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
  • 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: [email protected].
  • 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: [email protected].
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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