A Quantitative Glycomics and Proteomics Combined Purification Strategy

1. Protein and Glycoprotein Denaturation and Alkylation

1. For standard preparations, load 2.5 μl of a 0.1 μg/μl solution of glycoprotein (e.g. Fetuin or RNase B) onto a 30 kDa or 10 kDa molecular weight (MW) cut-off filter.
   1. For biological plasma samples, check the protein concentration by a standard Bradford¹⁹ or bicinchoninic acid²⁰ (BCA) protein assay. Dilute the plasma, if necessary, with 100 mM ammonium bicarbonate (NH₄HCO₃) in H₂O (PNGase Digest Buffer) at a pH of ~7 to contain between 10-100 mg/ml total protein. Load 2.5 μl plasma (25-250 μg protein) onto a filter.
      Note: This protocol has only been tested on plasma from blood samples collected in the presence of ethylenediaminetetraacetic acid (EDTA).
2. Add 2 μl of 1 M Dithiothreitol Solution (DTT) to each sample in the filter.
3. Dilute the sample with 200 μl PNGase digest buffer.
4. Cap the filter sample tube and lightly vortex, being careful not to disturb the filter from its seat. Incubate the sample at 56 °C for 30 min to denature the proteins and glycoproteins.
5. To alkylate the samples, add 50 μl 1 M Iodoacetamide, to give a final concentration of ~200 mM, and incubate at 37 °C for 60 min.
   NOTE: If proteomics analysis is not desired, step 1.5 may be skipped.
6. Concentrate the denatured glycoprotein onto the filter by centrifuging samples at 14,000 x g for 40 min. Discard flow through.

2. N-linked Glycan Enzymatic Digestion

1. Wash the sample with 100 μl PNGase digest buffer. Concentrate the glycoprotein on the filter at 14,000 x g for 20 min and discard flow through.
   1. Repeat the wash and concentrate step twice, for a total of three times, yielding a concentrate in the filter dead volume (~5 μl). Discard all flow-through.
   2. Discard collection vial once washes are complete. Collect all future eluents and washes in a new collection vial.
      NOTE: PNGase digest buffer in H₂¹⁸O may be used during the PNGase F digestion step for stable isotope labeling of de-glycosylated asparagine sites, which become chemically deamidated.
2. Add 2 μl of glycerol-free PNGase F (75,000 x units/ml) to filter after transferring to a fresh collection vial. Add 98 μl of PNGase digest buffer, bringing total volume to 100 μl, and gently pipette up and down on the filter to mix. Enzymatically digest the glycans under the conditions in steps 2.2.1, 2.2.2, OR 2.2.3.
   NOTE: Three methods for the deglycosylation of proteins may be used. For solely glycomics analysis (no proteomics), the long incubation in step 2.2.1 is recommended. For glycomics and proteomics research, steps 2.2.2 and 2.2.3 will produce high quality peptides with minimal non-specific deamidation.
   1. Glycomics-only digestion protocol (18hr): Incubate samples at 37 °C for 18 hr to enzymatically cleave all N-glycans.
   2. Spike-in digestion protocol (SPI): Incubate samples at 50 °C for 2 hr. Working quickly, remove samples and spike in an additional 2 μl of glycerol-free PNGase F (75,000 x units/ml). Lightly vortex the samples for 1-2 sec to mix. Incubate the samples at 50 °C for an additional 2 hr.
   3. Microwave digestion protocol (MD): Place samples in a microcentrifuge tube floating rack. Float samples in a 1 L beaker filled with 1 L of deionized (DI) water. Place the beaker in the center of a microwave oven (950 W) with a rotating plate. Microwave at 60% power (570 W) for 5 min. To cool, remove samples and hold at room temperature for 2 min. Then microwave for an additional 5 min at 60% power.
      NOTE: Microwave power settings will need to be adjusted based on the maximum power output of the microwave. Average power should be held constant between models.

