title: Quantitative Secretome Analysis of Activated Jurkat Cells Using Click Chemistry-Based Enrichment of Secreted Glycoproteins.
abstract: Quantitative secretome analyses are a high-performance tool for the discovery of physiological and pathophysiological changes in cellular processes. However, serum supplements in cell culture media limit secretome analyses, but serum depletion often leads to cell starvation and consequently biased results. To overcome these limiting factors, we investigated a model of T cell activation (Jurkat cells) and performed an approach for the selective enrichment of secreted proteins from conditioned medium utilizing metabolic marking of newly synthesized glycoproteins. Marked glycoproteins were labeled via bioorthogonal click chemistry and isolated by affinity purification. We assessed two labeling compounds conjugated with either biotin or desthiobiotin and the respective secretome fractions. 356 proteins were quantified using the biotin probe and 463 using desthiobiotin. 59 proteins were found differentially abundant (adjusted p-value ≤0.05, absolute fold change ≥1.5) between inactive and activated T cells using the biotin method and 86 using the desthiobiotin approach, with 31 mutual proteins cross-verified by independent experiments. Moreover, we analyzed the cellular proteome of the same model to demonstrate the benefit of secretome analyses and provide comprehensive data sets of both. 336 proteins (61.3%) were quantified exclusively in the secretome. Data are available via ProteomeXchange with identifier PXD004280.
authors.affiliation: Medizinisches Proteom-Center, Ruhr-Universität Bochum , 44801 Bochum, Germany.
authors.forename: Kathrin E
authors.initials: KE
authors.lastname: Witzke
authors.affiliation: Medizinisches Proteom-Center, Ruhr-Universität Bochum , 44801 Bochum, Germany.
authors.forename: Kristin
authors.initials: K
authors.lastname: Rosowski
authors.affiliation: Medizinisches Proteom-Center, Ruhr-Universität Bochum , 44801 Bochum, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Müller
authors.affiliation: Medizinisches Proteom-Center, Ruhr-Universität Bochum , 44801 Bochum, Germany.
authors.forename: Maike
authors.initials: M
authors.lastname: Ahrens
authors.affiliation: Medizinisches Proteom-Center, Ruhr-Universität Bochum , 44801 Bochum, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Eisenacher
authors.affiliation: Medizinisches Proteom-Center, Ruhr-Universität Bochum , 44801 Bochum, Germany.
authors.forename: Dominik A
authors.initials: DA
authors.lastname: Megger
authors.affiliation: Medical Clinic III for Pneumology, Allergology, Sleep and Respiratory Medicine, Bergmannsheil University Hospital, Ruhr-Universität Bochum , 44801 Bochum, Germany.
authors.forename: Jürgen
authors.initials: J
authors.lastname: Knobloch
authors.affiliation: Medical Clinic III for Pneumology, Allergology, Sleep and Respiratory Medicine, Bergmannsheil University Hospital, Ruhr-Universität Bochum , 44801 Bochum, Germany.
authors.forename: Andrea
authors.initials: A
authors.lastname: Koch
authors.affiliation: Medizinisches Proteom-Center, Ruhr-Universität Bochum , 44801 Bochum, Germany.
authors.forename: Thilo
authors.initials: T
authors.lastname: Bracht
authors.affiliation: Medizinisches Proteom-Center, Ruhr-Universität Bochum , 44801 Bochum, Germany.
authors.forename: Barbara
authors.initials: B
authors.lastname: Sitek
mesh_terms: D001710:Biotin / Q000031:analogs & derivatives / Q000737:chemistry; D002846:Chromatography, Affinity; D057930:Click Chemistry / Q000379:methods*; D017077:Culture Media, Conditioned / Q000737:chemistry; D015870:Gene Expression; D063990:Gene Ontology; D006023:Glycoproteins / Q000096:biosynthesis / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D019169:Jurkat Cells; D008213:Lymphocyte Activation; D058977:Molecular Sequence Annotation; D014176:Protein Biosynthesis; D020543:Proteome / Q000096:biosynthesis / Q000302:isolation & purification* / Q000378:metabolism; D013194:Staining and Labeling / Q000379:methods*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D017077:Culture Media, Conditioned; D006023:Glycoproteins; D020543:Proteome; D001710:Biotin; C004749:desthiobiotin; D013755:Tetradecanoylphorbol Acetate
keywords: Jurkat cells; SPECS; T cell activation; affinity purification; biotin; click chemistry; desthiobiotin; label-free proteomics; quantitative secretomics