The authors would like to thank Drs. Amanda Del Rosario and Guillaume Carmona for critical reading of the manuscript. This work was supported by grants from the Department of Defense&#160;DOD Innovator Award (W81XWH-14-1-0240&#160;to ROH); the National Cancer Institute (U54 CA126515/CA163109); the Broad Institute of MIT and Harvard; the Howard Hughes Medical Institute, of which ROH is an Investigator; and in part by the Support Grant from the National Cancer Institute to the Koch Institute for Integrative Cancer Research at MIT (P30-CA14051). AN received postdoctoral fellowships from the Howard Hughes Medical Institute and the Ludwig Center for Cancer Research at MIT.

NOTE: The procedure can be conducted on fresh or flash frozen tissues. We recommend perfusing highly vascularized tissues with PBS at the time of dissection to eliminate red blood cells and plasma proteins. We do not recommend conducting the procedure on fixed tissues as fixation (i.e., chemical crosslinking) interferes with decellularization and can also significantly compromise subsequent mass spectrometry analysis. For the entire procedure, we recommend the use of low-retention tubes and pipette tips to maximize protein and peptide recovery.
1. Decellularization of Tissues or Tumors
NOTE: Before starting, prepare the reagents and add protease inhibitors (provided with the Compartment Protein Extraction kit) to the desired volume of each buffer. All the buffers and samples should be kept on ice for the duration of the experiment except the Buffer CS that needs to be kept at RT to prevent SDS precipitation. &#160; 
NOTE: The protocol uses a series of incubation in buffers of different pH and containing different amount of salts and detergents to sequentially extract intracellular proteins and enrich for insoluble ECM proteins (Figure 1, Table 1; see also Discussion for alternatives to the use of the commercial kit detailed here).&#160; The volumes of reagents given below are for 100 mg of tissues or tumors (see Table 1) and need to be adjusted appropriately.
<ol>
	<li>Homogenize 100 mg of tissue in 500 &#956;l of Buffer C containing protease inhibitors using a tissue homogenizer until the tissue is completely disrupted and a homogenous suspension is obtained. 
	NOTE: Add deoxyribonuclease I (final concentration: 200 &#956;g/ml, reconstituted according to the manufacturer&#8217;s instructions) and ribonuclease A (final concentration: 20 &#956;g/ml, reconstituted according to the manufacturer&#8217;s instructions) to Buffer N.</li>
	<li>Sequential extraction of intracellular soluble proteins
	<ol>
		<li>Extraction of cytosolic proteins.&#160; Incubate the homogenate on a tube rotator for 20 min at 4 &#176;C. Save a small aliquot (10 &#956;l to 20 &#956;l) of the homogenate for subsequent western blot analysis (see Expected Results section and Figure 2).</li>
		<li>Centrifuge the homogenate at 16,000 x g for 20 min at 4 &#176;C. Collect the supernatant in a clean tube, this will constitute the cytosolic (C) fraction of the western blot analysis. Flash freeze this fraction and store at -80 &#176;C.</li>
		<li>To wash, resuspend the pellet in 400 &#956;l of Buffer W containing protease inhibitors and incubate the sample on a tube rotator for 20 min at 4 &#176;C. Centrifuge the homogenate at 16,000 x g for 20 min at 4 &#176;C. Discard the supernatant.</li>
		<li>To extract, nuclear proteins, resuspend the pellet in 150 &#956;l of Buffer N containing protease inhibitors, deoxyribonuclease I and ribonuclease A and incubate the sample on a tube rotator for 30 min at 4 &#176;C.</li>
		<li>Centrifuge the sample at 16,000 x g for 30 min at 4 &#176;C and collect the supernatant in a clean tube. Repeat this step once: after centrifuging the sample for the second time, add the supernatant to the previous supernatant: this will constitute the nuclear (N) fraction of the western blot analysis. Flash freeze this fraction and store at -80 &#176;C. Then, perform washings as per step 1.2.3.</li>
		<li>To extract membrane proteins, resuspend the pellet in 100 &#956;l of Buffer M containing protease inhibitors and incubate the sample on a tube rotator for 30 min at 4 &#176;C. Centrifuge the sample at 16,000 x g for 30 min at 4 &#176;C</li>
		<li>Collect the supernatant in a clean tube: this will constitute the membrane (M) fraction of the western blot analysis. Flash freeze this fraction and store at -80 &#176;C.</li>
		<li>To extract cytoskeletal proteins, resuspend the pellet in 200 &#956;l of Buffer CS containing protease inhibitors and incubate the sample on a tube rotator for 30 min at RT. 
		Note that the pellet will not fully dissolve. We suggest disrupting the pellet by pipetting up and down until observing disruption of the pellet.</li>
		<li>Centrifuge the sample at 16,000 x g for 30 min at RT. Collect the supernatant in a clean tube. Note at this point a further marked decrease in the size of the pellet (see video).</li>
		<li>Resuspend the pellet in 150 &#956;l of Buffer C containing protease inhibitors and incubate the sample on a tube rotator for 20 min at 4 &#176;C. Centrifuge the sample at 16,000 x g for 20 min at 4 &#176;C.</li>
		<li>Collect the supernatant and add it to the previous supernatant: this will constitute the cytoskeletal (CS) fraction of the western blot analysis. Flash freeze this fraction and store at -80 &#176;C.</li>
		<li>Perform additional washes. Resuspend the pellet in 500 &#956;l of PBS containing protease inhibitors and incubate the sample on a tube rotator for 5 min at 4 &#176;C. Centrifuge the sample at 16,000 x g for 5 min at 4 &#176;C. Discard the supernatant. Repeat this step three times. 
		NOTE: All traces of detergents need to be removed by extensive washes prior to digestion of the proteins into peptides (see Section 3). At this point, the ECM-enriched pellet can be flash-frozen and kept at -80 &#176;C. Note that the size of the pellet will depend on the amount of insoluble (ECM) proteins in the starting material and the efficiency of the decellularization.</li>
	</ol>
	</li>
</ol>
2. Monitoring the Quality of the Decellularization/ECM Enrichment by SDS-PAGE and Western Blot
<ol>
	<li>Mix 10&#8211;20 &#956;l aliquot of total tissue extract and 50 &#956;l aliquots of intermediate fractions with Laemmli Buffer containing 100 mM DTT. Resuspend the ECM-enriched, insoluble fraction in 3x Laemmli Buffer containing 100 mM DTT. 
	Note: he elevated concentrations of DTT and SDS assist in solubilization of ECM proteins that are relatively insoluble.</li>
	<li>Separate the proteins by SDS-PAGE and transferred onto nitrocellulose membranes.</li>
	<li>Perform immune-blots using antibodies to monitor proteins representative of each of the cytosolic, nuclear, membrane, cytoskeletal and ECM fractions (see Representative Results section, Table 2 and Figure 2).</li>
</ol>
3. In-solution Digestion of Proteins to Peptides for Mass Spectrometry Analysis

