title: Application of SILAC labeling to primary bone marrow-derived dendritic cells reveals extensive GM-CSF-dependent arginine metabolism.
abstract: Although dendritic cells (DCs) control the priming of the adaptive immunity response, a comprehensive description of their behavior at the protein level is missing. The introduction of the quantitative proteomic technique of metabolic labeling (SILAC) into the field of DC research would therefore be highly beneficial. To achieve this, we applied SILAC labeling to primary bone marow-derived DCs (BMDCs). These cells combine both biological relevance and experimental feasibility, as their in vitro generation permits the use of (13)C/(15)N-labeled amino acids. Interestingly, BMDCs appear to exhibit a very active arginine metabolism. Using standard cultivation conditions, ∼20% of all protein-incorporated proline was a byproduct of heavy arginine degradation. In addition, the dissipation of (15)N from labeled arginine to the whole proteome was observed. The latter decreased the mass accuracy in MS and affected the natural isotopic distribution of peptides. SILAC-connected metabolic issues were shown to be enhanced by GM-CSF, which is used for the differentiation of DC progenitors. Modifications of the cultivation procedure suppressed the arginine-related effects, yielding cells with a proteome labeling efficiency of ≥90%. Importantly, BMDCs generated according to the new cultivation protocol preserved their resemblance to inflammatory DCs in vivo, as evidenced by their response to LPS treatment.
authors.affiliation: Institute of Molecular Pathology, Faculty of Military Health Sciences, University of Defence , Trebesska 1575, 500 01 Hradec Kralove, Czech Republic.
authors.forename: Ivo
authors.initials: I
authors.lastname: Fabrik
authors.forename: Marek
authors.initials: M
authors.lastname: Link
authors.forename: Anetta
authors.initials: A
authors.lastname: Härtlova
authors.forename: Vera
authors.initials: V
authors.lastname: Dankova
authors.forename: Pavel
authors.initials: P
authors.lastname: Rehulka
authors.forename: Jiri
authors.initials: J
authors.lastname: Stulik
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000378:metabolism*; D001854:Bone Marrow Cells / Q000378:metabolism*; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D003713:Dendritic Cells / Q000378:metabolism*; D005260:Female; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D011392:Proline / Q000378:metabolism; D020543:Proteome; D053719:Tandem Mass Spectrometry
chemical_list: D020543:Proteome; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D001120:Arginine; D011392:Proline