JBB is a Career Establishment Fellow in the King's British Heart Foundation Centre. MM is a Senior Fellow of the British Heart Foundation (FS/13/2/29892). The study was supported by an excellence initiative (Competence Centers for Excellent Technologies - COMET) of the Austrian Research Promotion Agency FFG: "Research Center of Excellence in Vascular Ageing - Tyrol, VASCage" (K-Project number 843536) and the NIHR Biomedical Research Center based at Guy's and St. Thomas' National Health Service Foundation Trust and King's College London in partnership with King's College Hospital.

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&#39;s Hospital, London, UK. All tissue samples must be frozen at -80 &#176;C. Do not use samples preserved with fixatives, such as paraformaldehyde, that cross-link proteins.
<ol>
	<li>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 &#176;C) to ensure consistency between extractions.</li>
	<li>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.</li>
	<li>Add 500 &#181;L of ice-cold phosphate-buffered saline (PBS; see Table 1 and the Table of Materials) and perform five washes to minimize blood contamination.</li>
	<li>Extraction Step 1: Incubation with NaCl Buffer
	<ol>
		<li>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.</li>
		<li>Transfer the extracts to new tubes and centrifuge at 16,000 x g for 10 min at 4 &#176;C. Store the extracts at &#8722;20 &#176;C until use. Briefly wash the remaining tissue pellets with fresh NaCl buffer. Use the same type of buffer (i.e., 100 &#181;L of NaCl buffer) for washing to prevent the extraction of other proteins with different solubilities (i.e., proteins not extractable with NaCl).</li>
		<li>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 &#181;g/&#181;L after extraction.</li>
	</ol>
	</li>
	<li>Extraction Step 2: Decellularization with SDS Buffer
	<ol>
		<li>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.</li>
		<li>Transfer the extracts to new tubes. Centrifuge at 16,000 x g for 10 min at 4 &#176;C; store at -20 &#176;C until use. Briefly wash the remaining tissue pellets with ddH2O to remove the SDS. Ensure the complete removal of the liquid after washing.</li>
	</ol>
	</li>
	<li>Extraction Step 3: Incubation with GuHCl Buffer
	<ol>
		<li>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.</li>
		<li>Transfer the extracts to new tubes. Centrifuge at 16,000 &#215; g for 10 min at 4 &#176;C and store at -20 &#176;C until use.</li>
	</ol>
	</li>
</ol>
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.
<ol>
	<li>Quantification.
	<ol>
		<li>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.</li>
		<li>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 &#62;4 M GuHCl.</li>
		<li>Use a bicinchoninic acid (BCA)-based colorimetric assay17 (see the Table of Materials), following the manufacturer&#39;s instructions for assays in 96-well plates; it is recommended to perform at least duplicate measurements.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Protein Precipitation
	<ol>
		<li>Thaw GuHCl extracts at RT. Aliquot 10 &#181;g of protein for each sample into new tubes. For a direct glycopeptide analysis, aliquot 50 &#181;g. Add 10 times the volume of ethanol and incubate overnight at -20 &#176;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.</li>
		<li>Centrifuge the samples at 16,000 &#215; g for 30 min at 4 &#176;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 &#176;C until use.</li>
		<li>Optionally, run a gel electrophoresis as quality control (QC, see the Supplemental Methods).</li>
	</ol>
	</li>
</ol>
3. Sequential Deglycosylation for the Assessment of N-glycosylation Site Occupancy
<ol>
	<li>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.</li>
	<li>Add 10 &#181;L 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.</li>
	<li>Incubate for 2 h at 25 &#176;C to allow for the removal of heparan sulfate by heparinase II.Increase the temperature to 37 &#176;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&#176;.</li>
	<li>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.</li>
	<li>Resuspend the dried samples with 10 &#181;L 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.</li>
	<li>Incubate for 36 h at 37 &#176;C under constant agitation in the incubator shaker.</li>
</ol>
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).
<ol>
	<li>Use 10 &#181;g 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 &#181;g of protein as the starting amount. 
	NOTE: The following steps are described for 10 &#181;g of protein. Scale up th evolumes (i.e., 5 times for 50 &#181;g) as required.</li>
	<li>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 &#181;L sample, 20 &#181;L of urea/thiourea).</li>
	<li>Reduce the proteins by adding 100 mM dithiotreitol (DTT, 3.33 &#181;L, final concentration: 10 mM). Incubate at 37 &#176;C for 1 h with agitation at 240 rpm.</li>
	<li>Cool down the samples to RT before performing the alkylation by adding 0.5 M iodoacetamide (3.7 &#181;L, final concentration: 50 mM). Incubate in the dark for 1 h.</li>
	<li>Use pre-chilled (-20 &#176;C) acetone (6 times the volume of the sample) to incubate the samples overnight at -20 &#176;C. Precipitate by centrifugation at 14,000 x g for 25 min at 4 &#176;C.</li>
	<li>Aspirate the supernatant. Take care not to disturb the precipitated pellet. Dry the protein pellets using a vacuum concentrator for 30 min at RT.</li>
	<li>Resuspend in 20 &#181;L of 0.1 M triethylammonium bicarbonate (TEAB) buffer, pH 8.2, containing trypsin (0.01 &#181;g/&#181;L) and digest overnight at 37 &#176;C and 240 rpm.</li>
	<li>Stop the digestion by acidifying the samples with 10% trifluoroacetic acid (TFA, 2 &#181;L for a final concentration of 1% TFA).</li>
</ol>
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.
<ol>
	<li>Activate the resin on the C18 spin plate (see the Table of Materials) using 200 &#181;L of methanol per well and centrifuge at 1,000 x g for 1 min.</li>
	<li>Wash by adding 200 &#181;L per well of 80% acetonitrile (ACN) and 0.1% TFA in H2O. Centrifuge at 1,000 x g for 1 min.</li>
	<li>Equilibrate by adding 200 &#181;L 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.</li>
	<li>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.</li>
	<li>Wash by adding 200 &#181;L 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.</li>
	<li>Elute the samples with 170 &#181;L 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.</li>
	<li>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 &#176;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.</li>
	<li>Thaw and resuspend the deglycosylated samples in 2% ACN and 0.05% TFA in H2O to a final protein concentration of 0.5 &#181;g/&#181;L. Proceed with step 6 for the direct glycopeptide analysis of non-deglycosylated samples.</li>
	<li>Optionally, filter the peptides prior to labeling with tandem mass tags (TMT); see the Supplemental Methods for peptide fitration.</li>
</ol>
6. Labeling with TMT (for Direct Glycopeptide Analysis Only)
<ol>
	<li>Thaw and resuspend the dried pellets in 50 &#181;L of 50 mM TEAB to obtain a concentration of 1 &#181;g/&#181;L.</li>
	<li>Resusped the 0.8-mg vial of TMT Zero (TMT0, see the Table of Materials) reagent in 41 &#181;L of ACN. Follow the manufacturer&#39;s recommendantions for resuspension.</li>
	<li>Label the peptide samples at a ratio of 50 &#181;g peptides to 0.4 mg TMT0 ( i.e., 50 &#181;L of peptides to 20.5 &#181;L of TMT0). Incubate at RT for 1 h.</li>
	<li>Quench the labeling reaction by adding 5% hydroxylamine at a ratio 6:100 (i.e., 4.23 &#181;L of 5% hydroxylamine). Incubate at RT for 15 min.</li>
	<li>Dry the TMT0-labelled peptide samples for 1 h at RT using a vacuum concentrator. Resuspend in 10 &#181;L 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.</li>
</ol>
7. Glycopeptide Enrichment
<ol>
	<li>Use reaction buffers provided in the kit (see the Table of Materials).</li>
	<li>Add 50 &#181;L of binding buffer to each 10 &#181;L 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.</li>
	<li>Aliquot 50 &#181;L 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 &#181;L 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.</li>
	<li>Centrifuge for 2 min at 2,000 x g and transfer the supernatant to new tubes. Keep the tubes. Add 150 &#181;L of wash buffer to the resin tubes. Mix using a pipette and incubate at RT for 10 min in agitation at 1,200 rpm.</li>
	<li>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.</li>
	<li>Add 75 &#181;L 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.</li>
	<li>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.</li>
	<li>Dry the total 150 &#181;L of eluate using a vacuum concentrator for approximately 2 h at RT. Resuspend the dried-down glycopeptides in 15 &#181;L of 2% ACN and 0.05% TFA in ddH2O.</li>
	<li>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.</li>
</ol>
8. Mass Spectrometry Analysis
<ol>
	<li>Perform LC-MS/MS using HCD fragmentation to analyse the ECM protein composition; see the Supplemental Methods for details.</li>
	<li>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 publications3,11,15.</li>
</ol>

