A Dual SILAC Proteomic Labeling Strategy for Quantifying Constitutive and Cell–Cell Induced Protein Secretion

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.