Glycomics-Guided Glycoproteomics Facilitates Comprehensive Profiling of the Glycoproteome in Complex Tumor Microenvironments

So we study complex carbohydrates, or glycans, in the innate immune system and immune-related disorders, focusing currently on sepsis and cancer. Our work involves analytical methods and systems glycobiology, including glycomics and glycoproteomics methods, for the detailed characterization of the human glycoprotein. Glycoproteomics has experienced tremendous advances over the past decades, and progress is no longer limited by the shortage of high quality mass spectrometry data available.

Rather, the challenge is to interpret and generate biological knowledge from the spectral data. The glycomics-guided glycoproteomics method enables glyco scientists to obtain deep and accurate insight into the glycoproteome and hard barriers with human health indices, Glycoproteomics is uniquely capable of generating site-specific insights into the glycoproteome. But glycopeptide rates remain high.

To tackle these reproducibility issues, Our glycomics glycoproteomics method enables precision glycoproteome profiling by defining constraints in the glycan search space through the glycomics survey of the same sample. Ultimately, it enables quicker and more accurate glycopeptide searches. Our glycomics-guided glycoproteomics method allows for a comprehensive mapping of the heterogeneity and dynamics of the glycoproteome indices from a variety of different human specimens.

This method can therefore help us with better understanding of the disease progression and can also help us find new diagnostic biomarkers for therapeutic targets against a wide variety of human diseases. The glycomics-guided glycoproteomics method allows us to decipher the complexity of human glycoproteome, which, in turn, open new biological questions such as how glycan structures affect the protein function, cell communication, or immune responses. It also raises questions on how and why the glycosylation pattern changed in certain diseases.

And these insights might lead to discoveries and new diagnostic markers and personalized therapies in complex diseases. To immobilize the protein of interest on a PVDF membrane, using a single hole puncture, cut the membrane based on the number of samples to be spotted. Wet the excised membranes with methanol, then transfer them to a flat-bottom 96-well plate.

Once the membranes are near dry, apply 2.5 microliters of protein samples per spot. Air dry the membranes at 20 to 25 degrees Celsius for at least three hours, or preferably overnight, taking care to avoid contamination. Add 100 microliters of 1%PDP-40 in methanol to each well containing the spotted membranes.

Shake the plate in the PDP-40 solution at 20 to 25 degrees Celsius. After five minutes, discard the PDP-40 solution. Next, wash each well containing the blocked protein spots with 100 microliters of ultrapure water and shake the plate for five minutes.

Add 15 microliters of PNGase F solution to each well. To prevent dehydration, add water to the surrounding empty wells. Seal the plate carefully with transparent film and incubate for at least eight hours at 37 degrees Celsius.

After incubation, sonicate the plate for five minutes to help any evaporated droplets on the sides of the wells collect at the bottom. Transfer the released N-glycan samples to individual 1.5 milliliter micro-centrifuge tubes. Wash each sample well twice with 20 microliters of ultrapure water, aspirating and combining all remaining samples into the new tubes.

Next, add 10 microliters of 100-millimolar ammonium acetate pH 5 and incubate the samples for one hour at 20 to 25 degrees Celsius. Dry the N-glycan samples in a vacuum concentrator system. To prepare homemade porous graphite carbon, or PGC-C18 solid phase extraction, or SPE micro-columns, use a syringe to plug and transfer C18 discs into 10-microliter pipette tips.

Pipette a 10 microliter of PGC resin slurry suspended in 50%methanol into the C18 SPE micro-columns. Place the micro-columns into 2-milliliter tubes with micro-centrifuge adapters. Spin the tubes to create PGC-C18 SPE micro-columns with an approximate column height of 0.5 centimeters.

Next, add 50 microliters of 0.1%trifluoroacetic acid, or TFA, in acetonitrile into the micro-columns and centrifuge at 2000 G for 60 seconds to facilitate the mobile phases to pass through the micro-columns. Similarly, wash and condition the micro-columns three times using 0.1%aqueous TFA. Apply the N-glycan samples to each micro-column in loading volumes of up to 40 microliters.

Spin the micro-column after each edition, repeating until the entire sample is loaded. Wash each micro-column with 20 microliters of ultrapure water. Transfer the micro-columns to new 1.5-milliliter micro-centrifuge tubes.

To elute the N-glycans, add 40 microliters of 0.1%TFA in 50%acetonitrile to each micro-column. Spin the tubes and combine the eluate from each spin. Dry the desalted N-glycans in a vacuum concentrators system.

Browse the generated LC-MS/MS raw data using appropriate software. Confirm high data quality by assessing narrow LC peak width, effective separation of N-glycan isomers, high MS accuracy, resolution, and signal to noise ratio, along with high CID MS/MS fragmentation efficiency. Manually identify N-glycans in the sample based on monoisotopic precursor mass, CID MS/MS fragmentation patterns, and relative and absolute PGC-LC retention times.

For relative N-glycan quantitation, generate a transition list containing the monoisotopic precursor mass to charge ratio for all identified N-glycan isomers. Determine the area under the curve values from extracted ion chromatograms of all N-glycan precursor ions. To prepare ZIC-HILIC-C8-SPE micro-columns, using a syringe, plug and transfer C8 discs into 10-microliter pipette tips.

Deposit a slurry of ZIC-HILIC resin suspended in methanol into the C8-SPE micro-columns. Place the micro-columns in 2-milliliter tubes with micro-centrifuge adapters, ensuring the columns are suspended at the center. Spin to create C8-SPE HPLC micro-columns with an approximate height of one centimeter.

Add 50 microliters of methanol into the micro-columns and centrifuge at 2000 G for 60 seconds, allowing the mobile phases to pass through the column. Similarly, wash the micro-columns three times, each with 50 microliters of ultrapure water followed by 50 microliters of 1%TFA in 80%acetonitrile. Resuspend the dried peptide mixtures designated for N-glycopeptide enrichment in 50 microliters of 1%TFA in 80%acetonitrile, and mix thoroughly.

Place the condition ZIC-HILIC-C8-SPE micro-columns into new 1.5-milliliter tubes with micro-centrifuge adapters. Apply the resuspended peptide mixtures to each micro-column and spin at 2000 G for 60 seconds. Collect the flow through fractions and reapply each fraction to the same ZIC-HILIC-C8-SPE micro-column.

Then elute the N-glycopeptide with the sequential addition of 50 microliters of 25-millimolar ammonium bicarbonate and 50 microliters of 50%acetonitrile. Transfer the micro-columns to new 1.5-milliliter low binding micro-centrifuge tubes. To sequentially elute retained N-glycopeptide, add 50 microliters of 0.1%aqueous TFA into the micro-columns and centrifuge at 2000 G for 60 seconds.

Then elute the N-glycopeptide with the sequential addition of 50 microliters of 0.1%aqueous TFA followed by 50 microliters of 25-millimolar ammonium bicarbonate and 50 microliters of 50%acetonitrile. Collect, combine, and dry the enriched N-glycopeptides and the non-modified peptide flow through fractions in a vacuum concentrator system. Analysis of the N-glycomics data identified 76 N-glycan isomers in colorectal cancer tissues with 70%complex/hybrid glycans, 20%oligomannose, and 10%paucimannose.

All N-glycan structures were confirmed with spectral evidence, quantified using area under the curve values from extracted ion chromatograms.