3. Elution of N-glycans

1. Elute glycans by centrifuging the sample at 14,000 x g for 20 min at 20 °C.
2. Add 100 μl of PNGase digest buffer to the filter and centrifuge at 14,000 x g for 20 min at 20 °C. Collect wash containing any remaining N-linked glycan, in the same collection vial as the eluent. Repeat twice. Remove filter and place in new collection vial.
3. Incubate glycan samples in the -80 °C freezer until frozen (30-60 min). Dry to completion, at room temperature, in vacuum concentrator (4-6 hr).
   NOTE: N-glycans may be stored at -20 °C for up to six months prior to derivatization.

4. Protein FASP Digestion

NOTE: If proteomics or deamidation site analysis is not desired, section 4 of the protocol may be skipped.

1. Rinse the filter, containing the deamidated peptides, with 400 μl of 8 M urea buffer. Cap the collection vial and lightly vortex to mix. Concentrate on the filter at 14,000 x g for 15 min. Discard all flow-through.
   1. Repeat step 4.1 two additional times, for a total of three urea buffer washes.
2. Rinse the filter, containing the deamidated peptides, with 400 μl of 2 M urea, 10 mM CaCl₂ buffer (trypsin digest buffer). Cap the collection vial and lightly vortex to mix. Concentrate on the filter at 14,000 x g for 15 min. Discard all flow-through.
   1. Repeat step 4.1 two additional times, for a total of three trypsin digest buffer washes.
   2. Discard collection vial once washes are complete. Collect all future eluents and washes in a new collection vial.
3. Add 50 μl porcine modified trypsin in a 1:50 or 1:5 trypsin: protein (w/w) ratio for discovery or quantitative proteomics experiments, respectively. Cap the collection vial and lightly vortex to mix.
4. Incubate samples at 37 °C for 2 hr. Working quickly, remove samples and spike in an additional 50 μl of trypsin at the 1:50 or 1:5 trypsin: protein ratio. Lightly vortex the samples for 1-2 sec to mix. Incubate the samples at 50 °C for an additional 2 hr.
5. Elute the peptides by spinning at 14,000 x g for 15 min.
6. Rinse the remaining peptides from the filter with 400 μl 1% formic acid and 0.001% Zwittergent 3-16 (quench buffer). Elute off the filter by spinning at 14,000 x g for 15 min.
7. Quantify the peptide concentration in the samples by Bradford¹⁹ or BCA assay²⁰.
8. Incubate peptide samples in the -80 °C freezer until frozen (30-60 min). Dry to completion, at room temperature, in vacuum concentrator (4-6 hr).
   NOTE: Dried peptides may be stored at -20 °C for up to three months.
9. Resuspend tryptic proteins just prior to liquid chromatography-mass spectrometry (LC-MS) analysis in 2% acetonitrile, 98% H₂O, and 0.1% formic acid (mobile phase A) to desired concentration. Tryptic peptide concentrations from 2.5 μl plasma range from 100 – 300 ng/μl.

5. Derivatization of N-linked Glycans with Hydrophobic Hydrazide Tags

1. Reconstitute dried heavy (SIL) or light (NAT) P2GPN reagents in 1 ml of 75:25 MeOH:Acetic Acid (Derivatization solution), for a final concentration of 0.25 mg/ml. Reagents will take up to 10 min to fully solubilize. Extensively vortex to ensure complete solubilization.
   Note: In the event this derivatization protocol is used with standard N-glycans, versus purified glycans, the molar ratio of P2GPN: glycan should be approximately (and no less) than 17: 1.
2. Tag dried N-glycans with 200 μl (50 μg) of SIL or NAT P2GPN reagent. Pipette up and down to resuspend dried glycans. Vortex samples and then spin down samples for ~5 sec on a bench-top centrifuge.
3. React the glycans with the reagent for 3 hr at 56 °C.
4. Immediately move glycans from the incubator to the vacuum concentrator. Dry to completion at 55 °C (3-5 hr).
   NOTE: This step quenches the derivatization reaction; the sample must be completely dry to prevent cross-reactivity in the subsequent steps.
   NOTE: Dried, tagged samples may be stored at -20 °C for up to six months.
5. Resuspend tagged N-glycans in 25-50 μl of H₂O just prior to LC-MS analysis. Pipette up and down to ensure N-glycans are fully solubilized.
   NOTE: The volume for resuspension is dependent on the starting concentration of glycoprotein, which may be estimated from the starting protein concentration. However, the range will vary per sample type and should be individually optimized.
6. Centrifuge samples at 14,000 x g for 5 min. Remove the supernatant, being careful not to let the tip of the pipette touch the bottom of the centrifuge tube.
   Note: Excess tag may not be visible to the eye, but will be reduced by centrifugation.
7. Combine a pair of NAT and SIL samples, if desired for relative quantification, in a 1:1 ratio for tandem analysis.