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Proteome Res. 12 (12), 5791-5800 (2013).</li><li>Li, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+analysis+of+glycosylated+and+sialylated+prostate-specific+antigen+revealing+differential+distribution+of+glycosylated+prostate-specific+antigen+isoforms+in+prostate+cancer+tissues.">Simultaneous analysis of glycosylated and sialylated prostate-specific antigen revealing differential distribution of glycosylated prostate-specific antigen isoforms in prostate cancer tissues.</a> Anal. Chem. 83 (1), 240-245 (2011).</li><li>Rienks, M., Papageorgiou, A. P., Frangogiannis, N. G., Heymans, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myocardial+extracellular+matrix:+an+ever-changing+and+diverse+entity.">Myocardial extracellular matrix: an ever-changing and diverse entity.</a> Circ. Res. 114 (5), 872-888 (2014).</li></ol>

This proteomics protocol has been optimized over the last few years in our laboratory. Here, we used cardiac tissue, but only minor adjustments may be required for its application to other tissues. For example, the extraction protocol needs to take the cellularity of the tissue into consideration. Cardiac tissue is highly cellular compared to vascular tissue. When using vascular tissue, the SDS concentration can be lower (i.e. 0.08%) and the decellularization time is shorter (i.e. 4 h)11,12,13. The use of deglycosylation enzymes is crucial for LC-MS/MS analysis of ECM composition. However, incubation times need to be adjusted for different tissue types. For example, heparinase II required extended incubation times at 25 &#176;C when using samples such as skin, which are rich in basement membrane proteins (e.g. agrin, perlecan) (data not shown). Direct glycopeptide analysis can be performed on conditioned media from cells in culture15. Enrichment steps may not be required for the analysis of this simplified subproteome. Similar to GuHCl extracts, NaCl extracts are also amenable for glycoproteomics analysis with minor modifications. Other extraction protocols for enrichment of ECM proteins can be adapted to characterize ECM glycopeptides19,20.
Glycosylation is the most complex PTM5. Indirect identification of glycopeptides is achieved by the detection of deamidated Asn with incorporated 18O at a NxT/S sequon. Deamidated Asn at other positions may represent false positives. Likewise, N-glycosylation must be considered in the context of protein ontologies: intracellular proteins containing a NxT/S sequon will not be glycosylated but might give rise to false positives. As current search algorithms do not allow for the screening of PTMs at pre-determined sequences only (i.e. Asn at NxT/S), manual filtering of the data is required. Identification of presence/absence of glycosylation at these positions can be compared between disease and control samples. There is no enzyme equivalent to PNGase F for O-deglycosylation (i.e. introducing a mass shift on threonine or serine). Therefore, the identification of O-glycosylation is restricted to direct glycopeptide analysis. Direct glycopeptide analysis is used to obtain compositional information of sugars attached to proteins, but does not provide structural information of the glycan. Moreover, the glycan composition is the result of glycan synthesis and processing after secretion.
Our 3-step extraction method for ECM proteins (&#34;English Quickstep&#34;)6 has allowed characterizing the ECM in a variety of cardiovascular tissues. Fractionation of the tissue into several extracts is required to obtain a simplified ECM proteome as discussed elsewhere6. Intracellular proteins would otherwise contribute to an excessive dynamic range of protein abundances within the extracts that would hinder identification of less abundant ECM proteins. Moreover, intracellular proteins carry O-glycosylations5 that would complicate ECM glycopeptide enrichment and subsequent MS analysis. Other authors applied similar extraction methodologies to characterize for example lung21 and cartilage tissues10, however they did not pursue the analysis of glycosylation. Previous analysis of glycosylation focused on identification of glycosites only, require removal of the glycan from the protein core, and cannot assess O-glycosylation22,23. Lectin arrays and chemical enrichment are available for assessment of glycan types on biological samples based on their binding specificity, but these techniques cannot assign glycan types to specific proteins24 nor can they assess glycosylation sites.
Initially, we used gel electrophoresis prior to LC-MS/MS of ECM proteins. Although gel separation is useful in obtaining simplified protein fractions amenable to LC-MS/MS analysis, the latest instruments offer faster scan speeds. Thus, the electrophoretic separation step can be omitted. This provides an additional advantage as large ECM proteins, which are retained on top of the gel, are analyzed more efficiently. However, information regarding the Mw of the intact proteins is lost. The evaporation step prior to PNGase F deglycosylation ensures complete removal of regular H2O to minimize false negatives. Sugar residues (i.e. variable glycan masses) interfere with the separation by LC and compromise subsequent peptide identification by MS/MS. A pan-deglycosylation protocol is also recommended for proteomics analysis of ECM proteins not focused on glycosylation.
Proteomics can provide unprecedented insights into the ECM. Beyond structural support, glycans attached to the ECM are essential for host-pathogen interaction, cell-cell communication and the immune response25, i.e. allograft rejection after organ transplantation. Glycoproteomics will be an essential tool in glycobiology.

