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 an...
| Main Authors: | , , |
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| Other Authors: | |
| Format: | Article |
| Language: | en_US |
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MyJoVE Corporation
2017
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| Online Access: | http://hdl.handle.net/1721.1/106549 https://orcid.org/0000-0001-7603-8396 |
| _version_ | 1826207453566468096 |
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| author | Clauser, Karl R. Naba, Alexandra Hynes, Richard O. |
| author2 | Massachusetts Institute of Technology. Department of Biology |
| author_facet | Massachusetts Institute of Technology. Department of Biology Clauser, Karl R. Naba, Alexandra Hynes, Richard O. |
| author_sort | Clauser, Karl R. |
| collection | MIT |
| description | 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. |
| first_indexed | 2024-09-23T13:49:53Z |
| format | Article |
| id | mit-1721.1/106549 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T13:49:53Z |
| publishDate | 2017 |
| publisher | MyJoVE Corporation |
| record_format | dspace |
| spelling | mit-1721.1/1065492022-09-28T16:28:40Z Enrichment of Extracellular Matrix Proteins from Tissues and Digestion into Peptides for Mass Spectrometry Analysis Clauser, Karl R. Naba, Alexandra Hynes, Richard O. Massachusetts Institute of Technology. Department of Biology Koch Institute for Integrative Cancer Research at MIT Naba, Alexandra Hynes, Richard O. 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. United States. Department of Defense (DOD Innovator Award W81XWH-14-1-0240) National Cancer Institute (U.S.) (Grant U54 CA126515/CA163109) Broad Institute of MIT and Harvard Howard Hughes Medical Institute National Cancer Institute (U.S.) (David H. Koch Institute for Integrative Cancer Research at MIT. Grant P30-CA14051) 2017-01-20T15:11:20Z 2017-01-20T15:11:20Z 2015-07 Article http://purl.org/eprint/type/JournalArticle 1940-087X http://hdl.handle.net/1721.1/106549 Naba, Alexandra, Karl R. Clauser, and Richard O. Hynes. “Enrichment of Extracellular Matrix Proteins from Tissues and Digestion into Peptides for Mass Spectrometry Analysis.” Journal of Visualized Experiments 101 (2015): n. pag. https://orcid.org/0000-0001-7603-8396 en_US http://dx.doi.org/10.3791/53057 Journal of Visualized Experiments Creative Commons Attribution-NonCommercial-NoDerivs License http://creativecommons.org/licenses/by-nc-nd/3.0/ application/pdf MyJoVE Corporation Journal of Visualized Experiments (JoVE) |
| spellingShingle | Clauser, Karl R. Naba, Alexandra Hynes, Richard O. Enrichment of Extracellular Matrix Proteins from Tissues and Digestion into Peptides for Mass Spectrometry Analysis |
| title | Enrichment of Extracellular Matrix Proteins from Tissues and Digestion into Peptides for Mass Spectrometry Analysis |
| title_full | Enrichment of Extracellular Matrix Proteins from Tissues and Digestion into Peptides for Mass Spectrometry Analysis |
| title_fullStr | Enrichment of Extracellular Matrix Proteins from Tissues and Digestion into Peptides for Mass Spectrometry Analysis |
| title_full_unstemmed | Enrichment of Extracellular Matrix Proteins from Tissues and Digestion into Peptides for Mass Spectrometry Analysis |
| title_short | Enrichment of Extracellular Matrix Proteins from Tissues and Digestion into Peptides for Mass Spectrometry Analysis |
| title_sort | enrichment of extracellular matrix proteins from tissues and digestion into peptides for mass spectrometry analysis |
| url | http://hdl.handle.net/1721.1/106549 https://orcid.org/0000-0001-7603-8396 |
| work_keys_str_mv | AT clauserkarlr enrichmentofextracellularmatrixproteinsfromtissuesanddigestionintopeptidesformassspectrometryanalysis AT nabaalexandra enrichmentofextracellularmatrixproteinsfromtissuesanddigestionintopeptidesformassspectrometryanalysis AT hynesrichardo enrichmentofextracellularmatrixproteinsfromtissuesanddigestionintopeptidesformassspectrometryanalysis |