NOTE: The pellet obtained after the decellularization procedure and removal of SDS is highly enriched in insoluble ECM proteins For further analysis by mass spectrometry these proteins need to be digested into peptides. Note that, as a consequence of ECM protein insolubility, it is not possible at this step to measure the protein concentration of the sample. We thus provide volumes of reagents to digest the ECM-enriched samples into peptides based on the size (mm) or dry weight of the ECM-enriched pellet (Table 3). The solutions of ammonium bicarbonate, urea, DTT, iodoacetamide and trifluoroacetic acid all need to be freshly prepared.
<ol>
	<li>Resuspend the ECM-enriched sample by adding the appropriate volume of 8 M urea to the ECM-enriched pellet and add DTT at a final concentration of 10mM (see Table 3). Incubate with continuous agitation at 1,400 rpm for 2 hr at 37 &#176;C. 
	NOTE: at this point the ECM proteins will not be fully dissolved and the visibly large protein particles should not be discarded by centrifugation or filtration. The suspension will clear upon deglycosylation and digestion (see video).</li>
	<li>Alkylation
	<ol>
		<li>Prepare the iodoacetamide solution in HPLC-grade water. Cool the sample to RT and add the iodoacetamide to a final concentration of 25 mM. For complete alkylation, the DTT:iodoacetamide ratio should be between 1:2.5 and 1:3.</li>
		<li>Incubate in the dark for 30 min at RT.</li>
	</ol>
	</li>
	<li>Deglycosylation: 
	Note: deglycosylation is needed to remove carbohydrate side chains that interfere with identification of peptides modified by N-linked glycosylation.
	<ol>
		<li>Dilute to 2 M urea with 100 mM ammonium bicarbonate pH 8.0 and add the appropriate amount of PNGaseF (see Table 3). Incubate with continuous agitation at 1,400 rpm for 2 hr at 37 &#176;C.</li>
	</ol>
	</li>
	<li>Digestion
	<ol>
		<li>Add Lys-C and incubate with continuous agitation at 1,400 rpm for 2 hr at 37 &#176;C. Add the trypsin and incubate with continuous agitation at 1,400 rpm O/N at 37 &#176;C. &#160; 
		NOTE: the ECM-rich suspension that began cloudy upon initial reconstitution in 8M urea appears clear after O/N digestion (see video).</li>
		<li>Add a second aliquot of trypsin to the sample and incubate with continuous agitation at 1,400 rpm for an additional 2 hr at 37 &#176;C.</li>
	</ol>
	</li>
	<li>Acidification
	<ol>
		<li>Upon completion of the digestion, inactivate the trypsin by acidifying the sample with freshly prepared 50% trifluoro-acetic acid (TFA). The sample should reach pH &#60; 2. We suggest adding 1&#8211;1.5 &#956;l of 50% TFA at a time and using 1 &#956;l of the peptide solution to measure the pH of the solution using pH paper (see video).</li>
		<li>Centrifuge the acidified sample at 16,000 x g for 5 min at RT. Collect the supernatant in a clean low-retention tube. At this point, the peptide solution can be stored at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Desalting 
	Note: that this last step is usually conducted at a mass spectrometry facility according to their own preferred methods.
	<ol>
		<li>Prior to proteomics analysis, desalt the samples and peptides eluted with freshly prepared 60% acetonitrile, 0.1% trifluoroacetic acid, followed by concentration in a vacuum concentrator. Resuspend the peptides in freshly prepared 3% acetonitrile, 0.1% trifluoroacetic acid8. 
		NOTE: that after desalting, the concentration of the peptide solution can be measured by spectrophotometry (see Expected Results section).</li>
		<li>Now analyze the sample by mass spectrometry, again according to the optimal procedures of the mass spectrometry facility. 
		NOTE: We encourage researchers interested in characterizing the composition of ECMs using mass spectrometry to refer to our other publications8&#8211;10 and website that provide further detailed explanation, including LC-MS/MS parameters, mass spectrometry data search for protein identification and data analysis using the in silico matrisome annotation tool we developped8.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Hynes, R. O., Naba, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview+of+the+matrisome--an+inventory+of+extracellular+matrix+constituents+and+functions.">Overview of the matrisome--an inventory of extracellular matrix constituents and functions.</a> Cold Spring Harb Perspect Biol. 4 (1), a004903 (2012).</li><li>Mouw, J. K., Ou, G., Weaver, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+matrix+assembly:+a+multiscale+deconstruction.">Extracellular matrix assembly: a multiscale deconstruction.</a> Nat. Rev. Mol. Cell Biol. 15 (12), (2014).</li><li>Hynes, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+extracellular+matrix:+not+just+pretty+fibrils.">The extracellular matrix: not just pretty fibrils.</a> Science. 326 (5957), 1216-1219 (2009).</li><li>Bonnans, C., Chou, J., Werb, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remodelling+the+extracellular+matrix+in+development+and+disease.">Remodelling the extracellular matrix in development and disease.</a> Nat. Rev. Mol. Cell Biol. 15 (12), 786-801 (2014).</li><li>Dvir, T., Timko, B. P., Kohane, D. S., Langer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanotechnological+strategies+for+engineering+complex+tissues.">Nanotechnological strategies for engineering complex tissues.</a> Nat Nanotechnol. 6 (1), 13-22 (2011).</li><li>Wilson, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+extracellular+matrix:+an+underexplored+but+important+proteome.">The extracellular matrix: an underexplored but important proteome.</a> Expert Rev Proteomics. 7 (6), 803-806 (2010).</li><li>Naba, A., Hoersch, S., Hynes, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+definition+of+an+ECM+parts+list:+an+advance+on+GO+categories.">Towards definition of an ECM parts list: an advance on GO categories.</a> Matrix Biol. 31 (7-8), 371-372 (2012).</li><li>Naba, A., Clauser, K. R., Hoersch, S., Liu, H., Carr, S. A., Hynes, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+matrisome:+in+silico+definition+and+in+vivo.+characterization+by+proteomics+of+normal+and+tumor+extracellular+matrices.">The matrisome: in silico definition and in vivo. characterization by proteomics of normal and tumor extracellular matrices.</a> Mol. Cell Proteomics. 11 (4), M111.014647 (2012).</li><li>Naba, A., Clauser, K. R., Whittaker, C. A., Carr, S. A., Tanabe, K. K., Hynes, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+matrix+signatures+of+human+primary+metastatic+colon+cancers+and+their+metastases+to+liver.">Extracellular matrix signatures of human primary metastatic colon cancers and their metastases to liver.</a> BMC Cancer. 14 (1), 518 (2014).</li><li>Naba, A., Clauser, K. R., Lamar, J. M., Carr, S. A., Hynes, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+matrix+signatures+of+human+mammary+carcinoma+identify+novel+metastasis+promoters.">Extracellular matrix signatures of human mammary carcinoma identify novel metastasis promoters.</a> eLife. 3, e01308 (2014).</li><li>Naba, A., Ding, H., Whittaker, C. A., Hynes, R. O., 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=Proteomic+analysis+of+altered+extracellular+matrix+turnover+in+bleomycin-induced+pulmonary+fibrosis.">Proteomic analysis of altered extracellular matrix turnover in bleomycin-induced pulmonary fibrosis.</a> Mol. Cell Proteomics. 13 (7), 1741-1752 (2014).</li><li>Tsuchiya, T., Balestrini, J. L., Mendez, J. J., Calle, E. A., Zhao, L., Niklason, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+pH+on+extracellular+matrix+preservation+during+lung+decellularization.">Influence of pH on extracellular matrix preservation during lung decellularization.</a> Tissue Eng Part C Methods. 20 (12), 1028-1036 (2014).</li><li>Byron, A., Humphries, J. D., Humphries, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+the+extracellular+matrix+using+proteomics.">Defining the extracellular matrix using proteomics.</a> Int J Exp Pathol. 94 (2), 75-92 (2013).</li><li>Wilson, R., Diseberg, A. F., 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=Comprehensive+profiling+of+cartilage+extracellular+matrix+formation+and+maturation+using+sequential+extraction+and+label-free+quantitative+proteomics.">Comprehensive profiling of cartilage extracellular matrix formation and maturation using sequential extraction and label-free quantitative proteomics.</a> Mol. Cell Proteomics. 9 (6), 1296-1313 (2010).</li><li>Belluoccio, D., Wilson, R., Thornton, D. J., Wallis, T. P., Gorman, J. J., Bateman, J. 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C., Kiemele, L., 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=An+in-solution+ultrasonication-assisted+digestion+method+for+improved+extracellular+matrix+proteome+coverage.">An in-solution ultrasonication-assisted digestion method for improved extracellular matrix proteome coverage.</a> Mol. Cell Proteomics. 8 (7), 1648-1657 (2009).</li><li>Didangelos, A., Yin, X., Mandal, K., Baumert, M., Jahangiri, M., Mayr, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomics+characterization+of+extracellular+space+components+in+the+human+aorta.">Proteomics characterization of extracellular space components in the human aorta.</a> Mol. Cell Proteomics. 9 (9), 2048-2062 (2010).</li><li>Lennon, R., Byron, A., 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=Global+analysis+reveals+the+complexity+of+the+human+glomerular+extracellular+matrix.">Global analysis reveals the complexity of the human glomerular extracellular matrix.</a> J. Am. Soc. Nephrol. 25 (5), 939-951 (2014).</li></ol>