6. Ultra-high Pressure Liquid Chromatography and Mass Spectrometry Analysis

NOTE: The LC and MS conditions described for an Easy nLC-1000 and a Q Exactive High Field, respectively, were optimized in-house for proteomics analysis and for glycan analysis²¹. These conditions may be adapted to other ultra-high pressure- or nano- LC systems and other high-resolving power mass spectrometers but may require slight modifications. The use of high resolving power mass spectrometry is necessary for glycan identification and deamidation analysis^22,23.

NOTE: Steps 6.1-6.3 can be skipped if an appropriate reverse-phase chromatography or hydrophilic interaction commercial trap and column are used.

1. Synthesize a frit in 100 μM ID capillary according to Meiring et al.²⁴ for use as a trap.
2. Pack the trap under pressure with 2.6 μM, 100 Å, C₁₈ packing material and cut to a length of 5 cm.
3. Pack a 75 μM ID emitter column under pressure with 2.6 μM, 100 Å, C₁₈ packing material and cut to a length of 30 cm.
4. Prepare two different LC-MS methods for the proteomics (6.4.1) and glycomics (6.4.2) workflows.
   Note: Protein and glycan samples may be run on the same trap/column setup in any order.
   1. For protein analysis, inject ~400 ng of protein onto the column in mobile phase A (MPA). Run the sample at 300 nl/min, according to Table 1, with a 1 μl pre-column equilibration (500 bar) and a 5 μl analytical column equilibration (500 bar). Ionize proteins under the MS conditions provided in Table 2.
   2. For glycan analysis, inject 5 μl of resuspended, P2GPN tagged NAT/SIL equimolar samples onto the column. Run the sample at 300 nl/min, according to Table 3, with a 2 μl pre-column equilibration (500 bar) and a 5 μl analytical column equilibration (500 bar). Ionize glycans under the MS conditions provided in Table 4.

Table 1. LC conditions for proteomic analysis. A gradient elution with mobile phase B (MPB, 98% acetonitrile, 2% H₂O, 0.1% formic acid) was performed to elute peptides for shot-gun proteomics, data-dependent-acquisition, MS experiments.

Table 2: MS conditions for proteomic analysis. The parameters for electrospray ionization, MS¹ acquisition, and MS² acquisition in an orbitrap instrument using higher-energy dissociation (HCD) are given.

Table 3: LC conditions for glycan analysis. A gradient elution was performed to elute hydrazide tagged N-glycans for MS analysis.

Table 4: MS conditions for hydrazide tagged N-glycan analysis. The parameters for electrospray ionization, MS¹ acquisition, and MS² acquisition in an orbitrap instrument using higher-energy dissociation (HCD) are given.

