The overall goal of this protocol is to analyze the extracellular matrix protein composition including the identification of glycosylation sites and compositional characterization of glycan forms in cardiovascular tissues. This method can help answer key questions in the process of tissue fibrosis, such as how a structural matrix glycostlation affects tissue fibrosis during cardiovascular disease. The main advantage of this technique is that unlike previous protocols which analyze overall glycosylation changes it allows for the characterization of glycosylation at particular sites of a structural matrix proteins, providing a tool for more targeted follow up mechanistic studies.
Demonstrating the procedures will be Dr.Marika Fava, and Ms.Ferheen Baig from our laboratory. To begin this procedure, prepare all extraction buffers as outlined in the text protocol. Next, use a scalpel to dice 20 to 50 milligrams of tissue into three or four smaller pieces about two millimeters in size.
Place the diced tissue into a 1.5 milliliter tube and add 500 microliters of ice cold PBS. Wash the tissue five times using 500 microliters of PBS for each wash to minimize blood contamination. Transfer the washed samples into a 1.5 milliliter tube with a screw cap and add a volume of sodium chloride buffer 10 times the tissue weight.
Vortex at minimum speed for one hour at room temperature. Then, transfer the extract to new centrifuge tubes and centrifuge at 16, 000 times G for 10 minutes at four degrees Celsius. Briefly wash the resulting tissue pellets with fresh sodium chloride buffer.
After this, add a volume of SDS buffer 10 times the tissue weight and vortex at a minimum speed for 16 hours at room temperature. Transfer the extract to new centrifuge tubes and centrifuge at 16, 000 times G for 10 minutes at four degrees Celsius. Wash the resulting pellets with double distilled water.
Add a volume of guanidine hydrochloride buffer five times the tissue weight and vortex at maximum speed for 72 hours at room temperature. Transfer the resulting extract to new tubes and centrifuge at 16, 000 times G for 10 minutes at four degrees Celsius. After quantifying the protein, aliquot 10 micrograms to a new tube for each sample, for the assessment of glycosylation site occupancy and 50 micrograms to a separate tube for direct glycopeptide analysis.
Add 10 times the volume of ethanol to each and incubate overnight at 20 degrees Celsius. The next day, centrifuge the samples at 16, 000 times G for 30 minutes at four degrees Celsius. Aspirate the supernatant, taking care not to disturb the precipitated pellet.
Then use a vacuum concentrator to dry the pellets for 15 minutes at room temperature. Add 10 microliters of prepared deglycosylation buffer to each sample. Pack the sample tubes inside a 50 milliliter conical tube and incubate at a 45 degree angle for two hours at 25 degrees Celsius.
After this, increase the temperature to 37 degrees Celsius and incubate for 36 hours with gentle agitation. After centrifuging at 16, 000 times G for one minute use a vacuum concentrator at room temperature for 45 minutes to evaporate the water. Resuspend the dried samples with 10 microliters of O-18 labeled water containing 50 units per milliliter PNGase F.Incubate for 36 hours at 37 degrees Celsius under constant agitation then perform in-solution trypsin digestion for all samples and proceed with C-18 clean up.
To begin, add 200 microliters of methanol to each well of the C-18 spin plate to activate the resin. Centrifuge at 1, 000 times G for one minute. Then add 200 microliters of 80%ACN and 0.1%TFA in water to each well and centrifuge at 1, 000 times G for one minute.
Next, add 200 microliters of a solution containing 1%ACN and 0.1%TFA in water to each well, and centrifuge for one minute at 1, 000 times G.Repeat the addition of this solution and centrifugation twice. After this, load the samples into the wells of the spin plate and centrifuge at 1500 times G for one minute. Reload the flow through and repeat the centrifugation.
Then add 200 microliters of a solution containing 1%ACN and 0.1%TFA in water to each well, and centrifuge at 1500 times G for one minute. Repeat the addition of this solution and centrifugation twice. Next, add 170 microliters of a solution containing 50%ACN and 0.1%TFA in water to each well and centrifuge at 1500 times G for one minute.
Repeat this addition and centrifugation once more. Use a vacuum concentrator to dry the combined elute at room temperature for two hours. Store the dried samples at 80 degrees Celsius until ready to use.
After this, perform liquid chromatography tandem mass spectrometry analysis on the samples intended for glycosite occupancy assessment. For samples intended for direct glycopeptide analysis proceed first with the standard TMT zero labeling as outlined in the text protocol. To begin add to each 10 microliters of TMT zero labeled sample 50 microliters of provided binding buffer.
Then, aliquot 50 microliters of homogeneous glyco capture resin suspension to new 1.5 milliliter tubes and centrifuge for one minute at 2500 times G.Remove the supernatant and add 60 microliters of the sample and binding buffer solution to the tubes containing the resin pellets using a pipette to mix. Incubate at room temperature for 20 minutes while agitating at 1200 RPM. After centrifuging for two minutes at 2, 000 times G transfer the supernatant to new tubes.
After this, add 150 microliters of wash buffer to each resin tube using the pipette to mix to begin the washing process. Incubate at room temperature for 10 minutes while agitating at 1200 RPM. After centrifuging at 2500 times G for two minutes transfer each supernatant to the corresponding supernatant storage tube to complete the wash.
Repeat this washing process two more times. Next, add 75 microliters of elution buffer and mix with a pipette. Incubate at room temperature for five minutes while agitating at 1200 RPM.
Centrifuge at 2500 times G for two minutes and transfer each supernatant to a new 1.5 milliliter tube. Add 75 microliters of elution buffer using a pipette to mix and then repeat the incubation and centrifugation of the samples. Transfer each elute to the corresponding tube.
After centrifuging the combined elute for two minutes at 2500 times G transfer each supernatant to new tube to ensure complete removal of the resin. After this, use a vacuum concentrator to dry the elute for two hours at room temperature. Resuspend the dried down glycopeptides in 15 microliters of 2%ACN and 05%TFA in double distilled water.
Then, perform mass spectrometry analysis as outlined in the text protocol. In this study, ECM proteins from cardiovascular tissue are prepared for liquid chromatography tandem mass spectrometry analysis of ECM composition as well as indirect and direct glycopeptide analysis. The core strategy is based on sequential incubations with sodium chloride, SDS, and guanadine hydrochloride followed by indirect or direct glycopeptide analysis.
The efficiency of the extraction is monitored on bis-tris acrylamide gels with silver staining for visualization. Of the three extracts run, the majority of the ECM glycoproteins are seen in the guanadine hydrochloride aliquots. ECM glycoproteins are decorated with large and repetitive glycosamine glycan chains along with short and diverse N and O-linked oligosaccharides.
The efficiency of deglycosylation is seen with the addition of enzymes that digest glycosaminic glycans and enzymes that target smaller N and O-linked oligosaccharides. Glycoproteins are then identified by the presence of deamidated asparagines labeled with oxygen 18 within NXT or NXS sequons. A representative HCD tandem mass spectrum for a peptide of decorin with a deamidated asparagine at position two 11 is shown here.
HCD ETD fragmentation is then used to analyze the glycoprotein enriched ECM extract. The ETD tandem mass spectrum allows for the characterization of glycan composition. Once mastered, this technique should take approximately two to three weeks.
This glycoproteomics allow us to explore physiological and pathology of fibrosis function not only on changes in proteomics but also on the composition of the thought sugars. After watching this video, you should have a good understanding on how to prepare samples for a study in protein glycosylation by mass spectrometry. Although this method can provide insight into cardiovascular disease it can also be applied to other pathologies in volume fibrosis such as respiratory diseases or cancer.