Fibrosis is a hallmark of many diseases. Fibroblasts proliferate and differentiate towards highly synthetic phenotypes, which are associated with the exacerbated secretion and deposition of extracellular matrix (ECM)1. Excessive ECM deposition can continue, even after the initial injury has abated, leading to functional impairment. Using proteomics, we have previously identified more than 150 ECM and ECM-associated proteins in cardiac tissue2,3. They are not only structural proteins, but also matricellular proteins and proteases that contribute to the continuous remodeling and dynamic adaptation of the heart. Notably, many ECM proteins are glycosylated4. This post-translational modification (PTM) involves the addition of sugar residues to certain amino acid positions, and it affects protein folding, solubility, binding, and degradation5.
There are two main glycosylation types that occur in mammals. (1) N-glycosylation occurs at the carboxamido nitrogen of asparagine residues (Asn) within the consensus sequence Asn-Xaa-Thr/Ser, where Xaa is any amino acid except for proline. (2) In O-glycosylation, sugar residues attach to serine and threonine residues (Ser, Thr) or, to a much lesser extent, to hydroxyproline and hydroxylysine. While O-glycosylation can occur in a variety of protein groups, N-glycosylation is restricted to secreted proteins or extracellular domains of membrane proteins5. This makes N-glycosylation an attractive target when studying the ECM.
Proteomics sets a new standard for the analysis of protein changes in disease. Thus far, most proteomics studies have been focused on intracellular proteins6. This is mainly due to the following reasons. First, abundant intracellular proteins hamper the identification of scarce ECM components. This is particularly crucial in cardiac tissue, in which mitochondrial and myofilament proteins account for a large proportion of the protein content7. Second, integral ECM proteins are heavily cross-linked and difficult to solubilize. Lastly, the presence of abundant PTMs (i.e., glycosylation) alters the molecular mass, charge, and electrophoretic properties of peptides, affecting both the separation and the identification by liquid chromatography tandem mass spectrometry (LC-MS/MS). Over recent years, we have developed and improved a sequential extraction and enrichment method for ECM proteins that is compatible with subsequent mass spectrometry (MS) analysis. The strategy is based on sequential incubations.
The first step is performed with NaCl, an ionic buffer that facilitates the extraction of ECM-associated and loosely bound ECM proteins, as well as newly synthesized ECM proteins. It is detergent free, non-denaturing, non-disruptive of cell membranes, and amenable for further biochemical assays8. Then, decellularization is achieved with sodium dodecyl sulfate (SDS). At this step, a low SDS concentration ensures membrane destabilization and the release of intracellular proteins whilst preventing the disruption of the more soluble non-integral ECM components. Finally, ECM proteins are extracted with a guanidine hydrochloride buffer (GuHCl). GuHCl is effective in extracting heavily cross-linked proteins and proteoglycans from tissues such as tendons9, cartilage10, vessels11,12,13 and the heart2,3. We applied this biochemical fractionation, in combination with LC-MS/MS, to explore ECM remodeling in cardiovascular disease2,3,11,12,13,14. Recent advances in MS include novel fragmentation methods, such as combinations of higher-energy collision dissociation (HCD) and electron transfer dissociation (ETD), which allow for the direct analysis of intact glycopeptides3,15.
Here we describe a methodology to prepare ECM for MS analysis that allows for the analysis of protein composition, the identification of glycosylation sites, and the characterization of glycan forms. Compared to previous analyses of ECM glycosylation16, this methodology allows for the direct assessment of compositional changes in glycosylation profiles in a site-specific manner using MS. We have applied this methodology to cardiovascular tissues. However, it can also be applied, with minor modifications, to the study of the ECM in other tissue specimens and can provide unprecedented insights into ECM biology.

<table><tbody><tr><td>&lt;strong&gt;A. Chemicals&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Acetonitrile, MS-grade&amp;nbsp;(ACN, C&lt;sub&gt;2&lt;/sub&gt;H&lt;sub&gt;3&lt;/sub&gt;N)</td><td>Thermo Scientific</td><td>51101</td><td>5.2-5.8, 6.2, 7.11, Supp 2, 3, 4</td></tr><tr><td>Cocktail of proteinase inhibitors</td><td>Sigma-Aldrich</td><td>P8340</td><td>1.3,&amp;nbsp; 1.4.1, 1.5.1, 1.6.1</td></tr><tr><td>Disodium phosphate (Na&lt;sub&gt;2&lt;/sub&gt;H&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>S7907</td><td>3.1</td></tr><tr><td>Dithiotreitol (DTT, C&lt;sub&gt;4&lt;/sub&gt;H&lt;sub&gt;10&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;S&lt;sub&gt;2&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>D0632</td><td>4.3</td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA, C&lt;sub&gt;10&lt;/sub&gt;H&lt;sub&gt;16&lt;/sub&gt;N&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;8&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>E9884</td><td>1.3,&amp;nbsp; 1.4.1, 1.5.1, 1.6.1</td></tr><tr><td>Ethanol (C&lt;sub&gt;2&lt;/sub&gt;H&lt;sub&gt;6&lt;/sub&gt;O)</td><td>VWR</td><td>437433T</td><td>2.2.1</td></tr><tr><td>Guanidine hydrochloride (GuHCl, CH&lt;sub&gt;6&lt;/sub&gt;ClN&lt;sub&gt;3&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>G3272</td><td>1.6.1</td></tr><tr><td>Glycerol (C&lt;sub&gt;3&lt;/sub&gt;H&lt;sub&gt;8&lt;/sub&gt;O&lt;sub&gt;3&lt;/sub&gt;)</td><td>Acros organics</td><td>158920025</td><td>Suppl 1.1</td></tr><tr><td>H&lt;sub&gt;2&lt;/sub&gt;O LC-MS Cromasolv</td><td>Sigma-Aldrich</td><td>39253-1L-R</td><td>Throughout the protocol</td></tr><tr><td>H&lt;sub&gt;2&lt;/sub&gt;&lt;sup&gt;18&lt;/sup&gt;O</td><td>Taiyo Nippon Sanso</td><td>FO3-0027</td><td>3.5</td></tr><tr><td>Hydroxylamine (HA, H&lt;sub&gt;3&lt;/sub&gt;NO)</td><td>Sigma-Aldrich</td><td>467804</td><td>6.4</td></tr><tr><td>Iodoacetamide (IAA, C&lt;sub&gt;2&lt;/sub&gt;H&lt;sub&gt;4&lt;/sub&gt;INO)</td><td>Sigma-Aldrich</td><td>A3221</td><td>4.4</td></tr><tr><td>Phosphate-buffered Saline (PBS), 10x</td><td>Lonza</td><td>51226</td><td>1.3</td></tr><tr><td>Sodium acetate (C&lt;sub&gt;2&lt;/sub&gt;H&lt;sub&gt;3&lt;/sub&gt;NaO&lt;sub&gt;2&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>S7545</td><td>1.6.1</td></tr><tr><td>Sodium chloride (NaCl)</td><td>Sigma-Aldrich</td><td>S9888</td><td>1.4.1, 3.2</td></tr><tr><td>Sodium dodecyl sulfate (SDS, NaC&lt;sub&gt;12&lt;/sub&gt;H&lt;sub&gt;25&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>466143</td><td>1.5.1</td></tr><tr><td>Triethylammonium bicarbonate (TEAB, C&lt;sub&gt;7&lt;/sub&gt;H&lt;sub&gt;17&lt;/sub&gt;NO&lt;sub&gt;3&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>11268</td><td>4.7, 6.1</td></tr><tr><td>Trifluoroacetic acid (TFA, C&lt;sub&gt;2&lt;/sub&gt;HF&lt;sub&gt;3&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>T62200</td><td>4.8, 5.2-5.8, 7.11, Supp 2, 3</td></tr><tr><td>Thiourea (CH&lt;sub&gt;4&lt;/sub&gt;N&lt;sub&gt;2&lt;/sub&gt;S)</td><td>Sigma-Aldrich</td><td>T8656</td><td>4.2</td></tr><tr><td>Tris-hydrochloride (Tris-HCl, NH&lt;sub&gt;11&lt;/sub&gt;C&lt;sub&gt;4&lt;/sub&gt;O&lt;sub&gt;3&lt;/sub&gt;[HCl])</td><td>Sigma-Aldrich</td><td>T3253</td><td>1.4.1, Suppl 1.</td></tr><tr><td>Urea (CH&lt;sub&gt;4&lt;/sub&gt;N&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Sigma-Aldrich</td><td>U1250</td><td>4.2</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;B. Enzymes&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&amp;alpha;2-3,6,8,9-Neuraminidase (Sialidase)</td><td>EDM Millipore</td><td>362280 (KP0012)</td><td>3.1</td></tr><tr><td>&amp;beta;1,4-Galactosidase</td><td>EDM Millipore</td><td>362280 (KP0004)</td><td>3.1</td></tr><tr><td>&amp;beta;-N-Acetylglucosaminidase</td><td>EDM Millipore</td><td>362280 (KP0013)</td><td>3.1</td></tr><tr><td>Chondroitinase ABC</td><td>Sigma-Aldrich</td><td>C3667</td><td>3.1</td></tr><tr><td>Endo-&amp;alpha;-N-acetylgalactosaminidase (O-glycosidase)</td><td>EDM Millipore</td><td>362280 (KP0011)</td><td>3.1</td></tr><tr><td>Heparinase II</td><td>Sigma-Aldrich</td><td>H6512</td><td>3.1</td></tr><tr><td>Keratanase</td><td>Sigma-Aldrich</td><td>G6920</td><td>3.1</td></tr><tr><td>PNGase-F (N-Glycosidase F)</td><td>EDM Millipore</td><td>362280 (KP0001)</td><td>3.5</td></tr><tr><td>Trypsin</td><td>Thermo Scientific</td><td>90057</td><td>4.7</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;C. Reagent kits&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>30 kDa MWCO spin filters&amp;nbsp;</td><td>Amicon, Millipore</td><td>10256744</td><td>5.9, Suppl 2</td></tr><tr><td>Macro SpinColumn C-18, 96-Well Plate</td><td>Harvard Apparatus</td><td>74-5657</td><td>5.1</td></tr><tr><td>NuPAGE Novex BisTris Acrylamide Gels</td><td>Thermo-Scientific</td><td>NP0322PK2</td><td>Suppl 1</td></tr><tr><td>Pierce BCA Protein Assay Kit</td><td>Thermo Scientific</td><td>23227</td><td>2.1.3</td></tr><tr><td>ProteoExtract Glycopeptide Enrichment Kit</td><td>Merk Millipore</td><td>72103</td><td>7</td></tr><tr><td>Tandem mass tag 0 (TMT&lt;sup&gt;0&lt;/sup&gt;)</td><td>Thermo Scientific</td><td>900067</td><td>6.2, 6.3</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;D. Equipment and software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Acclaim PepMap100 C18 Trap, 5 mm x 300 &amp;micro;m, 5 &amp;micro;m, 100 &amp;Aring;</td><td>Thermo Scientific</td><td>160454</td><td>Suppl 3, 4</td></tr><tr><td>Acclaim PepMap100 C18, 50 cm x 75 &amp;micro;m, 3 &amp;micro;m, 100 &amp;Aring;</td><td>Thermo Scientific</td><td>164570</td><td>Suppl 3</td></tr><tr><td>Byonic Search Engine</td><td>Protein Metrics</td><td>Version 2.9.30</td><td>Suppl 5</td></tr><tr><td>Dionex UltiMate 3000 RSLCnano</td><td>Thermo Scientific</td><td>n/a</td><td>Suppl 3, 4</td></tr><tr><td>EASY-Spray Ion Source&amp;nbsp;</td><td>Thermo Scientific</td><td>ES081</td><td>Suppl 4</td></tr><tr><td>EASY-Spray PepMap RSLC C18,&amp;nbsp; 50 cm x 75 &amp;micro;m, 2 &amp;mu;m, 100 &amp;Aring;</td><td>Thermo Scientific</td><td>ES803</td><td>Suppl 4</td></tr><tr><td>Mascot Search Engine</td><td>Matrix Science</td><td>Version 2.3.01</td><td>Suppl 3</td></tr><tr><td>Orbitrap Fusion Lumos Tribrid Mass Spectrometer</td><td>Thermo Scientific</td><td>IQLAAEGAAPFADBMBHQ</td><td>Suppl 4</td></tr><tr><td>Proteome Discoverer Software</td><td>Thermo Scientific</td><td>Version 2.1.1.21</td><td>Suppl 3, 5</td></tr><tr><td>Picoview Nanospray Source</td><td>New Objective</td><td>550</td><td>Suppl 3</td></tr><tr><td>Q Exactive HF Mass Spectrometer</td><td>Thermo Scientific</td><td>IQLAAEGAAPFALGMBFZ</td><td>Suppl 3</td></tr><tr><td>Savant SpeedVac Concentrator</td><td>Thermo Scientific</td><td>SPD131DDA</td><td>2.2.2, 3.4, 4.6, 5.7, 6.5, 7.11</td></tr><tr><td>Scaffold Software</td><td>Proteome Software&amp;nbsp;</td><td>Version 4.3.2</td><td>Suppl 3</td></tr></tbody></table>

