title: Selective enrichment of newly synthesized proteins for quantitative secretome analysis.
abstract: Secreted proteins constitute a large and biologically important subset of proteins that are involved in cellular communication, adhesion and migration. Yet secretomes are understudied because of technical limitations in the detection of low-abundance proteins against a background of serum-containing media. Here we introduce a method that combines click chemistry and pulsed stable isotope labeling with amino acids in cell culture to selectively enrich and quantify secreted proteins. The combination of these two labeling approaches allows cells to be studied irrespective of the complexity of the background proteins. We provide an in-depth and differential secretome analysis of various cell lines and primary cells, quantifying secreted factors, including cytokines, chemokines and growth factors. In addition, we reveal that serum starvation has a marked effect on secretome composition. We also analyze the kinetics of protein secretion by macrophages in response to lipopolysaccharides.
authors.affiliation: EMBL, Genome Biology Unit, Heidelberg, Germany.
authors.forename: Katrin
authors.initials: K
authors.lastname: Eichelbaum
authors.forename: Markus
authors.initials: M
authors.lastname: Winter
authors.forename: Mauricio
authors.initials: M
authors.lastname: Berriel Diaz
authors.forename: Stephan
authors.initials: S
authors.lastname: Herzig
authors.forename: Jeroen
authors.initials: J
authors.lastname: Krijgsveld
mesh_terms: D002460:Cell Line; D057930:Click Chemistry / Q000379:methods*; D003470:Culture Media; D016895:Culture Media, Serum-Free; D022781:Hepatocytes / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D007553:Isotope Labeling / Q000379:methods*; D007700:Kinetics; D008070:Lipopolysaccharides / Q000494:pharmacology; D008262:Macrophage Activation / Q000187:drug effects; D014176:Protein Biosynthesis / Q000187:drug effects; D060066:Protein Interaction Maps / Q000187:drug effects; D011506:Proteins / Q000378:metabolism*
chemical_list: D003470:Culture Media; D016895:Culture Media, Serum-Free; D008070:Lipopolysaccharides; D011506:Proteins