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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>A. Chemicals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Acetonitrile, MS-grade&#160;(ACN, C2H3N)</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,&#160; 1.4.1, 1.5.1, 1.6.1</td>
		</tr>
		<tr>
			<td>Disodium phosphate (Na2H2PO4)</td>
			<td>Sigma-Aldrich</td>
			<td>S7907</td>
			<td>3.1</td>
		</tr>
		<tr>
			<td>Dithiotreitol (DTT, C4H10O2S2)</td>
			<td>Sigma-Aldrich</td>
			<td>D0632</td>
			<td>4.3</td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA, C10H16N2O8)</td>
			<td>Sigma-Aldrich</td>
			<td>E9884</td>
			<td>1.3,&#160; 1.4.1, 1.5.1, 1.6.1</td>
		</tr>
		<tr>
			<td>Ethanol (C2H6O)</td>
			<td>VWR</td>
			<td>437433T</td>
			<td>2.2.1</td>
		</tr>
		<tr>
			<td>Guanidine hydrochloride (GuHCl, CH6ClN3)</td>
			<td>Sigma-Aldrich</td>
			<td>G3272</td>
			<td>1.6.1</td>
		</tr>
		<tr>
			<td>Glycerol (C3H8O3)</td>
			<td>Acros organics</td>
			<td>158920025</td>
			<td>Suppl 1.1</td>
		</tr>
		<tr>
			<td>H2O LC-MS Cromasolv</td>
			<td>Sigma-Aldrich</td>
			<td>39253-1L-R</td>
			<td>Throughout the protocol</td>
		</tr>
		<tr>
			<td>H218O</td>
			<td>Taiyo Nippon Sanso</td>
			<td>FO3-0027</td>
			<td>3.5</td>
		</tr>
		<tr>
			<td>Hydroxylamine (HA, H3NO)</td>
			<td>Sigma-Aldrich</td>
			<td>467804</td>
			<td>6.4</td>
		</tr>
		<tr>
			<td>Iodoacetamide (IAA, C2H4INO)</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 (C2H3NaO2)</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, NaC12H25SO4)</td>
			<td>Sigma-Aldrich</td>
			<td>466143</td>
			<td>1.5.1</td>
		</tr>
		<tr>
			<td>Triethylammonium bicarbonate (TEAB, C7H17NO3)</td>
			<td>Sigma-Aldrich</td>
			<td>11268</td>
			<td>4.7, 6.1</td>
		</tr>
		<tr>
			<td>Trifluoroacetic acid (TFA, C2HF3O2)</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 (CH4N2S)</td>
			<td>Sigma-Aldrich</td>
			<td>T8656</td>
			<td>4.2</td>
		</tr>
		<tr>
			<td>Tris-hydrochloride (Tris-HCl, NH11C4O3[HCl])</td>
			<td>Sigma-Aldrich</td>
			<td>T3253</td>
			<td>1.4.1, Suppl 1.</td>
		</tr>
		<tr>
			<td>Urea (CH4N2O)</td>
			<td>Sigma-Aldrich</td>
			<td>U1250</td>
			<td>4.2</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>B. Enzymes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#945;2-3,6,8,9-Neuraminidase (Sialidase)</td>
			<td>EDM Millipore</td>
			<td>362280 (KP0012)</td>
			<td>3.1</td>
		</tr>
		<tr>
			<td>&#946;1,4-Galactosidase</td>
			<td>EDM Millipore</td>
			<td>362280 (KP0004)</td>
			<td>3.1</td>
		</tr>
		<tr>
			<td>&#946;-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-&#945;-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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>C. Reagent kits</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>30 kDa MWCO spin filters&#160;</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 (TMT0)</td>
			<td>Thermo Scientific</td>
			<td>900067</td>
			<td>6.2, 6.3</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>D. Equipment and software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Acclaim PepMap100 C18 Trap, 5mm x 300&#181;m, 5&#181;m, 100&#197;</td>
			<td>Thermo Scientific</td>
			<td>160454</td>
			<td>Suppl 3, 4</td>
		</tr>
		<tr>
			<td>Acclaim PepMap100 C18, 50cm x 75&#181;m, 3&#181;m, 100&#197;</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&#160;</td>
			<td>Thermo Scientific</td>
			<td>ES081</td>
			<td>Suppl 4</td>
		</tr>
		<tr>
			<td>EASY-Spray PepMap RSLC C18,&#160; 50cm x 75&#181;m, 2&#956;m, 100&#197;</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&#160;</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

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological processes. Affinity purification coupled to mass spectrometry (AP-MS) has become the method of choice for identifying protein-protein interactions. However, conventional AP-MS studies provide information on protein interactions, but the organizational information is lost. To address this issue, we developed a strategy to unravel the distinct functional assemblies a protein might be involved in, by resolving affinity-purified protein complexes prior to their characterization by mass spectrometry. Protein complexes isolated through affinity purification of a bait protein using an epitope tag and competitive elution are separated through blue native electrophoresis. Comparison of protein migration profiles through correlation profiling using quantitative mass spectrometry allows assignment of interacting proteins to distinct molecular entities. This method is able to resolve protein complexes of close molecular weights that might not be resolved by traditional chromatographic techniques such as gel filtration. With little more work than conventional AP-geLC-MS/MS, we demonstrate this strategy may in many cases be adequate for obtaining protein complex topological information concomitantly to identifying protein interactions.

