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  3. Metabolic labeling and LC-MS/MS-based identification of interleukin-1α-induced secreted proteomes from epithelial cells in the presence or absence of serum

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 Protoc. 2023 Mar 31;4(2):102195. doi: 10.1016/j.xpro.2023.102195.
Jasmin Priester 1,  Johanna Meier-Soelch 1,  Hendrik Weiser 1,  Daniel Heylmann 1,  Axel Weber 1,  Uwe Linne 2,  Michael Kracht 3
Affiliations

Affiliations

  • 1 Rudolf Buchheim Institute of Pharmacology, Justus Liebig University, 35392 Giessen, Germany.
  • 2 Mass Spectrometry Facility of the Department of Chemistry, Philipps University, 35032 Marburg, Germany. Electronic address: linneu@staff.uni-marburg.de.
  • 3 Rudolf 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. Electronic address: michael.kracht@pharma.med.uni-giessen.de.
PMID: 37004159 DOI: 10.1016/j.xpro.2023.102195
Abstract

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.

Keywords

Cell Biology; Cell Culture; Flow Cytometry/Mass Cytometry; Immunology; Molecular/Chemical Probes; Protein Biochemistry; Protein Expression and Purification; Proteomics.

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