The authors declare that they have no competing financial interests.

Modifications
Although we employed this exact procedure to enrich the ECM from ten tissues and tumor types8&#8211;10, modifications of the protocol should be considered in the following instances:
1) Detection of ECM proteins in the intermediate fractions.
Extracellular matrices from different tissues or tumor types may differ in their extractability/insolubility, as discussed above for fibronectin and laminins. For example, it is thought that the ECM of fibrotic tissues or remodeling tissues turns over very dynamically and thus one might observe a higher proportion of ECM proteins in those tissues to be more readily extractable12. Depending on the fraction in which ECM proteins are detected, we suggest reducing the incubation time of the step causing the extraction of ECM proteins or omitting that step.
2) Detection of a significant proportion of intracellular components in the ECM-enriched pellet. In some tissues or tumor types the ratio cells:ECM is particularly high (e.g., liver, spleen, non-desmoplastic tumors). In that case, a significant contamination of the ECM-enriched fraction by intracellular proteins (in particular cytoskeletal proteins and/or histones) can be observed. To deplete intracellular proteins efficiently, we suggest repeating twice the incubation in Buffer M and/or Buffer CS (both containing detergents, this usually depletes abundant intracellular proteins). Another alternative would be to use alternative buffers with higher detergent concentrations, with the caveat that this may lead to the depletion of relatively more soluble ECM proteins as well (see next paragraph).
3) Alternative to using a commercial kit.
We were unable, for proprietary reasons, to obtain the precise composition of the buffers from the supplier of the Compartmental Protein Extraction kit. However, we have included in Table 1 notes based on our own experience using home-made buffers with defined detergent (NP-40, sodium deoxycholate and SDS) concentrations to conduct similar extractions. A recent study also highlighted the importance of the pH of decellularization buffers to retain ECM proteins13.
Limitations of the technique
The method presented here relies on the fact that ECM proteins are intrinsically more insoluble than most intracellular proteins. However, the decellularization method described here certainly extracts soluble components present within the ECM such as some growth factors or ECM-remodeling enzymes. Although ECM-associated proteins tightly bound to ECM proteins were detected by proteomics in samples prepared as described, this method may be too stringent to fully profile the composition of matrisome-associated proteins.
Significance of the technique with respect to other methods
The advantage of the method presented here over other methods is that it can be tailored to the nature of the ECM of interest: intermediate steps can be omitted or repeated to prevent the loss of ECM proteins or increase the depletion of contaminating intracellular proteins respectively. This method also only uses minimal amounts of detergents that are rinsed off to prevent their interference with subsequent peptide preparation and mass spectrometry. Finally, the method described here to digest ECM-rich protein preparations into peptides has also the advantage of not requiring proteins to be soluble and can be conducted on &#8220;crude&#8221; ECM-enriched fractions.
Alternative decellularization methods utilizing chaotropes (such as guanidine hydrochloride) to study the composition of ECMs by mass spectrometry have been reported in the literature (reviewed in 14) and have been used in combination with mass spectrometry to characterize the ECM composition of cartilage15,16, heart17, mammary gland18 and vascular19 and glomerular20 basement membranes.
Recommendations
Decellularization methods employing trypsin to digest out cells should not be used if ECMs are enriched for subsequent proteomics analyses, as trypsinization will result in partial ECM digestion and loss of ECM proteins and peptides. Similarly, if collagenase digestion were to be used to aid tissue disruption, it would need to be monitored carefully as it causes ECM digestion and loss of ECM proteins and peptides.
In-solution vs. in-gel digestion? ECM proteins are cross-linked and highly insoluble and, even when resuspended in 3x Laemmli buffer (containing 6% SDS) and 100mM DTT, separate poorly on SDS gels. Thus in-gel digestion is not a preferred method.
Future applications
It is worth noting that, while not discussed here, the composition of each of the intermediate fractions collected during the decellularization could also be analyzed by mass spectrometry. This may be particularly valuable when studying very small samples (i.e., human biopsies) or when information is desired on other cellular fractions.

