The dual-functional Titanium immobilized metal affinity chromatography strategy can enrich both N-glycopeptides and phosphopeptides in the same workflow, increasing biomolecular information from the same analysis. The main advantage of this enrichment is two separation mechanisms, which enable the simultaneous separation of N-glycopeptides and phosphopeptides in separate fractions for downstream mass spectrometry analysis. Using this enrichment method can improve detection of glycosylation and phosphorylation in complex biological samples like pancreatic tissues toward disease biomarker discovery such as in diabetes and cancer.
Because this method relies on spin tips and centrifugation, it is important to control the centrifugation speed, and make sure that samples are free of particulates to prevent any clogging. To begin, pre-chill the parts of the tissue pulverizer that will come in contact with the pancreatic tissues, namely the chamber, pulverizer, and recovery spoon, along with any spatulas, in a polystyrene container. Next, transfer tissue pieces into the pre-chilled sample holder, and add a liquid nitrogen to the tissue until they are frozen.
After placing the pulverizer into the chamber, strike it using a mallet five to ten times to crush the sample, then remove the pulverizer from the chamber and scrape off adherent tissue powder and pieces. Next, add a spoonful of liquid nitrogen to the chamber if tissues begin to melt, and repeat the pulverization process until a fine powder is obtained and portion the samples into approximately 100-milligram aliquots into pre-chilled tubes. After preparing the lysis buffer, dissolve one tablet each of protease and phosphatase inhibitor in 500 microliters of water, for a stock of 20 times the desired final concentration, and add the required volume of stock of each inhibitor to the lysis buffer to achieve the final inhibitor concentrations.
After adding 600 microliters of lysis buffer to the tube, incubate at 95 degrees Celsius in the heating block for 10 minutes with shaking at 800 RPM, then remove the samples from the heating block and let them cool to room temperature. Next, sonicate the samples at 60-Watt energy for 45 seconds, using 15 second pulses with a 30-second rest in-between, and pellet the samples at 3000 times G for 15 minutes at four degrees Celsius. Add the supernatant to five times its volume of precipitation solvent.
For example, 300 microliters of lysis buffer to 1.5 milliliters of precipitation solvent containing 50%acetone, 49.9%ethanol, and 0.1%acetic acid, then chill overnight at 80 degrees Celsius. Pellet the samples again at 3000 times G for 15 minutes at four degrees Celsius, and remove the supernatant, then wash the pellet by breaking up with a spatula and mix with the same amount of precipitation solvent. After centrifuging, air dry the sample pellet in a fume hood for 15 minutes, and store at 80 degrees Celsius until ready to proceed.
First, re-suspend the protein pellet in 300 microliters of freshly-made digestion buffer containing 50 millimolar triethylammonium bicarbonate buffer and eight molar urea. To the protein in solution, add dithiothreitol to a final concentration of five millimolar, mix, and reduce at room temperature for one hour, then add iodoacetamide to a final concentration at 15 millimolar. Mix, and alkylate at room temperature for 30 minutes in the dark.
Next, add Lys-C/Trypsin at 1 to 100 enzyme-to-protein ratio, and incubate at 37 degrees Celsius in an incubator for four hours, then add 50 millimolar triethylammonium bicarbonate buffer to dilute the eight molar urea used to less than one molar. After adding trypsin at 1 to 100 enzyme-to-protein ratio, incubate at 37 degrees Celsius in an incubator overnight, subsequently quench the digestion with the addition of trifluoroacetic acid. For every one milligram of starting protein, condition a desalting cartridge with one milliliter of acetonitrile, and thrice with one milliliter of 0.1%trifluoroacetic acid, then load the digested mixture onto the desalting cartridge, and wash the mixture thrice using one milliliter of 0.1%trifluoroacetic acid.
Next, elute peptides using one milliliter of 60%acetonitrile and 0.1%formic acid solution, then dry eluted peptides at approximately 35 degrees Celsius using a centrifugal vacuum concentrator until the solvent has completely evaporated. After re-suspending the peptides in water, estimate peptide concentrations using a peptide assay according to the manufacturer's protocol, then portion the peptides into 500-microgram aliquots, and dry completely. Weigh approximately three milligrams of cotton wool and pack it into an empty spin tip.
After transferring one gram of Titanium immobilized metal affinity chromatography material into a tube, add 0.1%trifluoroacetic acid to a known concentration of material. After adding enough slurry to a spin tip to transfer 20 milligrams of material, wash the spin tip by centrifuging with 200 microliters of 0.1%trifluoroacetic acid. Re-suspend the samples in 200 microliters of loading/washing solvent, and flow through the spin tip.
After washing the spin tip by centrifugation with loading/washing solvent, wash the spin tip with 200 microliters of 80%acetonitrile 0.1 formic acid solution, then elute the peptides with 200 microliters of E1 and E2, and analyze each elution separately. Repeat the elution process using E3 and E4 solvents, and combine the appropriate elution fractions before downstream analysis. Before performing the elutions at basic pH, condition the material thrice using 200 microliters of 90%acetonitrile in 2.5%ammonium hydroxide, then elute the peptides with 200 microliters of E5 and E6 solvents, and analyze these elutions separately after desalting using a packed tip.
Next, elute the peptides with 200 microliters of different concentrations of acetonitrile and ammonium hydroxide. Afterward, combine these elutions, and analyze as one sample, E7, after desalting using a packed tip. Annotated high-confidence MS/MS spectra from N-glycosylated and phosphorylated peptides were obtained with rich fragmentation of precursors, increasing confidence in the identification, as assigned by the database-searching software.
Peptide identifications were found from samples enriched using the dual-functional Titanium immobilized metal affinity chromatography spin tip method over each elution fraction. The majority of glycopeptides elute in the first four fractions, while the majority of phosphopeptides elute in the last three fractions, lessening potential interferences in ionization. The glycoproteomics results from the dual titanium method were compared to an Ehrlich-glycopeptide-only enrichment, demonstrating that proportions of each type of glycan after binning into six categories based on their compositions, are similar between both methods.
Phosphoproteomics results from the dual titanium method were compared to a conventional immobilized metal affinity chromatography phosphopeptide-only enrichment or using both methods. The majority of phosphorylated amino acids were identified as serine and threonine, with around 1%phosphorylated tyrosine. Properly constructing the spin tip is important to ensure smooth elution.
Plug the cotton firmly into the tip, so that the enrichment material can be packed on top of the cotton. We have used the dual-functional titanium IMAC method for simultaneous characterization of glycosylation and phosphorylation in most tissues and the SARS-CoV-2 spike protein.