glycoproteomics of the extracellular matrix: a method for intact glycopeptide analysis using mass spectrometry

Fibrosis is a hallmark of many cardiovascular diseases and is associated with the exacerbated secretion and deposition of the extracellular matrix (ECM). Using proteomics, we have previously identified more than 150 ECM and ECM-associated proteins in cardiovascular tissues. Notably, many ECM proteins are glycosylated. This post-translational modification affects protein folding, solubility, binding, and degradation. We have developed a sequential extraction and enrichment method for ECM proteins that is compatible with the subsequent liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of intact glycopeptides. The strategy is based on sequential incubations with NaCl, SDS for tissue decellularization, and guanidine hydrochloride for the solubilization of ECM proteins. Recent advances in LC-MS/MS include fragmentation methods, such as combinations of higher-energy collision dissociation (HCD) and electron transfer dissociation (ETD), which allow for the direct compositional analysis of glycopeptides of ECM proteins. In the present paper, we describe a method to prepare the ECM from tissue samples. The method not only allows for protein profiling but also the assessment and characterization of glycosylation by MS analysis.

A schematic workflow of the protocol is provided in Figure 1.
ECM extraction protocol
The efficiency of the extraction can be monitored by running aliquots form each extract on Bis-Tris acrylamide gels and using silver staining for visualization. Figure 2A shows the complementarity of the NaCl, SDS and GuHCl extracts after sequential extraction. This QC allows for identification of potential issues with sample quality such as excessive protein degradation. After extraction, ECM glycoproteins are abundant in the GuHCl extracts (Figure 2B).
Deglycosylation
To assess the efficiency of deglycosylation, a non-deglycosylated control should be run in parallel. Deglycosylation times have to be suitable to achieve a complete and homogeneous removal of sugar residues, as exemplified in Figure 3A. Figure 3B shows a representative example of samples efficiently deglycosylated by the addition of enzymes for GAG removal and deglycosylation enzymes that target smaller N- and O-linked oligosaccharides.
Glycoproteomics
The protocol for assessment of the occupancy of NxT/S sequons improves protein sequence coverage for ECM glycoproteins after MS (Figure 3C) and allows for an initial screening of the presence of glycoproteins. This helps to reduce the search time for glycopeptides, as databases can be customized to contain previously identified glycoproteins. HCD-ETD fragmentation is used for identification and compositional characterization of oligosaccharides attached to ECM glycoproteins. Figure 4A displays a representative spectrum obtained for a peptide labeled with 18O after deglycosylation (indirect glycopeptide analysis). Figure 4B is a representative example of a spectrum obtained after analysis of intact glycopeptides from ECM extracts (direct glycopeptide analysis).
<img alt="Figure 1" src="/files/ftp_upload/55674/55674fig1.jpg" /> 
Figure 1: Method Overview. (A) After sequential enrichment for ECM proteins, LC-MS/MS analyses are performed on the deglycosylated extracts. (B) Alternatively, non deglycosylated ECM extracts are further enriched for glycopeptides. <a href="http://cloudflare.jove.com/files/ftp_upload/55674/55674fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55674/55674fig2.jpg" /> 
Figure 2: Extraction of ECM Proteins. (A) The 3 different extracts from the sequential extraction procedure (&#34;English Quickstep&#34;) are complementary in their protein content. While SDS extracts are enriched in intracellular proteins, GuHCl extracts contain the majority of ECM proteins. Successful fractionation is visualized by the different silver staining pattern. (B) ECM proteins such as the small leucine-rich proteoglycans decorin, biglycan and mimecan are predominantly detected in the GuHCl extracts, with little presence in the SDS and NaCl extracts. <a href="http://cloudflare.jove.com/files/ftp_upload/55674/55674fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55674/55674fig3.jpg" /> 
Figure 3. Analysis of Glycosylation. (A) Appropriate incubation times are required for complete deglycosylation. The example shows the effect of incubation time during removal of glycosaminoglycan chains from the glycoprotein decorin. (B) ECM glycoproteins are decorated with large and repetitive glycosaminoglycan chains and short and diverse N- and O-linked oligosaccharides. Lane 1 on each of the immunoblots represents untreated cardiac extracts. Lane 2 contains extracts treated with enzymes that digest glycosaminoglycans. Samples in lane 3 contain, in addition, enzymes for the removal of N- and O-linked oligossacharides. Galectin-1 is not glycosylated, hence there is no shift in protein size. Adapted from Lynch M, et al.4 (C) In LC-MS/MS analysis, samples treated with PNGase-F in the presence of H218O achieve better sequence coverage (%, on the right side) compared to non-deglycosylated samples. Dark green areas represent sequence coverage by LC-MS/MS. The red, dotted lines represent glycosites, with numbers indicating their amino acid position. Detection of glycosylation of decorin at position Asn211 (N, highlighted in bold) is shown in detail as an example in Figure 4. <a href="http://cloudflare.jove.com/files/ftp_upload/55674/55674fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/55674/55674fig4.jpg" /> 
Figure 4. Glycopeptide Analysis by MS. (A) Using a shotgun proteomics approach on ECM enriched extracts, glycopeptides can be identified by the presence of deamidated asparagines within NxT/S sequons and labeled with 18O. The example shows a HCD MS/MS spectrum for a peptide of decorin containing the previously glycosylated Asn211. The data obtained can be used to create a customized database of ECM glycoproteins. (B) HCD-ETD fragmentation is used to analyze the glycopeptide enriched ECM extract. The ETD MS/MS spectrum allows the characterization of glycan composition. <a href="http://cloudflare.jove.com/files/ftp_upload/55674/55674fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>A. Stock solutions</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT (Dithiotreitol, C4H10O2S2)</td>
			<td>100 mM DTT in ddH2O.1</td>
		</tr>
		<tr>
			<td>EDTA (Ethylenediaminetetraacetic acid, C10H16N2O8)</td>
			<td>250 mM EDTA in ddH2O, pH 8.0.</td>
		</tr>
		<tr>
			<td>GuHCl (Guanidine hydrochloride, CH6ClN3)</td>
			<td>8 M GuHCl in ddH2O.</td>
		</tr>
		<tr>
			<td>IAA (Iodoacetamide, C2H4INO)</td>
			<td>500 mM IAA in ddH2O.1,2</td>
		</tr>
		<tr>
			<td>Na acetate (Sodium acetate, C2H3NaO2)</td>
			<td>1 M Na acetate in ddH2O, pH 5.8.</td>
		</tr>
		<tr>
			<td>NaCl (Sodium chloride, NaCl)&#160;</td>
			<td>1 M NaCl in ddH2O.</td>
		</tr>
		<tr>
			<td>Na phosphate dibasic (Disodium phosphate, Na2H2PO4)</td>
			<td>1 M Na phosphate dibasic in ddH2O, pH 6.8.</td>
		</tr>
		<tr>
			<td>SDS (Sodium dodecyl sulfate, NaC12H25SO4)</td>
			<td>1% SDS (35 mM) in ddH2O.3</td>
		</tr>
		<tr>
			<td>TFA (Trifluoroacetic acid, C2HF3O2)</td>
			<td>10% TFA (1.2 M)&#160; in ddH2O.</td>
		</tr>
		<tr>
			<td>TEAB (Triethylammonium bicarbonate, C7H17NO3)</td>
			<td>1M TEAB in in ddH2O, pH 8.5</td>
		</tr>
		<tr>
			<td>Thiourea (Thiourea, CH4N2S)</td>
			<td>3 M thiourea in ddH2O.</td>
		</tr>
		<tr>
			<td>Tris-HCl (Tris-hydrochloride (NH11C4O3[HCl])</td>
			<td>100 mM Tris&#8211;HCl in ddH2O, pH 7.5.</td>
		</tr>
		<tr>
			<td>Urea (Urea, CH4N2O)</td>
			<td>9 M urea in ddH2O.</td>
		</tr>
		<tr>
			<td>B. Reaction buffers</td>
			<td></td>
		</tr>
		<tr>
			<td>C18 clean-up equilibration buffer</td>
			<td>1% ACN, 0.1% TFA in ddH2O</td>
		</tr>
		<tr>
			<td>C18 clean-up column wash buffer</td>
			<td>80% ACN, 0.1% TFA in H2O</td>
		</tr>
		<tr>
			<td>C18 clean-up elution buffer</td>
			<td>50% acetonitrile, 0.1% TFA in ddH2O</td>
		</tr>
		<tr>
			<td>Deglycosylation Buffer (4x)</td>
			<td>600 mM NaCl and 200 mM Na phosphate in ddH2O, pH 6.8.</td>
		</tr>
		<tr>
			<td>GuHCl buffer4</td>
			<td>4 M guanidine hydrochloride, 50 mM Na acetate and 25 mM EDTA in ddH2O, pH 5.8. Add 1:100 (v:v) of cocktail of proteinase inhibitors before use.</td>
		</tr>
		<tr>
			<td>NaCl buffer4</td>
			<td>0.5 M NaCl, 10 mM Tris-HCl and 25 mM EDTA in ddH2O, pH 7.5. Add 1:100 (v:v) of cocktail of proteinase inhibitors before use.</td>
		</tr>
		<tr>
			<td>PBS (1x)</td>
			<td>1.7 mM KH2PO4, 5 mM Na2HPO4, 150 mM NaCl, pH 7.4. Add 25 mM EDTA and 1:100 (v:v) of cocktail of proteinase inhibitors before use.</td>
		</tr>
		<tr>
			<td>Sample buffer (4x)</td>
			<td>100 mM Tris, 2% SDS, 40% Glycerol, 0.02% bromophenol blue in ddH2O, pH 6.8. Add 10% &#223;-mercaptoethanol before use.</td>
		</tr>
		<tr>
			<td>SDS buffer4&#160;</td>
			<td>0.1 % SDS and 25 mM EDTA in ddH2O. Add 1:100 (v:v) of cocktail of proteinase inhibitors before use.</td>
		</tr>
		<tr>
			<td>C. Enzymes</td>
			<td></td>
		</tr>
		<tr>
			<td>Chondroitinase ABC5&#160;</td>
			<td>0.5 U/mL in deglycosylation buffer (1x)</td>
		</tr>
		<tr>
			<td>Keratanase5</td>
			<td>0.1 U/mL in deglycosylation buffer (1x)</td>
		</tr>
		<tr>
			<td>Heparinase II5</td>
			<td>0.1 U/mL in deglycosylation buffer (1x)</td>
		</tr>
		<tr>
			<td>&#945;2-3,6,8,9-Neuraminidase (sialidase)5</td>
			<td>0.025 U/mL in deglycosylation buffer (1x)</td>
		</tr>
		<tr>
			<td>&#946;1,4-Galactosidase5</td>
			<td>0.015 U/mL in deglycosylation buffer (1x)</td>
		</tr>
		<tr>
			<td>&#946;-N-Acetylglucosaminidase5</td>
			<td>0.25 U/mL in deglycosylation buffer (1x)</td>
		</tr>
		<tr>
			<td>Endo-&#945;-N-acetylgalactosaminidase (O-glycosidase)5</td>
			<td>0.013 U/mL in deglycosylation buffer (1x)</td>
		</tr>
		<tr>
			<td>PNGase-F(N-glycosidase-F)6</td>
			<td>50 U/mL in H218O</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>0.01 &#181;g/&#181;L in TEAB buffer</td>
		</tr>
		<tr>
			<td>Table NOTES.</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">1 Keep stock solution frozen at -20 &#176;C.</td>
		</tr>
		<tr>
			<td colspan="2">2 IAA should be kept protected from light.</td>
		</tr>
		<tr>
			<td colspan="2">3 SDS readily crystallizes at &#60; 20 &#176;C. In order to facilitate solubilization of 1% SDS (stock solution), warm the buffer under hot tap water.&#160;</td>
		</tr>
		<tr>
			<td colspan="2">4 Extraction buffers can be stored at RT. Add broad-spectrum cocktail of proteinase inhibitors as indicated before use.</td>
		</tr>
		<tr>
			<td colspan="2">5 These enzymes should be added during the first deglycosylation step.</td>
		</tr>
		<tr>
			<td colspan="2">6 PNGase-F should be only added during the second deglycosylation step.</td>
		</tr>
	</tbody>
</table>
Table 1: Stock Solutions, Reaction Buffers and Enzymes. This table lists the composition of each stock solution and reaction buffer required for the extraction and subsequent processing (including enzymatic digestions) of cardiac ECM proteins prior to MS analysis.