The extracellular matrix (ECM) is a complex meshwork of cross-linked and glycosylated proteins that provides architectural support and anchorage for cells and defines, in part, tissues&#8217; biomechanical properties1,2. ECM proteins also signal to the cells either directly through their receptors (e.g., integrins, syndecans, etc.) or by modulating growth factor signaling3. The ECM thus provides biophysical and biochemical cues that are major regulators of cellular processes such as proliferation, survival, polarization, differentiation, migration, etc.
The ECM plays key roles in physiology, development and aging4. Moreover, several pathologies, such as cardio-vascular diseases, fibrosis, musculo-skeletal diseases, cancers, are caused by, or result in, ECM alterations. Furthermore, the ECM contributes to the maintenance of stem cell niches and identifying key ECM molecules that support stemness will have direct application in tissue engineering and regenerative medicine5. However, despite its importance, the ECM has remained, until recently, underexplored6.
In silico analysis has revealed that the matrisome, defined as the ensemble of ECM and ECM-associated proteins, comprises the products of several hundred genes in both the human and mouse genomes1,7,8. However, the insolubility of ECM proteins has hindered the systematic characterization of the composition of in vivo extracellular matrices of normal and pathological specimens. We recently demonstrated that this insolubility could be turned to advantage and be used to enrich ECM proteins8&#8211;10. We and others further demonstrated that mass spectrometry was a method of choice to characterize the composition of ECMs8&#8211;10, 13.
We describe here a decellularization procedure that consists of sequential incubations in buffers of different pH and salt and detergent concentrations. This procedure results in the extraction (or depletion) of cytosolic, nuclear, membrane and cytoskeletal proteins and the enrichment of ECM proteins. We then describe how to digest ECM-enriched protein preparations into peptides for subsequent analysis by mass spectrometry.
Using the procedures detailed and illustrated here, we have successfully enriched and characterized by mass spectrometry the extracellular matrices from ten different tissues and tumor types: normal murine lung8, human and murine colon8,9, human liver9, human colorectal tumors and derived liver metastases9, melanoma xenografts8, mammary tumor xenografts10, murine pancreatic islets and murine insulinomas (Naba et al., unpublished). Comparison of the different matrisomes revealed tissue- and tumor-specific ECM signatures that could further be used as potential diagnostic or prognostic biomarkers.
We believe that this procedure can be applied to other specimens with no or relatively minor modifications.

<table><tbody><tr><td>Tissue homogenizer: Bullet blender + beads</td><td>Next Advance</td><td>&lt;a href=&quot;http://www.nextadvance.com/api/index.cfm/products.info/c/421/Bullet-Blender&quot;&gt;BB24-AU&lt;/a&gt;</td><td></td></tr><tr><td>Compartmental Extraction kit</td><td>Millipore</td><td>&lt;a href=&quot;http://www.emdmillipore.com/US/en/product/Compartment-Protein-Extraction-Kit,MM_NF-2145&quot;&gt;2145&lt;/a&gt;</td><td></td></tr><tr><td>Deoxyribonuclease I</td><td>Sigma-Aldrich</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/catalog/product/sigma/dn25?lang=en&amp;amp;region=US&quot;&gt;DN25&lt;/a&gt;</td><td></td></tr><tr><td>Ribonuclease A</td><td>Qiagen</td><td>&lt;a href=&quot;http://www.qiagen.com/products/catalog/sample-technologies/dna-sample-technologies/plasmid-dna/rnase-a/&quot;&gt;19101&lt;/a&gt;</td><td></td></tr><tr><td>HPLC-grade water</td><td>Sigma-Aldrich</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/catalog/product/sial/34877?lang=en&amp;amp;region=US&quot;&gt;34877&lt;/a&gt;</td><td></td></tr><tr><td>Urea</td><td>Sigma-Aldrich</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/catalog/product/sigma/u4883?lang=en&amp;amp;region=US&quot;&gt;U4883&lt;/a&gt;</td><td></td></tr><tr><td>Dithiotreitol (DTT)</td><td>Thermo Scientific</td><td>&lt;a href=&quot;http://www.piercenet.com/product/dithiothreitol-dtt&quot;&gt;20291&lt;/a&gt;</td><td></td></tr><tr><td>Ammonium bicarbonate (NH&lt;sub&gt;4&lt;/sub&gt;HCO&lt;sub&gt;3&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/catalog/product/fluka/09830?lang=en&amp;amp;region=US&quot;&gt;9830&lt;/a&gt;</td><td></td></tr><tr><td>Iodoacetamide</td><td>Sigma-Aldrich</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/catalog/product/sigma/a3221?lang=en&amp;amp;region=US&quot;&gt;A3221&lt;/a&gt;</td><td></td></tr><tr><td>Peptide-&lt;em&gt;N&lt;/em&gt;-Glycosidase F (PNGaseF)</td><td>New England Biolabs</td><td>&lt;a href=&quot;https://www.neb.com/products/p0704-pngase-f&quot;&gt;P0704S&lt;/a&gt;</td><td></td></tr><tr><td>Endoproteinase LysC, mass spectrometry-grade&amp;nbsp;</td><td>Wako</td><td>&lt;a href=&quot;http://www.wako-chem.co.jp/english/labchem/product/life/Lys-C/index.htm&quot;&gt;125-05061&lt;/a&gt;</td><td></td></tr><tr><td>Trypsin, mass spectrometry-grade&amp;nbsp;</td><td>Promega</td><td>&lt;a href=&quot;https://www.promega.com/products/mass-spectrometry/proteases-and-surfactants/sequencing-grade-modified-trypsin/&quot;&gt;V5111&lt;/a&gt;</td><td></td></tr><tr><td>Trifluoro-acetic Acid</td><td>Sigma-Aldrich</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/catalog/product/sial/t6508?lang=en&amp;amp;region=US&quot;&gt;T6508&lt;/a&gt;</td><td></td></tr><tr><td>Acetonitrile, mass spectrometry-grade&amp;nbsp;</td><td>Thermo Scientific</td><td>&lt;a href=&quot;http://www.piercenet.com/product/acetonitrile-acn-lc-ms-grade&quot;&gt;51101&lt;/a&gt;</td><td></td></tr><tr><td>Desalting columns: Oasis HLB 1&amp;nbsp;cc, 10 mg Sorbent per Cartridge</td><td>Waters</td><td>&lt;a href=&quot;http://www.waters.com/waters/partDetail.htm?partNumber=186000383&quot;&gt;186000383&lt;/a&gt;</td><td></td></tr></tbody></table>

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.