Here we present protocols for affinity purification of protein complexes and their separation by blue native PAGE, followed by protein correlation profiling using label free quantitative mass spectrometry. This method is useful to resolve interactomes into distinct protein complexes.

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological ...

A Tandem Liquid Chromatography&#8211;Mass Spectrometry-based Approach for Metabolite Analysis of Staphylococcus aureus

In an effort to thwart bacterial pathogens, hosts often limit the availability of nutrients at the site of infection. This limitation can alter the abundances of key metabolites to which regulatory factors respond, adjusting cellular metabolism. In recent years, a number of proteins and RNA have emerged as important regulators of virulence gene expression. For example, the CodY protein responds to levels of branched-chain amino acids and GTP and is widely conserved in low G+C Gram-positive bacteria. As a global regulator in Staphylococcus aureus, CodY controls the expression of dozens of virulence and metabolic genes. We hypothesize that S. aureus uses CodY, in part, to alter its metabolic state in an effort to adapt to nutrient-limiting conditions potentially encountered in the host environment. This manuscript describes a method for extracting and analyzing metabolites from S. aureus using liquid chromatography coupled with mass spectrometry, a protocol that was developed to test this hypothesis. The method also highlights best practices that will ensure rigor and reproducibility, such as maintaining biological steady state and constant aeration without the use of continuous chemostat cultures. Relative to the USA200 methicillin-susceptible S. aureus isolate UAMS-1 parental strain, the isogenic codY mutant exhibited significant increases in amino acids derived from aspartate (e.g., threonine and isoleucine) and decreases in their precursors (e.g., aspartate and O-acetylhomoserine). These findings correlate well with transcriptional data obtained with RNA-seq analysis: genes in these pathways were up-regulated between 10- and 800-fold in the codY null mutant. Coupling global analyses of the transcriptome and the metabolome can reveal how bacteria alter their metabolism when faced with environmental or nutritional stress, providing potential insight into the physiological changes associated with nutrient depletion experienced during infection. Such discoveries may pave the way for the development of novel anti-infectives and therapeutics.

Here we describe a protocol for the extraction of metabolites from Staphylococcus aureus and their subsequent analysis via liquid chromatography and mass spectrometry.

In an effort to thwart bacterial pathogens, hosts often limit the availability of nutrients at the site of infection. This limitation can alter the ...

Lipid Droplet Isolation for Quantitative Mass Spectrometry Analysis

Lipid droplets are vital to the replication of a variety of different pathogens, most prominently the Hepatitis C Virus (HCV), as the putative site of virion morphogenesis. Quantitative lipid droplet proteome analysis can be used to identify proteins that localize to or are displaced from lipid droplets under conditions such as virus infections. Here, we describe a protocol that has been successfully used to characterize the changes in the lipid droplet proteome following infection with HCV. We use Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) and thus label the complete proteome of one population of cells with &#34;heavy&#34; amino acids to quantitate the proteins by mass spectrometry. For lipid droplet isolation, the two cell populations (i.e.&#160;HCV-infected/&#34;light&#34; amino acids and uninfected control/&#34;heavy&#34; amino acids) are mixed 1:1 and lysed mechanically in hypotonic buffer. After removing the nuclei and cell debris by low speed centrifugation, lipid droplet-associated proteins are enriched by two subsequent ultracentrifugation steps followed by three washing steps in isotonic buffer. The purity of the lipid droplet fractions is analyzed by western blotting with antibodies recognizing different subcellular compartments. Lipid droplet-associated proteins are then separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie staining. After tryptic digest, the peptides are quantified by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Using this method, we identified proteins recruited to lipid droplets upon HCV infection that might represent pro- or antiviral host factors. Our method can be applied to a variety of different cells and culture conditions, such as infection with pathogens, environmental stress, or drug treatment.

Lipid droplets are important organelles for the replication of several pathogens, including the Hepatitis C Virus (HCV). We describe a method to isolate lipid droplets for quantitative mass spectrometry of associated proteins; it can be used under a variety of conditions, such as virus infection, environmental stress, or drug treatment.