Watch this Scientific Journal Video about Glycoproteomics of the Extracellular Matrix: A Method for Intact Glycopeptide Analysis Using Mass Spectrometry at JoVE.com

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

This paper describes a methodology to prepare cardiovascular tissue samples for MS analysis that allows for (1) the analysis of ECM protein composition, (2) the identification of glycosylation sites, and (3) the compositional characterization of glycan forms. This methodology can be applied, with minor modifications, to the study of the ECM in other tissues.

Fibrosis is a hallmark of many cardiovascular diseases and is associated with the exacerbated secretion and deposition of the extracellular matrix (ECM). ...

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12.2K Views.  King's College London. This paper describes a methodology to prepare cardiovascular tissue samples for MS analysis that allows for (1) the analysis of ECM protein composition, (2) the identification of glycosylation sites, and (3) the compositional characterization of glycan forms. This methodology can be applied, with minor modifications, to the study of the ECM in other tissues.

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

Glycan Node Analysis: A Bottom-up Approach to Glycomics

Synthesized in a non-template-driven process by enzymes called glycosyltransferases, glycans are key players in various significant intra- and extracellular events. Many pathological conditions, notably cancer, affect gene expression, which can in turn deregulate the relative abundance and activity levels of glycoside hydrolase and glycosyltransferase enzymes. Unique aberrant whole glycans resulting from deregulated glycosyltransferase(s) are often present in trace quantities within complex biofluids, making their detection difficult and sometimes stochastic. However, with proper sample preparation, one of the oldest forms of mass spectrometry (gas chromatography-mass spectrometry, GC-MS) can routinely detect the collection of branch-point and linkage-specific monosaccharides ("glycan nodes") present in complex biofluids. Complementary to traditional top-down glycomics techniques, the approach discussed herein involves the collection and condensation of each constituent glycan node in a sample into a single independent analytical signal, which provides detailed structural and quantitative information about changes to the glycome as a whole and reveals potentially deregulated glycosyltransferases. Improvements to the permethylation and subsequent liquid/liquid extraction stages provided herein enhance reproducibility and overall yield by facilitating minimal exposure of permethylated glycans to alkaline aqueous conditions. Modifications to the acetylation stage further increase the extent of reaction and overall yield. Despite their reproducibility, the overall yields of N-acetylhexosamine (HexNAc) partially permethylated alditol acetates (PMAAs) are shown to be inherently lower than their expected theoretical value relative to hexose PMAAs. Calculating the ratio of the area under the extracted ion chromatogram (XIC) for each individual hexose PMAA (or HexNAc PMAA) to the sum of such XIC areas for all hexoses (or HexNAcs) provides a new normalization method that facilitates relative quantification of individual glycan nodes in a sample. Although presently constrained in terms of its absolute limits of detection, this method expedites the analysis of clinical biofluids and shows considerable promise as a complementary approach to traditional top-down glycomics.

This article presents an enhanced form of a novel bottom-up glycomics technique designed to analyze the pooled compositional profile of glycans in unfractionated biofluids through the chemical breakdown of glycans into their constituent linkage-specific monosaccharides for detection by GC-MS. Potential applications include early detection of cancer and other glycan-affective disorders.

Synthesized in a non-template-driven process by enzymes called glycosyltransferases, glycans are key players in various significant intra- and ...

Chemistry

Extracellular Protein Microarray Technology for High Throughput Detection of Low Affinity Receptor-Ligand Interactions

Secreted factors, membrane-tethered receptors, and their interacting partners are main regulators of cellular communication and initiation of signaling cascades during homeostasis and disease, and as such represent prime therapeutic targets. Despite their relevance, these interaction networks remain significantly underrepresented in current databases; therefore, most extracellular proteins have no documented binding partner. This discrepancy is primarily due to the challenges associated with the study of the extracellular proteins, including expression of functional proteins, and the weak, low affinity, protein interactions often established between cell surface receptors. The purpose of this method is to describe the printing of a library of extracellular proteins in a microarray format for screening of protein-protein interactions. To enable detection of weak interactions, a method based on multimerization of the query protein under study is described. Coupled to this microbead-based multimerization approach for increased multivalency, the protein microarray allows robust detection of transient protein-protein interactions in high throughput. This method offers a rapid and low sample consuming-approach for identification of new interactions applicable to any extracellular protein. Protein microarray printing and screening protocol are described. This technology will be useful for investigators seeking a robust method for discovery of protein interactions in the extracellular space.

Here, we present a protocol to screen extracellular protein microarrays for identification of novel receptor-ligand interactions in high throughput. We also describe a method to enhance detection of transient protein-protein interactions by using protein-microbead complexes.

Secreted factors, membrane-tethered receptors, and their interacting partners are main regulators of cellular communication and initiation of signaling ...

Mass Spectrometric Analysis of Glycosphingolipid Antigens

Glycosphingolipids (GSL's) belong to the glycoconjugate class of biomacromolecules, which bear structural information for significant biological processes such as embryonic development, signal transduction, and immune receptor recognition1-2. They contain complex sugar moieties in the form of isomers, and lipid moieties with variations including fatty acyl chain length, unsaturation, and hydroxylation. Both carbohydrate and ceramide portions may be basis of biological significance. For example, tri-hexosylceramides include globotriaosylceramide (Gal&alpha;4Gal&beta;4Glc&beta;1Cer) and isoglobotriaosylceramide (Gal&alpha;3Gal&beta;4Glc&beta;1Cer), which have identical molecular masses but distinct sugar linkages of carbohydrate moiety, responsible for completely different biological functions3-4. In another example, it has been demonstrated that modification of the ceramide part of alpha-galactosylceramide, a potent agonist ligand for invariant NKT cells, changes their cytokine secretion profiles and function in animal models of cancer and auto-immune diseases5. The difficulty in performing a structural analysis of isomers in immune organs and cells serve as a barrier for determining many biological functions6.
Here, we present a visualized version of a method for relatively simple, rapid, and sensitive analysis of glycosphingolipid profiles in immune cells7-9. This method is based on extraction and chemical modification (permethylation, see below Figure 5A, all OH groups of hexose were replaced by MeO after permethylation reaction) of glycosphingolipids10-15, followed by subsequent analysis using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) and ion trap mass spectrometry. This method requires 50 million immune cells for a complete analysis. The experiments can be completed within a week. The relative abundance of the various glycosphingolipids can be delineated by comparison to synthetic standards. This method has a sensitivity of measuring 1% iGb3 among Gb3 isomers, when 2 fmol of total iGb3/Gb3 mixture is present9.
Ion trap mass spectrometry can be used to analyze isomers. For example, to analyze the presence of globotriaosylceramide and isoglobtriaosylceramide in the same sample, one can use the fragmentation of glycosphingolipid molecules to structurally discriminate between the two (see below Figure 5). Furthermore, chemical modification of the sugar moieties (through a permethylation reaction) improves the ionization and fragmentation efficiencies for higher sensitivity and specificity, and increases the stability of sialic acid residues. The extraction and chemical modification of glycosphingolipids can be performed in a classic certified chemical hood, and the mass spectrometry can be performed by core facilities with ion trap MS instruments.