Quality control of the decellularization procedure
The efficiency of the decellularization can be monitored by analyzing the protein content of each fraction by western blot. Table 2 lists proteins of diagnostic value to assess the quality of the decellularization procedure. Figure 2A shows the efficient extraction in the intermediate fractions of cytosolic (GAPDH), nuclear (histones), membrane (&#946;1 integrin) and cytoskeletal (actin) proteins, whereas no ECM proteins (collagen I) is detected in these fractions (Figure 2). In turn, the final pellet is enriched for ECM proteins and largely depleted of intracellular proteins (Figure 2A). Figure 2B presents satisfactory intracellular protein depletion (no histone is detected in the ECM-rich fraction), although, actin can still be detected in the ECM-rich fraction and depletion of monomeric collagen I &#8212; apparent molecular weight ~110 kDa, presumptively corresponding to unassembled collagen I &#8212; can be observed in the CS fraction.
We also routinely monitor additional ECM proteins such as fibronectin and laminin, although, in some tissues these components can be partially solubilized in earlier fractions8-10. For example, fibronectin also occurs as soluble plasma fibronectin that is not incorporated into the ECM. Perfusion of the tissue prior to extraction reduces plasma fibronectin concentration but does not always eliminate it. In some tissues, laminins are found loosely associated with the cell surface or the ECM and extract in intermediate fractions. If this occurs it can be addressed by altering extraction conditions (see Discussion).
Note that the enrichment of ECM proteins and concomitant depletion of intracellular components is based on the relative solubility of proteins in the different buffers. This differs among different tissues &#8212; in some cases the histones and actin are more readily extracted than in others. Also note that, although histones are expected to be extracted in the N fraction (Figure 2B), we often observe a more complete depletion of histones in the M or CS fraction (Figure 2A and B).
Indicative peptide concentration expected
The concentration of the peptide solution obtained after digestion, acidification and desalting can be measured by spectrophotometry either by measuring the absorbance of the peptide solution using the 280 nm wavelength corresponding to tryptophan, tyrosine, or using the 205 nm wavelength corresponding to absorbance of the peptide bonds.
We measured the concentration of the peptide solutions obtained from the decellularization of three murine lungs samples (82 mg, 100 mg, and 100 mg, respectively) prepared in parallel and obtained from each: 424 ng/&#181;l, 450 ng/&#181;l, and 580 ng/&#181;l of peptides respectively.
Identification of ECM peptides by mass spectrometry
Mass spectrometric analysis of the composition of ECM-enriched protein samples, prepared as described here, showed that &#62;70% of the signal intensity corresponds to ECM and ECM-associated proteins8-10.
Table 1. Volume of reagents from Compartmental Extraction kit to decellularize 100 mg (wet weight) of tissue or tumor. This table lists the composition and the volume of each buffer used to conduct the decellularization of 100 mg of tissue or tumor. A cocktail of protease inhibitors is provided as a 50x solution and needs to be added to each buffer2.
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Reagents from Compartment Protein Extraction Kit</td>
			<td>Volume for 100 mg of tissue</td>
			<td>Composition (based on Millipore datasheet cat#21451)</td>
		</tr>
		<tr>
			<td>Buffer C</td>
			<td>500 &#956;l</td>
			<td>HEPES (pH 7.93), MgCl2, KCl, EDT4, Sucrose, Glycerol, Sodium Orthovanadate5</td>
		</tr>
		<tr>
			<td>Buffer W</td>
			<td>400 &#956;l</td>
			<td>HEPES (pH 7.9), MgCl2, KCl, EDTA, Sucrose, Glycerol, Sodium Orthovanadate</td>
		</tr>
		<tr>
			<td>Buffer N&#160;</td>
			<td>150 &#956;l x 2</td>
			<td>HEPES (pH 7.9), MgCl2, NaCl, EDTA, Glycerol, Sodium Orthovanadate</td>
		</tr>
		<tr>
			<td>Buffer W</td>
			<td>400 &#956;l</td>
			<td>HEPES (pH 7.9), MgCl2, KCl, EDTA, Sucrose, Glycerol, Sodium Orthovanadate</td>
		</tr>
		<tr>
			<td>Buffer M</td>
			<td>100 &#956;l</td>
			<td>HEPES (pH 7.9), MgCl2, KCl, EDTA, Sucrose, Glycerol, Sodium deoxycholate (DOC)6, NP-406, Sodium Orthovanadate</td>
		</tr>
		<tr>
			<td>Buffer CS</td>
			<td>200 &#956;l</td>
			<td>PIPES (pH 6.8), MgCl2, NaCl, EDTA, Sucrose, Sodium Dodecyl Sulfate (SDS)7, Sodium Orthovanadate</td>
		</tr>
		<tr>
			<td>Buffer C</td>
			<td>150 &#956;l</td>
			<td>HEPES (pH 7.9), MgCl2, KCl, EDTA, Sucrose, Glycerol, Sodium Orthovanadate</td>
		</tr>
		<tr>
			<td>1x PBS</td>
			<td>500 &#956;l/wash</td>
			<td>-</td>
		</tr>
	</tbody>
</table>
Notes: 
1 For proprietary reasons, we were unable to obtain the precise composition of the buffers from the supplier of the kit, but we include here some notes based on our own experience using home-made buffers to conduct similar extractions. 
2 Protease inhibitor: it is advisable to include a variety of inhibitors against cysteine, serine and threonine peptidases, serine esterases, divalent cation-dependent metalloproteinases etc. Many commercially available protease inhibitor cocktails exist. 
3 pH above 7.0 is an effective inhibitor of lysosomal proteases. 
4 EDTA (usually used at 2 mM) is an effective inhibitor of divalent cation-dependent proteases. 
5 Sodium orthovanadate is a phosphatase inhibitor. A typical effective concentration would be 0.5&#8211;5 mM. 
6 NP40 at 0.1%&#8211;0.5% is sufficient to solubilize most membrane lipids. The combination of NP40 and DOC &#8212; often used at equal concentrations (e.g., 0.5% of each)&#160;&#8212; is often used as a more stringent extraction that still leaves many protein-protein interactions intact. 
7 SDS is a more stringent ionic detergent (CMC 0.1%). It can also be used in combination with the other two detergents SDS/NP40/DOC 0.1/0.5/0.5% as an intermediate stringency buffer.
Table 2. Diagnostic proteins to monitor the quality of the decellularization procedure. This table lists examples of proteins that are characteristics of each subcellular compartment (cytosol, nucleus, plasma membrane, cytoskeleton and ECM) that can be used to monitor the quality of the decellularization procedure and the efficiency of the ECM-enrichment.
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Intracellular Compartment</td>
			<td>Diagnostic Proteins</td>
		</tr>
		<tr>
			<td>Cytosolic proteins</td>
			<td>Glyceraldehyde 3-phosphate dehydrogenase (GAPDH)</td>
		</tr>
		<tr>
			<td>Nuclear proteins</td>
			<td>Histones, Lamins, Nucleoporin</td>
		</tr>
		<tr>
			<td>Membrane proteins</td>
			<td>Integrins, Transferrin Receptor</td>
		</tr>
		<tr>
			<td>Cytoskeletal proteins</td>
			<td>Actin, Tubulin, Vimentin</td>
		</tr>
		<tr>
			<td>Basement membrane ECM proteins&#160;</td>
			<td>Collagen IV, Nidogens, Laminins</td>
		</tr>
		<tr>
			<td>Interstitial ECM proteins (interstitial)</td>
			<td>Collagen I, Collagen III, Collagen VI, Fibronectin</td>
		</tr>
	</tbody>
</table>
For recommendation on antibodies, see our publications8-10.
Table 3. Volume of reagents to digest ECM-enriched samples into peptides. This table lists the reagents used to resuspend ECM-enriched protein samples and reduce, alkylate, deglycosylate and digest proteins samples into peptides prior to mass spectrometry analysis.
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Reagents</td>
			<td>Preparation</td>
			<td>Final Concentration / Amount&#160; for 1&#160;mm-thick pellet (~5&#8211;10&#160;mg dry weight)</td>
			<td>Volume for 1&#160;mm-thick pellet (~5&#8211;10&#160;mg dry weight)</td>
		</tr>
		<tr>
			<td>Ammonium bicarbonate (NH4HCO3)</td>
			<td>100 mM solution in HPLC-grade water</td>
			<td>&#160;-&#160;</td>
			<td>&#160;-&#160;</td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>8 M solution in 100 mM&#160; ammonium bicarbonate</td>
			<td>8 M</td>
			<td>50 &#956;l</td>
		</tr>
		<tr>
			<td>Dithiothreitol</td>
			<td>Reconstitute in HPLC-grade water at 500 mM</td>
			<td>10 mM</td>
			<td>1 &#956;l</td>
		</tr>
		<tr>
			<td>Iodoacetamide&#160;</td>
			<td>Reconstitute in HPLC-grade water at 500 mM</td>
			<td>25 mM</td>
			<td>2.5 &#956;l</td>
		</tr>
		<tr>
			<td>Peptide-N-Glycosidase F (PNGaseF)</td>
			<td>Commercial solution at 500 U/&#956;l</td>
			<td>1,000 U</td>
			<td>2 &#956;l</td>
		</tr>
		<tr>
			<td>Endoproteinase LysC, mass spectrometry-grade&#160;</td>
			<td>Reconstitute in HPLC-grade water at 0.5 &#956;g/&#956;l</td>
			<td>1 &#956;g</td>
			<td>2 &#956;l</td>
		</tr>
		<tr>
			<td>Trypsin, mass spectrometry-grade (round 1)</td>
			<td>Commercial solution at 0.5 &#956;g/&#956;l</td>
			<td>3 &#956;g</td>
			<td>6 &#956;l</td>
		</tr>
		<tr>
			<td>Trypsin, mass spectrometry-grade (round 2)</td>
			<td>Commercial solution at 0.5 &#956;g/&#956;l</td>
			<td>1.5 &#956;g</td>
			<td>3 &#956;l</td>
		</tr>
		<tr>
			<td>Trifluoro-acetic acid (TFA)</td>
			<td>50% solution in HPLC-grade water</td>
			<td>&#160;-&#160;</td>
			<td>2&#8211;5 &#956;l</td>
		</tr>
		<tr>
			<td>Acetonitrile (elution)</td>
			<td>60% solution with 0.1% TFA in HPLC-grade water</td>
			<td>&#160;-&#160;</td>
			<td>500 &#956;l</td>
		</tr>
		<tr>
			<td>Acetonitrile (reconstitution)</td>
			<td>3% solution with 0.1% TFA in HPLC-grade water</td>
			<td>&#160;-&#160;</td>
			<td>100 &#956;l</td>
		</tr>
	</tbody>
</table>
<img alt="Figure 1" src="/files/ftp_upload/53057/53057fig1highres.jpg" width="600" /> 
Figure 1. Scheme of the experimental procedure. Schematic workflow of the protocol to decellularize tissues (Section 1), control the quality of the decellularization and evaluate the ECM enrichment (Section 2) and digest ECM-enriched protein samples into peptides prior to mass spectrometry analysis (Section 3).
<img alt="Figure 2" src="/files/ftp_upload/53057/53057fig2highres.jpg" width="500" /> 
Figure 2. Quality control of the decellularization procedure by western blot. Western blots were performed on murine lung (A) and human mammary carcinoma xenograft (B) samples using the following antibodies: rabbit anti-actin (clone 14-1) and rabbit anti-&#946;1 integrin antibodies produced in our laboratory; rabbit anti-collagen I, mouse anti-GAPDH and rabbit anti-pan-histones antibodies were from Millipore. Following primary antibody incubation, the membranes were washed and incubated in the presence of HRP-conjugated goat anti-rabbit or goat anti-mouse secondary antibody from Jackson ImmunoResearch Laboratory. Finally, the membranes were washed and incubated in Western Lightning&#8482; Chemiluminescence Reagent (PerkinElmer LAS). * indicates minimal residual histone contamination of the ECM-rich fraction. ** indicates partial depletion of monomeric (presumptively unassembled) collagen I in the CS fraction. *** indicates residual actin contamination of the ECM-rich fraction. The smear detected with the anti-collagen antibody represents different levels of post-translational modifications (e.g., cross-linking, glycosylation).

