Glycoproteomics of the Extracellular Matrix: A Method for Intact Glycopeptide Analysis Using Mass Spectrometry

The study was approved by the Wandsworth Local Research Ethics Committee (reference number: 06/Q0803/37) and received institutional approval from the research and development office. All patients gave written informed consent.

1. Extraction of Extracellular Matrix Proteins

NOTE: The human atrial tissues used for these experiments were obtained from the atrial appendages during cardiopulmonary bypass, just after the cardioplegic arrest of the heart. All samples were collected at St George's Hospital, London, UK. All tissue samples must be frozen at -80 °C. Do not use samples preserved with fixatives, such as paraformaldehyde, that cross-link proteins.

1. Prepare all extraction buffers in advance of experiments, as per Table 1, in order to minimize the time between the extraction steps. Perform all incubations at room temperature (RT) in a temperature-controlled environment (i.e., ~20 °C) to ensure consistency between extractions.
2. Weigh 20-50 mg of tissue. If several samples are to be extracted, cut and weigh them one by one to avoid the complete thawing of the tissues. Using a scalpel, dice the tissue into 3-4 smaller pieces (i.e., 2-3 mm) and place them together into 1.5 mL tubes.
3. Add 500 µL of ice-cold phosphate-buffered saline (PBS; see Table 1 and the Table of Materials) and perform five washes to minimize blood contamination.
4. Extraction Step 1: Incubation with NaCl Buffer
   1. After washing with PBS, place the samples into 1.5 mL tubes with screw caps. Add NaCl buffer (see Table 1) at 10 times (v:w) the tissue weight. Vortex the tubes at RT for 1 h at minimum speed (i.e., 600 rpm).
      NOTE: A low vortex speed is critical to avoid the mechanical disruption of the tissue during this step. Use a foam adaptor to place all tubes during the extraction.
   2. Transfer the extracts to new tubes and centrifuge at 16,000 x g for 10 min at 4 °C. Store the extracts at −20 °C until use. Briefly wash the remaining tissue pellets with fresh NaCl buffer. Use the same type of buffer (i.e., 100 µL of NaCl buffer) for washing to prevent the extraction of other proteins with different solubilities (i.e., proteins not extractable with NaCl).
   3. After washing, ensure the complete removal of the buffer to minimize overlap in protein content with subsequent extraction steps. Discard the NaCl buffer used for washing.
      NOTE: The ratio between the buffer volume and tissue weight is important for a reproducible extraction. A 10:1 ratio (v:w) for the NaCl and SDS extractions and 5:1 for the GuHCl step provide sufficient amounts of protein without saturating the buffer. Protein concentrations are approximately 1-2 µg/µL after extraction.
5. Extraction Step 2: Decellularization with SDS Buffer
   1. Add SDS buffer (Table 1) at ten times (v:w) the tissue weight; the use of low SDS concentrations (i.e., 0.1%) is critical to avoid the loss of ECM proteins during decellularization. Vortex the tubes at RT for 16 h at minimum speed (i.e., 600 rpm).
      NOTE: A low vortex speed minimizes mechanical disruption of the ECM.
   2. Transfer the extracts to new tubes. Centrifuge at 16,000 x g for 10 min at 4 °C; store at -20 °C until use. Briefly wash the remaining tissue pellets with _ddH₂O to remove the SDS. Ensure the complete removal of the liquid after washing.
6. Extraction Step 3: Incubation with GuHCl Buffer
   1. Add GuHCl buffer (Table 1) at five times (v:w) the tissue weight. Vortex the tubes at RT for 72 h at maximum speed (i.e., 3,200 rpm); vigorous vortexing facilitates the mechanical disruption of the ECM.
   2. Transfer the extracts to new tubes. Centrifuge at 16,000 × g for 10 min at 4 °C and store at -20 °C until use.

2. Protein Quantification and Precipitation

NOTE: Due to the presence of detergents, the SDS buffer is incompatible with direct protein quantification based on measurements of absorbance at 280 nm. To ensure reproducible quantification, use colorimetric assays for all protein extracts¹⁷.

1. Quantification.
   1. Prepare standards for a calibration curve using bovine serum albumin (BSA) serially diluted in the appropriate extraction buffer (i.e., NaCl, SDS, or GuHCl)^17. During this time, thaw the sample extracts.
   2. Dilute the samples in extraction buffer to obtain concentrations within the linear range of absorbance; a 1:10 dilution (v:v) yields satisfactory results. Dilute the GuHCl samples with an equal amount of ddH₂O for quantification, as the colorimetric assay is not compatible with concentrations of >4 M GuHCl.
   3. Use a bicinchoninic acid (BCA)-based colorimetric assay¹⁷ (see the Table of Materials), following the manufacturer's instructions for assays in 96-well plates; it is recommended to perform at least duplicate measurements.
   4. After incubating for 30 min, take absorbance readings at a wavelength of 570 nm in order to calculate the protein concentration using the BSA standard calibration curve¹⁷.
2. Protein Precipitation
   1. Thaw GuHCl extracts at RT. Aliquot 10 µg of protein for each sample into new tubes. For a direct glycopeptide analysis, aliquot 50 µg. Add 10 times the volume of ethanol and incubate overnight at -20 °C.
      NOTE: GuHCl is not compatible with further enzymatic reactions and most electrophoretic applications. The removal of GuHCl is required before deglycosylation and trypsin digestion. The solubility of GuHCl in ethanol and, conversely, the low solubility of proteins allow for the effective removal of GuHCl while yielding approximately a 98% recovery of proteins¹⁸.
   2. Centrifuge the samples at 16,000 × g for 30 min at 4 °C and aspirate the supernatant. Take care not to disturb the precipitated pellet. Dry the pellets for 15 min using a vacuum concentrator (see the Table of Materials) at RT.
      NOTE: The protocol can be stopped here and the dried pellets stored at -20 °C until use.
   3. Optionally, run a gel electrophoresis as quality control (QC, see the Supplemental Methods).