Lipid droplets are vital to the replication of a variety of different pathogens, most prominently the Hepatitis C Virus (HCV), as the putative site of ...

Optimal Preparation of Formalin Fixed Samples for Peptide Based Matrix Assisted Laser Desorption/Ionization Mass Spectrometry Imaging Workflows

The use of matrix-assisted laser desorption/ionization, mass spectrometry imaging (MALDI MSI) has rapidly expanded, since this technique analyzes a host of biomolecules from drugs and lipids to N-glycans. Although various sample preparation techniques exist, detecting peptides from formaldehyde preserved tissues remains one of the most difficult challenges for this type of mass spectrometric analysis. For this reason, we have created and optimized a robust methodology that preserves the spatial information contained within the sample, while eliciting the greatest number of ionizable peptides. We have also aimed to achieve this in a cost effective and simple way, thereby eliminating potential bias or preparation error, which can occur when using automated instrumentation. The end result is a reproducible and inexpensive protocol.

This protocol describes a reproducible and reliable method for the sublimation-based preparation of formalin fixed tissue destined for imaging mass spectrometry.

The use of matrix-assisted laser desorption/ionization, mass spectrometry imaging (MALDI MSI) has rapidly expanded, since this technique analyzes a host ...

Proofreading and DNA Repair Assay Using Single Nucleotide Extension and MALDI-TOF Mass Spectrometry Analysis

The maintenance of the genome and its faithful replication is paramount for conserving genetic information. To assess high fidelity replication, we have developed a simple non-labeled and non-radio-isotopic method using a matrix-assisted laser desorption ionization with time-of-flight (MALDI-TOF) mass spectrometry (MS) analysis for a proofreading study. Here, a DNA polymerase [e.g., the Klenow fragment (KF) of Escherichia coli DNA polymerase I (pol I) in this study] in the presence of all four dideoxyribonucleotide triphosphates is used to process a mismatched primer-template duplex. The mismatched primer is then proofread/extended and subjected to MALDI-TOF MS. The products are distinguished by the mass change of the primer down to single nucleotide variations. Importantly, a proofreading can also be determined for internal single mismatches, albeit at different efficiencies. Mismatches located at 2-4-nucleotides (nt) from the 3' end were efficiently proofread by pol I, and a mismatch at 5 nt from the primer terminus showed only a partial correction. No proofreading occurred for internal mismatches located at 6 - 9 nt from the primer 3' end. This method can also be applied to DNA repair assays (e.g., assessing a base-lesion repair of substrates for the endo V repair pathway). Primers containing 3' penultimate deoxyinosine (dI) lesions could be corrected by pol I. Indeed, penultimate T-I, G-I, and A-I substrates had their last 2 dI-containing nucleotides excised by pol I before adding a correct ddN 5'-monophosphate (ddNMP) while penultimate C-I mismatches were tolerated by pol I, allowing the primer to be extended without repair, demonstrating the sensitivity and resolution of the MS assay to measure DNA repair.

A non-labeled, non-radio-isotopic method for DNA polymerase proofreading and a DNA repair assay was developed by using high-resolution MALDI-TOF mass spectrometry and a single nucleotide extension strategy. The assay proved to be very specific, simple, rapid, and easy to perform for proofreading and repair patches shorter than 9-nucleotides.

The maintenance of the genome and its faithful replication is paramount for conserving genetic information. To assess high fidelity replication, we have ...

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ubiquitinated proteins, peptides with a diglycine remnant conjugated to the epsilon amino group of lysine (&#39;K-&#949;-diglycine&#39; or simply &#39;diGly&#39;) can be used to track back the original modification site. Efficient immunopurification of diGly peptides combined with sensitive detection by mass spectrometry has resulted in a huge increase in the number of ubiquitination sites identified up to date. We have made several improvements to this workflow, including offline high pH reverse-phase fractionation of peptides prior to the enrichment procedure, and the inclusion of more advanced peptide fragmentation settings in the ion routing multipole. Also, more efficient cleanup of the sample using a filter-based plug in order to retain the antibody beads results in a greater specificity for diGly peptides. These improvements result in the routine detection of more than 23,000 diGly peptides from human cervical cancer cells (HeLa) cell lysates upon proteasome inhibition in the cell. We show the efficacy of this strategy for in-depth analysis of the ubiquitinome profiles of several different cell types and of in vivo samples, such as brain tissue. This study presents an original addition to the toolbox for protein ubiquitination analysis to uncover the deep cellular ubiquitinome.

We present a method for the purification, detection, and identification of diGly peptides that originate from ubiquitinated proteins from complex biological samples. The presented method is reproducible, robust, and outperforms published methods with respect to the level of depth of the ubiquitinome analysis.

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ...