Watch this Scientific Journal Video about Enrichment of Extracellular Matrix Proteins from Tissues and Digestion into Peptides for Mass Spectrometry Analysis at JoVE.com

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

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 ...

enrichment-extracellular-matrix-proteins-from-tissues-digestion-into

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

26.9K Views.  Massachusetts Institute of Technology. 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. 

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

Matrix-assisted Laser Desorption/Ionization Time of Flight (MALDI-TOF) Mass Spectrometric Analysis of Intact Proteins Larger than 100 kDa

Effectively determining masses of proteins is critical to many biological studies (e.g. for structural biology investigations). Accurate mass determination allows one to evaluate the correctness of protein primary sequences, the presence of mutations and/or post-translational modifications, the possible protein degradation, the sample homogeneity, and the degree of isotope incorporation in case of labelling (e.g. 13C labelling).
Electrospray ionization (ESI) mass spectrometry (MS) is widely used for mass determination of denatured proteins, but its efficiency is affected by the composition of the sample buffer. In particular, the presence of salts, detergents, and contaminants severely undermines the effectiveness of protein analysis by ESI-MS. Matrix-assisted laser desorption/ionization (MALDI) MS is an attractive alternative, due to its salt tolerance and the simplicity of data acquisition and interpretation. Moreover, the mass determination of large heterogeneous proteins (bigger than 100 kDa) is easier by MALDI-MS due to the absence of overlapping high charge state distributions which are present in ESI spectra.
Here we present an accessible approach for analyzing proteins larger than 100 kDa by MALDI-time of flight (TOF). We illustrate the advantages of using a mixture of two matrices (i.e. 2,5-dihydroxybenzoic acid and &#945;-cyano-4-hydroxycinnamic acid) and the utility of the thin layer method as approach for sample deposition. We also discuss the critical role of the matrix and solvent purity, of the standards used for calibration, of the laser energy, and of the acquisition time. Overall, we provide information necessary to a novice for analyzing intact proteins larger than 100 kDa by MALDI-MS.