A specific and sensitive method to gain insight into the expression profile of glycosphingolipid antigens in immune organs and cells is described. The method takes advantage of the ion trap mass spectrometry allowing step-wise fragmentation of glycosphingolipid molecules for structural analysis in comparison to chemically synthesized standards.

Glycosphingolipids (GSL's) belong to the glycoconjugate class of biomacromolecules, which bear structural information for significant biological processes ...

Immunology and Infection

N-glycan Profiling of Glycoproteins by Hydrophilic Interaction Liquid Chromatography with Fluorescence and Mass Spectrometric Detection

Glycosylation is a vital modification found in proteins. N-glycan profiling of glycoproteins is required to detect novel biomarker candidates and determine glycan alterations in diseases. Most commercially available biopharmaceutical proteins are glycoproteins. The efficacy of these drugs is affected by glycosylation patterns. Therefore, an in-depth characterization method for the N-glycans is necessary. Here, we present a comprehensive approach for qualitative and quantitative analysis of N-glycans using hydrophilic interaction liquid chromatography equipped with fluorescence detection and tandem mass spectrometry (HILIC-FLD-MS/MS). N-glycans were released from glycoproteins with a facile method and labeled by a procainamide fluorophore tag in the strategy. Subsequently, the procainamide labeled N-glycans were analyzed by a HILIC-FLD-MS/MS technique. In this approach, N-glycan structures were confirmed by the tandem mass spectrometric analysis, whereas fluorescence detection was used for the quantitative analysis. An application for data analysis of the detected N-glycan peaks is described in the study. This protocol can be applied to any glycoprotein extracted from various species.

N-glycan Profiling of Glycoproteins by Hydrophilic Interaction Liquid Chromatography with Fluorescence and Mass Spectrometric Detection

N-glycan profiling of glycoproteins is essential for discovering novel biomarkers and understanding glycan functions in cellular events. Additionally, N-glycan analysis of protein biopharmaceuticals is very important for human use. In this current article, a high-throughput strategy for identifying and quantifying N-glycan structures was presented using the HILIC-FLD-MS/MS technique.

Glycosylation is a vital modification found in proteins. N-glycan profiling of glycoproteins is required to detect novel biomarker candidates and ...

A Spin-Tip Enrichment Strategy for Simultaneous Analysis of N-Glycopeptides and Phosphopeptides from Human Pancreatic Tissues

Mass spectrometry can provide deep coverage of post-translational modifications (PTMs), although enrichment of these modifications from complex biological matrices is often necessary due to their low stoichiometry in comparison to non-modified analytes. Most enrichment workflows of PTMs on peptides in bottom-up proteomics workflows, where proteins are enzymatically digested before the resulting peptides are analyzed, only enrich one type of modification. It is the entire complement of PTMs, however, that leads to biological functions, and enrichment of a single type of PTM may miss such crosstalk of PTMs. PTM crosstalk has been observed between protein glycosylation and phosphorylation, the two most common PTMs in human proteins and also the two most studied PTMs using mass spectrometry workflows. Using the simultaneous enrichment strategy described herein, both PTMs are enriched from post-mortem human pancreatic tissue, a complex biological matrix. Dual-functional Ti(IV)-immobilized metal affinity chromatography is used to separate various forms of glycosylation and phosphorylation simultaneously in multiple fractions in a convenient spin tip-based method, allowing downstream analyses of potential PTM crosstalk interactions. This enrichment workflow for glyco- and phosphopeptides can be applied to various sample types to achieve deep profiling of multiple PTMs and identify potential target molecules for future studies.

Post-translational modifications (PTMs) change protein structures and functions. Methods for the simultaneous enrichment of multiple PTM types can maximize coverage in analyses. We present a protocol using dual-functional Ti(IV)-immobilized metal affinity chromatography followed by mass spectrometry for the simultaneous enrichment and analysis of protein N-glycosylation and phosphorylation in pancreatic tissues.

Mass spectrometry can provide deep coverage of post-translational modifications (PTMs), although enrichment of these modifications from complex biological ...

OLIgo Mass Profiling (OLIMP) of Extracellular Polysaccharides

The direct contact of cells to the environment is mediated in many organisms by an extracellular matrix. One common aspect of extracellular matrices is that they contain complex sugar moieties in form of glycoproteins, proteoglycans, and/or polysaccharides. Examples include the extracellular matrix of humans and animal cells consisting mainly of fibrillar proteins and proteoglycans or the polysaccharide based cell walls of plants and fungi, and the proteoglycan/glycolipid based cell walls of bacteria. All these glycostructures play vital roles in cell-to-cell and cell-to-environment communication and signalling.
An extraordinary complex example of an extracellular matrix is present in the walls of higher plant cells. Their wall is made almost entirely of sugars, up to 75% dry weight, and consists of the most abundant biopolymers present on this planet. Therefore, research is conducted how to utilize these materials best as a carbon-neutral renewable resource to replace petrochemicals derived from fossil fuel. The main challenge for fuel conversion remains the recalcitrance of walls to enzymatic or chemical degradation due to the unique glycostructures present in this unique biocomposite.
Here, we present a method for the rapid and sensitive analysis of plant cell wall glycostructures. This method OLIgo Mass Profiling (OLIMP) is based the enzymatic release of oligosaccharides from wall materials facilitating specific glycosylhydrolases and subsequent analysis of the solubilized oligosaccharide mixtures using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS)1 (Figure 1). OLIMP requires walls of only 5000 cells for a complete analysis, can be performed on the tissue itself2, and is amenable to high-throughput analyses3. While the absolute amount of the solubilized oligosaccharides cannot be determined by OLIMP the relative abundance of the various oligosaccharide ions can be delineated from the mass spectra giving insights about the substitution-pattern of the native polysaccharide present in the wall.
OLIMP can be used to analyze a wide variety of wall polymers, limited only by the availability of specific enzymes4. For example, for the analysis of polymers present in the plant cell wall enzymes are available to analyse the hemicelluloses xyloglucan using a xyloglucanase5, 11, 12, 13, xylan using an endo-&beta;-(1-4)-xylanase 6,7, or for pectic polysaccharides using a combination of a polygalacturonase and a methylesterase 8. Furthermore, using the same principles of OLIMP glycosylhydrolase and even glycosyltransferase activities can be monitored and determined 9.

OLIgo Mass Profiling OLIMP of Extracellular Polysaccharides

A rapid way is described to gain insights into the structure of polysaccharides in an extracellular matrix. The method takes advantage of the specificity of glycosylhydrolases and the sensitivity of mass spectrometry allowing minute amounts of materials to be analyzed. This technique is adaptable to be used directly on tissue itself.