7. Proteomics and Deamidation Analysis

1. Identify proteins/peptides using standard bioinformatics search tools.
   Note: The following analysis protocol was designed using the Swiss-Prot/TrEMBL databases in UniprotKB and searching via SEQUEST in the Proteome Discoverer 1.4 software, however, the protocol may be directly translated to any protein search and scoring engine using a GUI software. All commands given below may be accessed in software interfaces through drop-down menus.
   1. Create or download an organism appropriate protein sequence database from genomic data or available proteome databases as described by Apweiler et al.²⁵
      Note: Common proteome databases include Swiss-Prot, TrEMBL, and NCBI, but may be built from in-house data as well.
   2. Within the software, choose a database search engine and select parameters according to the quality of the data acquired and the rigorousness of the search desired, as described by Perkins et al. for MASCOT²⁶ or Eng et al. for SEQUEST²⁷.
      1. For high-mass accuracy data, set parameters for the search in the software as follows: max 2 missed cleavages, 5 ppm precursor mass tolerance, and 0.02 Da fragment mass tolerance (for optimized accurate deamidation detection²²) and include the following tryptic peptide modifications: "carbamidomethyl (C)" static modification and "deamidation (N/Q)" and "oxidation (M)" dynamic modifications. If using ¹⁸O digestion labeling, include "deamidation ¹⁸O(1)" as an additional dynamic modification.
      2. Search, using the selected engine, the raw peptide data against the protein database (7.1.1).
         Note: Mass tolerances should be appropriate for the instrument used and not arbitrarily low.
         Note: The search function is completed automatically by the software using various search engine specific algorithms. The percolator algorithm (MASCOT), SEQUEST, or other combinations of algorithms are employed to identify proteins from peptides and to score the validity of the hits; more information on the tools available are covered in a review from Gonzalez-Galarza et al.^28,29. Depending on the software, additional search parameters may need to be selected according to the discretion of the user.
   3. Export the identified peptides for manual curation and further analysis.
2. Manually curate a list of peptides containing an identified deamidation on the asparagine of the N-linked glycosylation motif (N-X-(S/T), where X ≠ P). Cross-validate these sites with known glycosylation motifs from the literature and create two pools of results (validated and theoretical).
3. Use spectral counting³⁰ to compare the percent occupancy of a given glycosylation site by comparing the abundance of deamidated and non-deamidated forms.

8. Glycan Relative Quantitation

Note: The following identification and quantification was completed using the XCalibur software. However, any software that analyzes raw data and automatically integrates chromatographic peaks may be substituted.

1. Generate a theoretical list of glycan compositions from the biologically possible combinations of hexoses, deoxy-hexoses, hexosamines, sialic acids, and other saccharides. For this purpose, a human glycan database has been published by Walker et al.¹⁵ and may be used as-is. For theoretical databases, species-specific biological constraints must be imposed on the combinatorial space, and these rules are described by Kronewitter et al.³¹.
2. Calculate the glycan monoisotopic masses (M) from the atomic masses for each composition for proton charge states +1-3. Modify the monoisotopic masses to include the addition of the NAT (254.14191 g/mol) or SIL (260.162042 g/mol) P2GPN tag.
3. Open the "Processing Setup" program from the roadmap view in XCalibur. Set up a processing method as exactly described in the Supplemental Material from Walker et al.¹⁵ using the list generated in step 8.2 containing the theoretical monoisotopic masses for the [M+H]¹⁺, [M+2H]²⁺, and [M+3H]³⁺ species.
   Note: To confirm glycan identity beyond accurate mass, for NAT/SIL duplexed runs, check that NAT and SIL N-glycan pairs co-elute with the same retention time. Qualitatively, cross-validate identifications with the MS² spectra, which must contain peaks belonging to the oxonium ion series³².
4. Export the integrated, extracted ion chromatogram (XIC) areas to Excel to obtain the SIL and NAT abundances as exactly described in the Supplemental Material from Walker et al.¹⁵
5. In Excel, program the cells in a new column to calculate the correction for the molecular weight overlap by adjusting the SIL abundance according to the equation published by Walker et al.¹⁵:
   ,
   where A is the monoisotopic peak and the theoretical isotope overlap, , may be independently calculated per composition.
6. In a second column, program the cells to normalize the NAT and SIL channels using the equation for the total normalized glycan factor (TGNF), per spectrum, as described by Walker et al.¹⁵:
   ,
   where N is the number of N-glycans above the limit of quantification.
7. In a new column, program the cells to take the ratio of SIL: NAT glycans (or vice versa) to calculate the fold-change in concentration between the two samples.