Matrix-assisted Laser Desorption/Ionization Time of Flight MALDI-TOF Mass Spectrometric Analysis of Intact Proteins Larger than 100 kDa

Accurate mass measurement represents an important step during the investigation of proteins. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) can be used for such determination and its key advantage is the tolerance to salts, detergents and contaminants. Here we illustrate an accessible approach for the analysis of proteins larger than 100 kDa by MALDI-MS.

Effectively determining masses of proteins is critical to many biological studies (e.g. for structural biology investigations). Accurate mass ...

Chemistry

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.

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). ...

Biochemistry

Low Molecular Weight Protein Enrichment on Mesoporous Silica Thin Films for Biomarker Discovery

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the monitoring of therapeutic efficiency.1-3 The circulating low molecular weight proteome (LMWP) composed of small proteins shed from tissues and cells or peptide fragments derived from the proteolytic degradation of larger proteins, has been associated with the pathological condition in patients and likely reflects the state of disease.4,5 Despite these potential clinical applications, the use of Mass Spectrometry (MS) to profile the LMWP from biological fluids has proven to be very challenging due to the large dynamic range of protein and peptide concentrations in serum.6 Without sample pre-treatment, some of the more highly abundant proteins obscure the detection of low-abundance species in serum/plasma. Current proteomic-based approaches, such as two-dimensional polyacrylamide gel-electrophoresis (2D-PAGE) and shotgun proteomics methods are labor-intensive, low throughput and offer limited suitability for clinical applications.7-9 Therefore, a more effective strategy is needed to isolate LMWP from blood and allow the high throughput screening of clinical samples. 
Here, we present a fast, efficient and reliable multi-fractionation system based on mesoporous silica chips to specifically target and enrich LMWP.10,11 Mesoporous silica (MPS) thin films with tunable features at the nanoscale were fabricated using the triblock copolymer template pathway. Using different polymer templates and polymer concentrations in the precursor solution, various pore size distributions, pore structures, connectivity and surface properties were determined and applied for selective recovery of low mass proteins. The selective parsing of the enriched peptides into different subclasses according to their physicochemical properties will enhance the efficiency of recovery and detection of low abundance species. In combination with mass spectrometry and statistic analysis, we demonstrated the correlation between the nanophase characteristics of the mesoporous silica thin films and the specificity and efficacy of low mass proteome harvesting. The results presented herein reveal the potential of the nanotechnology-based technology to provide a powerful alternative to conventional methods for LMWP harvesting from complex biological fluids. Because of the ability to tune the material properties, the capability for low-cost production, the simplicity and rapidity of sample collection, and the greatly reduced sample requirements for analysis, this novel nanotechnology will substantially impact the field of proteomic biomarker research and clinical proteomic assessment.

We developed a technology based on mesoporous silica thin film for the selective recovery of low molecular weight proteins and peptides from human serum. The physico-chemical properties of our mesoporous chips were finely tuned to provide substantial control in peptide enrichment and consequently profile the serum proteome for diagnostic purposes.

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the ...

Bioengineering

A Mass Spectrometry-Based Proteomics Approach for Global and High-Confidence Protein R-Methylation Analysis

Protein Arginine (R)-methylation is a widespread protein post-translational modification (PTM) involved in the regulation of several cellular pathways, including RNA processing, signal transduction, DNA damage response, miRNA biogenesis, and translation.
In recent years, thanks to biochemical and analytical developments, mass spectrometry (MS)-based proteomics has emerged as the most effective strategy to characterize the cellular methyl-proteome with single-site resolution. However, identifying and profiling in vivo protein R-methylation by MS remains challenging and error-prone, mainly due to the substoichiometric nature of this modification and the presence of various amino acid substitutions and chemical methyl-esterification of acidic residues that are isobaric to methylation. Thus, enrichment methods to enhance the identification of R-methyl-peptides and orthogonal validation strategies to reduce False Discovery Rates (FDR) in methyl-proteomics studies are required.
Here, a protocol specifically designed for high-confidence R-methyl-peptides identification and quantitation from cellular samples is described, which couples metabolic labeling of cells with heavy isotope-encoded Methionine (hmSILAC) and dual protease in-solution digestion of whole cell extract, followed by off-line High-pH Reversed Phase (HpH-RP) chromatography fractionation and affinity enrichment of R-methyl-peptides using anti-pan-R-methyl antibodies. Upon high-resolution MS analysis, raw data are first processed with the MaxQuant software package and the results are then analyzed by hmSEEKER, a software designed for the in-depth search of MS peak pairs corresponding to light and heavy methyl-peptide within the MaxQuant output files.

Protein Arginine (R)-methylation is a wide-spread post-translational modification regulating multiple biological pathways. Mass spectrometry is the best technology to globally profile the R-methyl-proteome, when coupled to biochemical approaches for modified peptide enrichment. The workflow designed for the high confidence identification of global R-methylation in human cells is described here.

Protein Arginine (R)-methylation is a widespread protein post-translational modification (PTM) involved in the regulation of several cellular pathways, ...

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 ...

Enrichment of Host Cell Proteins in Monoclonal Antibody Drug Products

Host Cell Protein Analysis using Enrichment Beads Coupled with Limited Digestion

Host cell proteins (HCPs) are impurities that can adversely affect therapeutic proteins, even in small quantities. To evaluate the potential risks associated with drug products, methods have been developed to identify low-abundance HCPs. A crucial approach for developing a sensitive HCP detection method involves enriching HCPs while simultaneously removing monoclonal antibodies (mAbs) before analysis, utilizing liquid chromatography-mass spectrometry (LC-MS).
This protocol offers detailed instructions for enriching host cell proteins using commercially available proteome enrichment beads. These beads contain a diverse library of hexapeptide ligands with specific affinities for different proteins. The protocol also incorporates limited digestion and subsequent peptide detection using nano LC-MS/MS. By employing these techniques, HCPs with low abundance can be enriched over 7000-fold, resulting in an impressive detection limit as low as 0.002 ppm. Significantly, this protocol enables the detection of 850 HCPs with a high level of confidence using a NIST mAb. Moreover, it is designed to be user-friendly and includes a video demonstration to assist with its implementation. By following these steps, researchers can effectively enrich and detect HCPs, enhancing the sensitivity and accuracy of risk assessment for drug products.

Author Spotlight: Detecting Low-Abundant Host Cell Proteins in Drug Products Using Enrichment Beads and Limited Digestion 

A protocol is presented for enriching host cell proteins (HCPs) from drug products (DP) and detecting peptides using proteome enrichment beads. The method is demonstrated using an in-house manufactured monoclonal antibody (mAb) drug substance (DS), which is a well-characterized reference material for evaluating and comparing different methods in terms of performance.

Host cell proteins (HCPs) are impurities that can adversely affect therapeutic proteins, even in small quantities. To evaluate the potential risks ...