A Plasma Sample Preparation for Mass Spectrometry using an Automated Workstation

Sample preparation for mass spectrometry analysis in proteomics requires enzymatic cleavage of proteins into a peptide mixture. This process involves numerous incubation and liquid transfer steps in order to achieve denaturation, reduction, alkylation, and cleavage. Adapting this workflow onto an automated workstation can increase efficiency and reduce coefficients of variance, thereby providing more reliable data for statistical comparisons between sample types. We previously described an automated proteomic sample preparation workflow1. Here, we report the development of a more efficient and better controlled workflow with the following advantages: 1) The number of liquid transfer steps is reduced from nine to six by combining reagents; 2) Pipetting time is reduced by selective tip pipetting using a 96-position pipetting head with multiple channels; 3) Potential throughput is increased by the availability of up to 45 deck positions; 4) Complete enclosure of the system provides improved temperature and environmental control and reduces the potential for contamination of samples or reagents; and 5) The addition of stable isotope labeled peptides, as well as &#946;-galactosidase protein, to each sample makes monitoring and quality control possible throughout the entire process. These hardware and process improvements provide good reproducibility and improve intra-assay and inter-assay precision (CV of less than 20%) for LC-MS based protein and peptide quantification. The entire workflow for digesting 96 samples in a 96-well plate can be completed in approximately 5 hours.

Here, we present an automated plasma protein digestion method for mass spectrometry-based quantitative proteomic analysis. In this protocol, the liquid transferring and incubation steps for protein denaturation, reduction, alkylation, and trypsin digestion reactions are streamlined and automated. It takes approximately five hours to prepare a 96-well plate with desired precision.

Sample preparation for mass spectrometry analysis in proteomics requires enzymatic cleavage of proteins into a peptide mixture. This process involves ...

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of biological membranes, energy storage and production, cellular signaling, vesicular transport of proteins, organelle biogenesis, and regulated cell death. Because the budding yeast Saccharomyces cerevisiae is a unicellular eukaryote amenable to thorough molecular analyses, its use as a model organism helped uncover mechanisms linking lipid metabolism and intracellular transport to complex biological processes within eukaryotic cells. The availability of a versatile analytical method for the robust, sensitive, and accurate quantitative assessment of major classes of lipids within a yeast cell is crucial for getting deep insights into these mechanisms. Here we present a protocol to use liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the quantitative analysis of major cellular lipids of S. cerevisiae. The LC-MS/MS method described is versatile and robust. It enables the identification and quantification of numerous species (including different isobaric or isomeric forms) within each of the 10 lipid classes. This method is sensitive and allows identification and quantitation of some lipid species at concentrations as low as 0.2 pmol/&#181;L. The method has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. The use of alternative mobile phase additives for electrospray ionization mass spectrometry in this method can increase the efficiency of ionization for some lipid species and can be therefore used to improve their identification and quantitation.

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

We present a protocol using liquid chromatography coupled with tandem mass spectrometry to identify and quantify major cellular lipids in Saccharomyces cerevisiae. The described method for a quantitative assessment of major lipid classes within a yeast cell is versatile, robust, and sensitive.

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of ...

Untargeted Liquid Chromatography-Mass Spectrometry-Based Metabolomics Analysis of Wheat Grain

Understanding the interactions between genes, the environment and management in agricultural practice could allow more accurate prediction and management of product yield and quality. Metabolomics data provides a read-out of these interactions at a given moment in time and is informative of an organism&#39;s biochemical status. Further, individual metabolites or panels of metabolites can be used as precise biomarkers for yield and quality prediction and management. The plant metabolome is predicted to contain thousands of small molecules with varied physicochemical properties that provide an opportunity for a biochemical insight into physiological traits and biomarker discovery. To exploit this, a key aim for metabolomics researchers is to capture as much of the physicochemical diversity as possible within a single analysis. Here we present a liquid chromatography-mass spectrometry-based untargeted metabolomics method for the analysis of field-grown wheat grain. The method uses the liquid chromatograph quaternary solvent manager to introduce a third mobile phase and combines a traditional reversed-phase gradient with a lipid-amenable gradient. Grain preparation, metabolite extraction, instrumental analysis and data processing workflows are described in detail. Good mass accuracy and signal reproducibility were observed, and the method yielded approximately 500 biologically relevant features per ionization mode. Further, significantly different metabolite and lipid feature signals between wheat varieties were determined.

A method for the untargeted analysis of wheat grain metabolites and lipids is presented. The protocol includes an acetonitrile metabolite extraction method and reversed phase liquid chromatography-mass spectrometry methodology, with acquisition in positive and negative electrospray ionization modes.

Understanding the interactions between genes, the environment and management in agricultural practice could allow more accurate prediction and management ...