3. Sequential Deglycosylation for the Assessment of N-glycosylation Site Occupancy

1. During sample drying (see step 2.2.2), prepare the deglycosylation buffer containing debranching deglycosylation enzymes, as per Table 1. See Table of Materials for product details.
2. Add 10 µL of deglycosylation buffer containing enzymes to each sample. Ensure the appropriate pellet resuspension by performing a quick vortex and spin-down of samples.
   NOTE: The removal of sugar monomers using debranching enzymes is essential for the subsequent and complete removal of O-linked complex saccharides and facilitates the later cleavage of N-linked sugars by PNGase-F.
3. Incubate for 2 h at 25 °C to allow for the removal of heparan sulfate by heparinase II.Increase the temperature to 37 °C and incubate for 36 h with gentle agitation.
   NOTE: Given the low reaction volumes and prolonged incubation times, use incubator shakers and tightly pack multiple 1.5 mL tubes inside a 50-mL conical tube leaning inside the incubator at approximately 45°.
4. After 36 h, centrifuge the samples for 1 min at 16,000 x g and evaporate the H₂O from the samples by using a vacuum concentrator at RT for approximately 45 min.
5. Resuspend the dried samples with 10 µL of H₂¹⁸O containing 50 U/mL PNGase-F, which cleaves all asparagine-linked glycans in a deamidation reaction.
   NOTE: The resulting aspartic acid carries an excess mass of 2.98 Da, indicative of the presence of N-glycosylation during MS analysis.
6. Incubate for 36 h at 37 °C under constant agitation in the incubator shaker.

4. In-solution Trypsin Digestion

NOTE: This step should be carried out for both non-deglycosylated (i.e., used for direct glycopeptide analysis) and deglycosylated samples (i.e., used for the assessment of glycan occupancy).

1. Use 10 µg of total protein for the assessment of N-glycosylation site occupancy (as indicated in the previous step). For samples intended for direct glycopeptide analysis, use 50 µg of protein as the starting amount.
   NOTE: The following steps are described for 10 µg of protein. Scale up th evolumes (i.e., 5 times for 50 µg) as required.
2. Denature the proteins on each sample aliquot using 9 M urea and 3 M thiourea, with final concentrations of 6 M urea and 2 M thiourea, respectively (e.g., for a 10 µL sample, 20 µL of urea/thiourea).
3. Reduce the proteins by adding 100 mM dithiotreitol (DTT, 3.33 µL, final concentration: 10 mM). Incubate at 37 °C for 1 h with agitation at 240 rpm.
4. Cool down the samples to RT before performing the alkylation by adding 0.5 M iodoacetamide (3.7 µL, final concentration: 50 mM). Incubate in the dark for 1 h.
5. Use pre-chilled (-20 °C) acetone (6 times the volume of the sample) to incubate the samples overnight at -20 °C. Precipitate by centrifugation at 14,000 x g for 25 min at 4 °C.
6. Aspirate the supernatant. Take care not to disturb the precipitated pellet. Dry the protein pellets using a vacuum concentrator for 30 min at RT.
7. Resuspend in 20 µL of 0.1 M triethylammonium bicarbonate (TEAB) buffer, pH 8.2, containing trypsin (0.01 µg/µL) and digest overnight at 37 °C and 240 rpm.
8. Stop the digestion by acidifying the samples with 10% trifluoroacetic acid (TFA, 2 µL for a final concentration of 1% TFA).

5. Peptide Cleanup Using C18 Columns

NOTE: The removal of interfering contaminants from the peptide mixture after digestion reduces ion suppression and improves signal-to-noise ratios and sequence coverage. This step should be carried out for both non-deglycosylated and deglycosylated samples.