Enrichment of Bacterial Lipoproteins and Preparation of N-terminal Lipopeptides for Structural Determination by Mass Spectrometry

Lipoproteins are important constituents of the bacterial cell envelope and potent activators of the mammalian innate immune response. Despite their significance to both cell physiology and immunology, much remains to be discovered about novel lipoprotein forms, how they are synthesized, and the effect of the various forms on host immunity. To enable thorough studies on lipoproteins, this protocol describes a method for bacterial lipoprotein enrichment and preparation of N-terminal tryptic lipopeptides for structural determination by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Expanding on an established Triton X-114 phase partitioning method for lipoprotein extraction and enrichment from the bacterial cell membrane, the protocol includes additional steps to remove non-lipoprotein contaminants, increasing lipoprotein yield and purity. Since lipoproteins are commonly used in Toll-like receptor (TLR) assays, it is critical to first characterize the N-terminal structure by MALDI-TOF MS. Herein, a method is presented to isolate concentrated hydrophobic peptides enriched in N-terminal lipopeptides suitable for direct analysis by MALDI-TOF MS/MS. Lipoproteins that have been separated by Sodium Dodecyl Sulfate Poly-Acrylamide Gel Electrophoresis (SDS-PAGE) are transferred to a nitrocellulose membrane, digested in situ with trypsin, sequentially washed to remove polar tryptic peptides, and finally eluted with chloroform-methanol. When coupled with MS of the more polar trypsinized peptides from wash solutions, this method provides the ability to both identify the lipoprotein and characterize its N-terminus in a single experiment. Intentional sodium adduct formation can also be employed as a tool to promote more structurally informative fragmentation spectra. Ultimately, enrichment of lipoproteins and determination of their N-terminal structures will permit more extensive studies on this ubiquitous class of bacterial proteins.

The enrichment of bacterial lipoproteins using a non-ionic surfactant phase partitioning method is described for direct use in TLR assays or other applications. Further steps are detailed to prepare N-terminal tryptic lipopeptides for structural characterization by mass spectrometry.

Lipoproteins are important constituents of the bacterial cell envelope and potent activators of the mammalian innate immune response. Despite their ...

Immunology and Infection

Scanning Electron Microscopy of Macerated Tissue to Visualize the Extracellular Matrix

Fibrosis is a component of all forms of heart disease regardless of etiology, and while much progress has been made in the field of cardiac matrix biology, there are still major gaps related to how the matrix is formed, how physiological and pathological remodeling differ, and most importantly how matrix dynamics might be manipulated to promote healing and inhibit fibrosis. There is currently no treatment option for controlling, preventing, or reversing cardiac fibrosis. Part of the reason is likely the sheer complexity of cardiac scar formation, such as occurs after myocardial infarction to immediately replace dead or dying cardiomyocytes. The extracellular matrix itself participates in remodeling by activating resident cells and also by helping to guide infiltrating cells to the defunct lesion. The matrix is also a storage locker of sorts for matricellular proteins that are crucial to normal matrix turnover, as well as fibrotic signaling. The matrix has additionally been demonstrated to play an electromechanical role in cardiac tissue. Most techniques for assessing fibrosis are not qualitative in nature, but rather provide quantitative results that are useful for comparing two groups but that do not provide information related to the underlying matrix structure. Highlighted here is a technique for visualizing cardiac matrix ultrastructure. Scanning electron microscopy of decellularized heart tissue reveals striking differences in structure that might otherwise be missed using traditional quantitative research methods.

Shown here is a method for visualizing extracellular matrix ultrastructure in decellularized cardiac tissues.

Fibrosis is a component of all forms of heart disease regardless of etiology, and while much progress has been made in the field of cardiac matrix ...

A Rapid, Scalable Method for the Isolation, Functional Study, and Analysis of Cell-derived Extracellular Matrix

The extracellular matrix (ECM) is recognized as a diverse, dynamic, and complex environment that is involved in multiple cell-physiological and pathological processes. However, the isolation of ECM, from tissues or cell culture, is complicated by the insoluble and cross-linked nature of the assembled ECM and by the potential contamination of ECM extracts with cell surface and intracellular proteins. Here, we describe a method for use with cultured cells that is rapid and reliably removes cells to isolate a cell-derived ECM for downstream experimentation.
Through use of this method, the isolated ECM and its components can be visualized by in situ immunofluorescence microscopy. The dynamics of specific ECM proteins can be tracked by tracing the deposition of a tagged protein using fluorescence microscopy, both before and after the removal of cells. Alternatively, the isolated ECM can be extracted for biochemical analysis, such as sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. At larger scales, a full proteomics analysis of the isolated ECM by mass spectrometry can be conducted. By conducting ECM isolation under sterile conditions, sterile ECM layers can be obtained for functional or phenotypic studies with any cell of interest. The method can be applied to any adherent cell type, is relatively easy to perform, and can be linked to a wide repertoire of experimental designs.

The extracellular matrix plays a major role in defining the microenvironment of cells and in modulating cell behavior and phenotype. We describe a rapid method for the isolation of cell-derived extracellular matrix, which can be adapted to different scales for microscopic, biochemical, proteomic, or functional studies.

The extracellular matrix (ECM) is recognized as a diverse, dynamic, and complex environment that is involved in multiple cell-physiological and ...

Combining Chemical Cross-linking and Mass Spectrometry of Intact Protein Complexes to Study the Architecture of Multi-subunit Protein Assemblies

Proteins interact with their ligands to form active and dynamic assemblies which carry out various cellular functions. Elucidating these interactions is therefore fundamental for the understanding of cellular processes. However, many protein complexes are dynamic assemblies and are not accessible by conventional structural techniques. Mass spectrometry contributes to the structural investigation of these assemblies, and particularly the combination of various mass spectrometric techniques delivers valuable insights into their structural arrangement.
In this article, we describe the application and combination of two complementary mass spectrometric techniques, namely chemical cross-linking coupled with mass spectrometry and native mass spectrometry. Chemical cross-linking involves the covalent linkage of amino acids in close proximity by using chemical reagents. After digestion with proteases, cross-linked di-peptides are identified by mass spectrometry and protein interactions sites are uncovered. Native mass spectrometry on the other hand is the analysis of intact protein assemblies in the gas phase of a mass spectrometer. It reveals protein stoichiometries as well as protein and ligand interactions. Both techniques therefore deliver complementary information on the structure of protein-ligand assemblies and their combination proved powerful in previous studies.

The architecture of protein complexes is essential for their function. Combining various mass spectrometric techniques proved powerful to study their assembly. We provide protocols for chemical cross-linking and native mass spectrometry and show how these complementary techniques help to elucidate the architecture of multi-subunit protein assemblies.

Proteins interact with their ligands to form active and dynamic assemblies which carry out various cellular functions. Elucidating these interactions is ...