title: A Dual SILAC Proteomic Labeling Strategy for Quantifying Constitutive and Cell-Cell Induced Protein Secretion.
abstract: Recent evidence suggests that the extracellular protein milieu is much more complex than previously assumed as various secretome analyses from different cell types described the release of hundreds to thousands of proteins. The extracellular function of many of these proteins has yet to be determined particularly in the context of three-dimensional tissues with abundant cell-cell contacts. Toward this goal, we developed a strategy of dual SILAC labeling astrocytic cultures for in silico exclusion of unlabeled proteins from serum or neurons used for stimulation. For constitutive secretion, this strategy allowed the precise quantification of the extra-to-intracellular protein ratio of more than 2000 identified proteins. Ratios covered 4 orders of magnitude indicating that the intracellular vs extracellular contributions of different proteins can be variable. Functionally, the secretome of labeled forebrain astrocytic cultures specifically changed within hours after adding unlabeled, "physiological" forebrain neurons. "Nonphysiological" cerebellar hindbrain neurons, however, elicited a different, highly repulsive secretory response. Our data also suggest a significant association of constitutive secretion with the classical secretion pathway and regulated secretion with unconventional pathways. We conclude that quantitative proteomics can help to elucidate general principles of cellular secretion and provide functional insight into the abundant extracellular presence of proteins.
authors.affiliation: †Max-Planck-Institute for Neurobiology, Am Klopferspitz 18, 82152 Munich-Martinsried, Germany.|‡Biozentrum, University of Basel, Klingelbergstrasse 50, 4056 Basel, Switzerland.
authors.forename: Michael
authors.initials: M
authors.lastname: Stiess
authors.affiliation: §Heidelberg University Biochemistry Center, Im Neuenheimer Feld 328, 69120 Heidelberg, Germany.
authors.forename: Sabine
authors.initials: S
authors.lastname: Wegehingel
authors.forename: Chuong
authors.initials: C
authors.lastname: Nguyen
authors.affiliation: §Heidelberg University Biochemistry Center, Im Neuenheimer Feld 328, 69120 Heidelberg, Germany.
authors.forename: Walter
authors.initials: W
authors.lastname: Nickel
authors.affiliation: †Max-Planck-Institute for Neurobiology, Am Klopferspitz 18, 82152 Munich-Martinsried, Germany.|⊥Axon Growth and Regeneration Group, Deutsches Zentrum für Neurodegenerative Erkrankungen, 51375 Bonn, Germany.
authors.forename: Frank
authors.initials: F
authors.lastname: Bradke
authors.affiliation: #Department of Functional Neuroanatomy, University of Heidelberg, Im Neuenheimer Feld 307, 69120 Heidelberg, Germany.
authors.forename: Sidney B
authors.initials: SB
authors.lastname: Cambridge
mesh_terms: D000596:Amino Acids / Q000378:metabolism*; D000818:Animals; D001253:Astrocytes / Q000166:cytology / Q000378:metabolism; D015153:Blotting, Western; D002450:Cell Communication; D002478:Cells, Cultured; D007553:Isotope Labeling / Q000379:methods; D013058:Mass Spectrometry; D009457:Neuroglia / Q000166:cytology / Q000378:metabolism; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D016548:Prosencephalon / Q000166:cytology; D020543:Proteome / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D051381:Rats; D015398:Signal Transduction
chemical_list: D000596:Amino Acids; D020543:Proteome
keywords: SILAC; astrocytes; extracellular matrix; proteomics; secretion