The overall goal of this single-filter based workflow is to easily and quantitatively purify N-glycans and proteins in tandem from biological samples from mass spectrometry analysis. The scalability, speed and analytical reproducability of this method makes it directly accessible in regards to resources and skill sets to biological mass spectrometry laboratories. The main advantage of this technique is it allows researchers to investigate glycomics, glycosylation site occupancy and proteomics questions simultaneously in large-scale high-throughput biological studies.

We've overcome the challenges of N-glycan measurements by coupling this method to a mass spectrometry tailored derivitization strategy resulting in accurate, relative quantification between cancer and control states. With reduced analytical variability new biological interactions may be elucidated between the glycome and proteome yielding key insights into disease states and new opportunities for biomarker discovery. To begin, load 2.5 microliters of plasma onto a filter.

Then, add 2 microliters of 1M dithiothreitol solution to each sample in the filter. Dilute the sample with 200 microliters of 100-mM Ammonium Bicarbonate PNGase digest buffer. Cap the filter sample tube and lightly vortex, being careful not to disturb the filter from its seat.

Incubate the sample at 56 degrees Celsius for 30 minutes to denature the proteins and glycoproteins. To alkilate the samples, add 50 microliters of 1M Iodoacetamide to give a final concentration of approximately 200 mM. Then, incubate at 37 degrees Celsius for 60 minutes.

Concentrate the denatured glycoprotein onto the filter by centrifuging the samples at 14, 000 x g for 40 minutes before discarding the flowthrough. Wash the sample with 100 microliters of PNGase digest buffer. Concentrate the glycoprotein on the filter and discard the flowthrough.

Repeat the wash and concentrate step twice for a total of three times, yielding a concentrate in the filter dead volume. Discard all the flowthrough and the collection vial once the washes are complete. Then, add 2 microliters of glycerol-free peptide N-glycosidase F or PNGase F to the filter after transferring to a fresh collection vial.

Add 98 microliters PNGase digest buffer bringing the total volume to 100 microliters, and gently pipette up and down on the filter to mix. For the spike in digestion protocol incubate the samples at 50 degrees Celsius for 2 hours. Working quickly, remove the samples and spike in an additional 2 microliters of glycerol-free PNGase F.Lightly vortex the samples for 1 to 2 seconds to mix.

Incubate the samples at 50 degrees Celsius for an additional 2 hours. After eluding glycans by centrifuging the sample at 20 degrees Celsius, add 100 microliters of PNGase digest buffer to the filter and centrifuge again. Collect the wash containing any remaining N-linked glycan in the same collection vial as the eluent.

After repeating twice, remove the filter and place in a new collection vial for proteomics preparation. Next, incubate the glycan samples in the 80 degrees Celsius freezer for 30 to 60 minutes until frozen. Then, dry to completion at room temperature in a vacuum concentrator for 4 to 6 hours.

Rinse the filter containing the deanimated peptides with 400 microliters of 8M urea buffer. Cap the collection vial and lightly vortex to mix. Concentrate the peptides on the filter before discarding all the flow through.

After repeating the urea buffer wash twice more, rinse the filter containing the deanimated peptides with Trypsin digest buffer. Cap the collection vial and lightly vortex to mix before concentrating on the filter again as before. Discard all flow through and repeat 2 additional times for a total of 3 Trypsin digest buffer washes.

Collect all future eluents and washes in a new collection vial. Then, add 50 microliters of porcine-modified Trypsin in a 1:50 Trypsin-to-protein ratio for discovery proteomics or a 1:5 Trypsin-to-protein ratio for quantitative proteomics experiments. Cap the collection vial and lightly vortex to mix.

After incubating the samples at 37 degrees Celsius for 2 hours, work quickly to remove the samples and spike in an additional 50 microliters of Trypsin at the appropriate Trypsin-to-protein ratio. Lightly vortex the samples for 1 to 2 seconds to mix. Then, incubate at 50 degrees Celsius for an additional 2 hours.