1. Activate the resin on the C18 spin plate (see the Table of Materials) using 200 µL of methanol per well and centrifuge at 1,000 x g for 1 min.
2. Wash by adding 200 µL per well of 80% acetonitrile (ACN) and 0.1% TFA in H₂O. Centrifuge at 1,000 x g for 1 min.
3. Equilibrate by adding 200 µL per well of 1% ACN and 0.1% TFA in H₂O. Centrifuge at 1,000 x g for 1 min. Repeat this step two more times.
4. Load the samples (the entire volume) from step 4 into the wells containing the resin and centrifuge at 1,500 x g for 1 min. Reload the flow-through a second time and repeat the centrifugation.
5. Wash by adding 200 µL per well of 1% ACN and 0.1% TFA in H₂O. Centrifuge at 1,500 x g for 1 min. Repeat this step two more times.
6. Elute the samples with 170 µL per well of 50% ACN, 0.1% TFA in H₂O. Centrifuge at 1,500 x g for 1 min. Repeat the previous step and combine the collected eluate.
7. Dry the eluate using a vacuum concentrator for 2 h at RT. If it is not being used immediately, keep the dried samples at -80 °C until use.
   NOTE: Deglycosylated samples intended for protein identification only are ready to use for LC-MS/MS after this step. Steps 6 and 7 are not required for these samples.
8. Thaw and resuspend the deglycosylated samples in 2% ACN and 0.05% TFA in H₂O to a final protein concentration of 0.5 µg/µL. Proceed with step 6 for the direct glycopeptide analysis of non-deglycosylated samples.
9. Optionally, filter the peptides prior to labeling with tandem mass tags (TMT); see the Supplemental Methods for peptide fitration.

6. Labeling with TMT (for Direct Glycopeptide Analysis Only)

1. Thaw and resuspend the dried pellets in 50 µL of 50 mM TEAB to obtain a concentration of 1 µg/µL.
2. Resusped the 0.8-mg vial of TMT Zero (TMT⁰, see the Table of Materials) reagent in 41 µL of ACN. Follow the manufacturer's recommendantions for resuspension.
3. Label the peptide samples at a ratio of 50 µg peptides to 0.4 mg TMT⁰ ( i.e., 50 µL of peptides to 20.5 µL of TMT⁰). Incubate at RT for 1 h.
4. Quench the labeling reaction by adding 5% hydroxylamine at a ratio 6:100 (i.e., 4.23 µL of 5% hydroxylamine). Incubate at RT for 15 min.
5. Dry the TMT⁰-labelled peptide samples for 1 h at RT using a vacuum concentrator. Resuspend in 10 µL of ddH₂O.
   NOTE: Due to the glycan residues, glycopeptides display a higher molecular mass than non-glycosylated peptides. TMT⁰ increases the charge state of glycopeptides. This reduces their relative mass to charge (m/z) ratios and facilitates ETD fragmentation.

7. Glycopeptide Enrichment

1. Use reaction buffers provided in the kit (see the Table of Materials).
2. Add 50 µL of binding buffer to each 10 µL sample from step 6.5. Vortex the glycocapture resin solution until it becomes homogeneous. Use a zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC)-based capture¹⁵.
3. Aliquot 50 µL of the resin suspension to new 1.5 mL tubes. Spin for 1 min at 2,500 x g and remove the supernatant. Add 60 µL of sample (i.e., the sample with binding buffer) to the tubes containing the resin pellets. Mix using a pipette and incubate at RT for 20 min in agitation at 1,200 rpm.
4. Centrifuge for 2 min at 2,000 x g and transfer the supernatant to new tubes. Keep the tubes. Add 150 µL of wash buffer to the resin tubes. Mix using a pipette and incubate at RT for 10 min in agitation at 1,200 rpm.
5. Spin for 2 min at 2,500 x g. Transfer the supernatant to the same tubes (from step 7.4). Repeat the washing steps two times.
6. Add 75 µL of elution buffer and mix using a pipette. Agitate at 1,200 rpm for 5 min at RT and then centrifuge the tubes for 2 min at 2,500 x g. Transfer the supernatants to new 1.5 mL tubes. Repeat the washing steps and then transfer the eluate supernatant to the same tube.
7. Centrifuge the tubes containing the eluate (i.e., glycopeptides) for 2 min at 2,500 x g. Transfer the supernatant to new tubes to ensure the removal of any remaining resin from the previous steps.
8. Dry the total 150 µL of eluate using a vacuum concentrator for approximately 2 h at RT. Resuspend the dried-down glycopeptides in 15 µL of 2% ACN and 0.05% TFA in ddH₂O.
9. Proceed to perform LC-MS/MS using HCD fragmentation to analyse the ECM protein composition, and LC-MS/MS using HCD and ETD fragmentation for glycopeptide characterization. See section 8.

8. Mass Spectrometry Analysis

1. Perform LC-MS/MS using HCD fragmentation to analyse the ECM protein composition; see the Supplemental Methods for details.
2. Perform LC-MS/MS using HCD and ETD fragmentation for glycopeptide characterization (see the Supplemental Methods for details); the enriched sample should be compared to the non-enriched input material¹⁵.
   NOTE: Detailed descriptions of LC-MS/MS methods for indirect glycopeptide analysis, direct glycopeptide analysis, and database search are provided in the Supplemental Methods. Researchers interested in characterizing ECM protein and glycan composition using mass spectrometry are encouraged to refer to previous publications^3,11,15.