The direct contact of cells to the environment is mediated in many organisms by an extracellular matrix. One common aspect of extracellular matrices is ...

Biology

Profiling of Colorectal Cancer Tissues Using Glycomics-Guided Glycoproteomics

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

Glycosylation is a common and structurally diverse protein modification that impacts a wide range of tumorigenic processes. Mass spectrometry-driven glycomics and glycoproteomics have emerged as powerful approaches to analyze liberated glycans and intact glycopeptides, respectively, offering a deeper understanding of the heterogeneous glycoproteome in the tumor microenvironment (TME). This protocol details the glycomics-guided glycoproteomics method, an integrated omics technology, which firstly employs porous graphitized carbon-LC-MS/MS-based glycomics to elucidate the glycan structures and their quantitative distribution in the glycome of tumor tissues, cell populations, or bodily fluids being investigated. This allows for a comparative glycomics analysis to identify altered glycosylation across patient groups, disease stages, or conditions, and, importantly, serves to enhance the downstream glycoproteomics analysis of the same sample(s) by creating a library of known glycan structures, thus reducing the data search time and the glycoprotein misidentification rate. Focusing on N-glycoproteome profiling, the sample preparation steps for the glycomics-guided glycoproteomics method are detailed in this protocol, and key aspects of the data collection and analysis are discussed. The glycomics-guided glycoproteomics method provides quantitative information on the glycoproteins present in the TME and their glycosylation sites, site occupancy, and site-specific glycan structures. Representative results are presented from formalin-fixed paraffin-embedded tumor tissues from colorectal cancer patients, demonstrating that the method is sensitive and compatible with tissue sections commonly found in biobanks. Glycomics-guided glycoproteomics, therefore, offers a comprehensive view into the heterogeneity and dynamics of the glycoproteome in complex TMEs, generating robust biochemical data required to better understand the glycobiology of cancers.

This protocol details the glycomics-guided glycoproteomics method, an integrated omics technology that offers comprehensive insights into the heterogeneous glycoproteome in complex tumor microenvironments required to better understand the glycobiology of cancers.

Glycosylation is a common and structurally diverse protein modification that impacts a wide range of tumorigenic processes. Mass spectrometry-driven ...

Cancer Research

A Streamlined Approach for Mass Spectrometry-Based Proteomics Using Selected Tissue Regions

Mass spectrometry (MS)-based proteomics enables comprehensive proteome analysis across a wide range of biological samples, including cells, tissues, and body fluids. Formalin-fixed, paraffin-embedded (FFPE) tissue sections, commonly used for long-term archiving, have emerged as valuable resources for proteomic studies. Beyond their storage benefits, researchers can isolate regions of interest (ROIs) from normal tissue regions through collaborative efforts with pathologists. Despite this potential, a streamlined approach for proteomic experiments encompassing ROI isolation, proteomic sample preparation, and MS analysis remains lacking. In this protocol, an integrated workflow that combines macrodissection of ROIs, suspension trapping-based sample preparation, and high-throughput MS analysis is presented. Through this approach, the ROIs of patients' FFPE tissues, consisting of benign serous cystic neoplasms (SCN) and precancerous intraductal papillary mucinous neoplasms (IPMN) diagnosed by pathologists, were macrodissected, collected, and analyzed, resulting in high proteome coverage. Furthermore, molecular differences between the two distinct pancreatic cystic neoplasms were successfully identified, thus demonstrating the applicability of this approach for advancing proteomic research with FFPE tissues.

Here, we establish a mass spectrometry-based proteomic method using isolated regions of interest in formalin-fixed, paraffin-embedded tissue sections. This protocol is used to analyze proteome from specific tissue areas in archived formalin-fixed, paraffin-embedded tissue sections.

Mass spectrometry (MS)-based proteomics enables comprehensive proteome analysis across a wide range of biological samples, including cells, tissues, and ...

Glycopeptide Capture for Cell Surface Proteomics

Cell surface proteins, including extracellular matrix proteins, participate in all major cellular processes and functions, such as growth, differentiation, and proliferation. A comprehensive characterization of these proteins provides rich information for biomarker discovery, cell-type identification, and drug-target selection, as well as helping to advance our understanding of cellular biology and physiology. Surface proteins, however, pose significant analytical challenges, because of their inherently low abundance, high hydrophobicity, and heavy post-translational modifications. Taking advantage of the prevalent glycosylation on surface proteins, we introduce here a high-throughput glycopeptide-capture approach that integrates the advantages of several existing N-glycoproteomics means. Our method can enrich the glycopeptides derived from surface proteins and remove their glycans for facile proteomics using LC-MS. The resolved N-glycoproteome comprises the information of protein identity and quantity as well as their sites of glycosylation. This method has been applied to a series of studies in areas including cancer, stem cells, and drug toxicity. The limitation of the method lies in the low abundance of surface membrane proteins, such that a relatively large quantity of samples is required for this analysis compared to studies centered on cytosolic proteins.

Cell surface proteins are biologically important and widely glycosylated. We introduce here a glycopeptide-capture approach to solubilize, enrich, and deglycosylate these proteins for facile LC-MS based proteomic analyses.

Cell surface proteins, including extracellular matrix proteins, participate in all major cellular processes and functions, such as growth, ...

Enrichment of Extracellular Matrix Proteins from Tissues and Digestion into Peptides for Mass Spectrometry Analysis

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of cell proliferation, survival, migration, etc. The ECM plays important roles in development and in diverse pathologies including cardio-vascular and musculo-skeletal diseases, fibrosis, and cancer. Thus, characterizing the composition of ECMs of normal and diseased tissues could lead to the identification of novel prognostic and diagnostic biomarkers and potential novel therapeutic targets. However, the very nature of ECM proteins (large in size, cross-linked and covalently bound, heavily glycosylated) has rendered biochemical analyses of ECMs challenging. To overcome this challenge, we developed a method to enrich ECMs from fresh or frozen tissues and tumors that takes advantage of the insolubility of ECM proteins. We describe here in detail the decellularization procedure that consists of sequential incubations in buffers of different pH and salt and detergent concentrations and that results in 1) the extraction of intracellular (cytosolic, nuclear, membrane and cytoskeletal) proteins and 2) the enrichment of ECM proteins. We then describe how to deglycosylate and digest ECM-enriched protein preparations into peptides for subsequent analysis by mass spectrometry.

This protocol describes a procedure for enriching ECM proteins from tissues or tumors and deglycosylating and digesting the ECM-enriched preparations into peptides to analyze their protein composition by mass spectrometry.

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of ...

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

Summary

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Chapters in this video

Glycan Node Analysis: A Bottom-up Approach to Glycomics

Extracellular Protein Microarray Technology for High Throughput Detection of Low Affinity Receptor-Ligand Interactions

Mass Spectrometric Analysis of Glycosphingolipid Antigens

N-glycan Profiling of Glycoproteins by Hydrophilic Interaction Liquid Chromatography with Fluorescence and Mass Spectrometric Detection

A Spin-Tip Enrichment Strategy for Simultaneous Analysis of N-Glycopeptides and Phosphopeptides from Human Pancreatic Tissues

OLIgo Mass Profiling (OLIMP) of Extracellular Polysaccharides

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

A Streamlined Approach for Mass Spectrometry-Based Proteomics Using Selected Tissue Regions

Glycopeptide Capture for Cell Surface Proteomics

Enrichment of Extracellular Matrix Proteins from Tissues and Digestion into Peptides for Mass Spectrometry Analysis