Following elution of the peptides by centrifuging as before for 20 minutes, rinse the remaining peptides from the filter with Quench buffer. Elute off the filter by centrifuging as before for 15 minutes. After quantifying the peptide concentration in the samples, incubate the peptide samples in the 80 degree Celsius freezer until frozen.

Then, dry to completion at room temperature in a vacuum concentrator for 4 to 6 hours. Then the tryptic proteins just prior to the liquid chromatography mass spectrometry or LCMS analysis in Mobile phase A to the desired concentration. Tryptic peptide concentrations from 2.5 microliters plasma range from 100 to 300 nanograms per microliter.

Prior to glycan analysis, reconstitute the dried stable isotope labeled 4-phenethyl benzohydrazide reagents, as well as the native P2GPN reagents in one milliliter of derivitization solution for a final concentration of 0.25 milligrams per milliliter. Reagents will take up to 10 minutes to fully solubilize. Extensively vortex to ensure complete solubilzation.

Tag the dried N-glycans with 200 microliters of stable-isotope-labeled or native P2GPN reagent. Pipette up and down to re-suspend the dried glycans. Then, vortex the samples and spin down for approximately 5 seconds on a bench top centrifuge.

React the glycans with the reagent for 3 hours at 56 degrees Celsius. Immediately transfer the glycans from the incubator to the vacuum concentrator and dry to completion at 55 degrees Celsius for 3 to 5 hours. Re-suspend the tagged N-glycan in 25 to 50 microliters of water just prior to liquid chromatography and mass spectrometry analysis.

Pipette up and down to ensure N-glycans are fully solubilized. After centrifuging samples as before for 5 minutes, remove the supernatant, being careful not to let the tip of pipette touch the bottom of the centrifuge tube. Combine a pair of native and stable-isotope-labeled samples in a 1:1 ratio for relative quantification tandem analysis.

Prepare 2 different LCMS methods for the proteomics and glycomics workflows. For protein analysis, inject approximately 400 nanograms of protein onto the column in NPA. Run the sample at 300 nanoliters per minute, with a 1 microliter pre-column equilibration and a 5 microliter analytical column equilibration.

Ionize the proteins under the MS conditions provided in the text protocol. For glycan analysis, inject 5 microliters of re-suspended P2GPN tagged native and stable-isotope-labeled equi-M samples onto the column. Run the sample at 300 nanoliters per minute with 2 microliter pre-column equilibration and a 5 microliter analytical column equilibration.

Then, ionize the glycans under the MS conditions provided in the text protocol. The filter-based purification protocol was compared to the standard solid-phase extraction method and the abundances of plasma glycans detected between the 2 protocols were not significantly different. In both methods, the same glycans found near the limit of detection where variability increases.

The intervariability for equiM mixures of plasma purified by the filter-based or standard solid-phase extraction protocol was assessed. Both protocols had Gaussian distributions whose means were not significantly different from zero. 80 percent of the glycans fell within a two-fold change.

The molecular weight distributions of glycans detected in the new and traditional purification protocols were not significantly different and covered the same range. Qualitatively, no bias between glycans was observed. Plasma proteins were prepared by standard filter-aided sample preparation and multiple in-line protocols.

The number of proteins identified was similar for the various protocols. Non-specific deamidation was controlled in microwave digestion and spike indigestion protocols to less than 10 percent. While attempting this procedure, it's important to remember that accurate glycosylation site occupancy is contingent on minimizing heat and time exposure.

Once mastered, the inline pecurikene N-glycan purification can be completed in 2 days. By following this procedure, accurate protein N-glycan quantification and glycosylation site occupancy can be integrated and modeled using higher-order statistical techniques. This technique affords the ability to quickly read back the complex language that cancer biology speaks, quantitatively compare differences in protein therapeutics, and truly reveal the importance of glycosylation in a biological context.