Metabolic labeling and LC-MS/MS-based identification of interleukin-1α-induced secreted proteomes from epithelial cells in the presence or absence of serum
Summary: The unbiased identification of cytokine-induced, secreted proteins from cells cultured in serum-containing medium is challenging. Here, we describe an experimental and bioinformatics workflow to label interleukin-1α-regulated proteins in living cells with the methionine analogue L-homopropa...
| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2023-06-01
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| Series: | STAR Protocols |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2666166723001533 |
| _version_ | 1797854266224803840 |
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| author | Jasmin Priester Johanna Meier-Soelch Hendrik Weiser Daniel Heylmann Axel Weber Uwe Linne Michael Kracht |
| author_facet | Jasmin Priester Johanna Meier-Soelch Hendrik Weiser Daniel Heylmann Axel Weber Uwe Linne Michael Kracht |
| author_sort | Jasmin Priester |
| collection | DOAJ |
| description | Summary: The unbiased identification of cytokine-induced, secreted proteins from cells cultured in serum-containing medium is challenging. Here, we describe an experimental and bioinformatics workflow to label interleukin-1α-regulated proteins in living cells with the methionine analogue L-homopropargylglycine. We detail their purification and identification by means of CLICK-chemistry-based biotinylation followed by nanoHPLC-MS/MS. A side-by-side comparison of enriched proteins and their ontologies to serum-free conditions demonstrates the sensitivity and specificity of this approach to study the inducible secreted proteomes of epithelial cells. : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. |
| first_indexed | 2024-04-09T20:04:15Z |
| format | Article |
| id | doaj.art-6971a1607610402ca927b0d82aaf7e32 |
| institution | Directory Open Access Journal |
| issn | 2666-1667 |
| language | English |
| last_indexed | 2024-04-09T20:04:15Z |
| publishDate | 2023-06-01 |
| publisher | Elsevier |
| record_format | Article |
| series | STAR Protocols |
| spelling | doaj.art-6971a1607610402ca927b0d82aaf7e322023-04-02T06:15:12ZengElsevierSTAR Protocols2666-16672023-06-0142102195Metabolic labeling and LC-MS/MS-based identification of interleukin-1α-induced secreted proteomes from epithelial cells in the presence or absence of serumJasmin Priester0Johanna Meier-Soelch1Hendrik Weiser2Daniel Heylmann3Axel Weber4Uwe Linne5Michael Kracht6Rudolf Buchheim Institute of Pharmacology, Justus Liebig University, 35392 Giessen, GermanyRudolf Buchheim Institute of Pharmacology, Justus Liebig University, 35392 Giessen, GermanyRudolf Buchheim Institute of Pharmacology, Justus Liebig University, 35392 Giessen, GermanyRudolf Buchheim Institute of Pharmacology, Justus Liebig University, 35392 Giessen, GermanyRudolf Buchheim Institute of Pharmacology, Justus Liebig University, 35392 Giessen, GermanyMass Spectrometry Facility of the Department of Chemistry, Philipps University, 35032 Marburg, Germany; Corresponding authorRudolf Buchheim Institute of Pharmacology, Justus Liebig University, 35392 Giessen, Germany; Universities of Giessen and Marburg Lung Center (UGMLC), Giessen, Germany; Cardio-Pulmonary Institute (CPI), Giessen, Germany; Corresponding authorSummary: The unbiased identification of cytokine-induced, secreted proteins from cells cultured in serum-containing medium is challenging. Here, we describe an experimental and bioinformatics workflow to label interleukin-1α-regulated proteins in living cells with the methionine analogue L-homopropargylglycine. We detail their purification and identification by means of CLICK-chemistry-based biotinylation followed by nanoHPLC-MS/MS. A side-by-side comparison of enriched proteins and their ontologies to serum-free conditions demonstrates the sensitivity and specificity of this approach to study the inducible secreted proteomes of epithelial cells. : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.http://www.sciencedirect.com/science/article/pii/S2666166723001533Cell BiologyCell CultureFlow Cytometry/Mass CytometryImmunologyMolecular/Chemical ProbesProtein Biochemistry |
| spellingShingle | Jasmin Priester Johanna Meier-Soelch Hendrik Weiser Daniel Heylmann Axel Weber Uwe Linne Michael Kracht Metabolic labeling and LC-MS/MS-based identification of interleukin-1α-induced secreted proteomes from epithelial cells in the presence or absence of serum STAR Protocols Cell Biology Cell Culture Flow Cytometry/Mass Cytometry Immunology Molecular/Chemical Probes Protein Biochemistry |
| title | Metabolic labeling and LC-MS/MS-based identification of interleukin-1α-induced secreted proteomes from epithelial cells in the presence or absence of serum |
| title_full | Metabolic labeling and LC-MS/MS-based identification of interleukin-1α-induced secreted proteomes from epithelial cells in the presence or absence of serum |
| title_fullStr | Metabolic labeling and LC-MS/MS-based identification of interleukin-1α-induced secreted proteomes from epithelial cells in the presence or absence of serum |
| title_full_unstemmed | Metabolic labeling and LC-MS/MS-based identification of interleukin-1α-induced secreted proteomes from epithelial cells in the presence or absence of serum |
| title_short | Metabolic labeling and LC-MS/MS-based identification of interleukin-1α-induced secreted proteomes from epithelial cells in the presence or absence of serum |
| title_sort | metabolic labeling and lc ms ms based identification of interleukin 1α induced secreted proteomes from epithelial cells in the presence or absence of serum |
| topic | Cell Biology Cell Culture Flow Cytometry/Mass Cytometry Immunology Molecular/Chemical Probes Protein Biochemistry |
| url | http://www.sciencedirect.com/science/article/pii/S2666166723001533 |
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