ensure the reaction is completed. When finished, terminate the digestion by acidifying the sample solution to pH one with 10 millimolar hydrochloric acid, a condition that also degrades the rapidest.
The detergent then degrade the rapidest at 37 degrees Celsius for one hour in an incubator. After incubation, remove the developed precipitation by centrifugation following centrifugation. Clean the SUP natin that contains the sample peptides by a C 18 solid phase extraction cartridge and dry the obtained sample by speed vac.
Perform an SDS page analysis of samples before and after trips and digestion to confirm the digestion efficiency. At this point, dissolve the cleaned peptides in 200 microliters of coupling buffer. After adding sodium puriate to the peptide solution to give a 10 millimolar final concentration.
Incubate the sample for 30 minutes in the dark at room temperature When the incubation is complete, quench the excessive prate with sodium sulfite to give a final concentration of 20 millimolar and pH of five following incubation for 10 minutes at room temperature, introduce 200 microliters of hydrocyte derivative resin solution, pree equilibrated and coupling buffer into the peptide solution. Incubate the reaction at 37 degrees Celsius for one to two days with end-to-end rotation for complete coupling. When finished, remove the unbound peptides by washing the resin twice with one milliliter of deionized water, one milliliter of 1.5 molar sodium chloride, one milliliter of methanol, and one milliliter of 80%aceto nitrile.
Next, collect the supernatant and the washes for the analysis of unbound peptides. Store the samples in a minus 80 degrees Celsius freezer until analysis. Then wash the resin three times with one milliliter of 100 millimolar ammonium bicarbonate at pH eight.
Finally, add 300 microliters of ammonium bicarbonate into the sample vial. To release the N glycopeptides from the resin. Add PNG ACE F and incubate the sample at 37 degrees Celsius with an end-to-end rotation.
Following this, collect the released peptides by centrifugation, then wash the resin with one milliliter of 80%acetyl nitrile. Combine the wash with the previously collected. Finally dry the obtained solution in the speed VAC for lc MS analysis.
Atypical glycopeptide spectrum taken after the enrichment method is shown here in the obtained glycopeptide. The n glycan was removed and the glycan attached disparaging was converted to an spartic acid by P-N-G-A-F. Therefore, the spectrum can be readily searched by any proteomic search engine against common protein sequence databases.
Atypical lc MS.Result of the captured n glycopeptides is shown here in which more than 100 glycoproteins can be identified from a single lc MS run of a cell microsomal fraction. The enrichment selectivity to both glycoproteins and glycopeptides is generally more than 90%A successful analysis can have an enrichment selectivity of 95%using an SCX column and step gradient to further fractionate. The samples prior to LCMS analysis will usually double the number of glycoprotein identifications.
Sometimes when the quantity of the obtained glycopeptides is low, the impurity accumulated from the vials can be observed in the final sample. These contaminants can be removed by an MCX column. So after watching this video, I hope you have a good understanding of how to use our glycopeptide capture methods to study self-service membrane protein.
While you're attempting this protocol, it is also important to set aside samples for each step for troubleshooting. The volume of the reagent can also be optimized for your specific study.
