Cell type-selective secretome profiling in vivo

Abstract

Secreted polypeptides are a fundamental biochemical axis of intercellular and endocrine communication. However, a global understanding of composition and dynamics of cellular secretomes in intact mammalian organisms has been lacking. Here, we introduce a proximity biotinylation strategy that enables labeling, detection, and enrichment of secreted polypeptides in a cell type-selective manner in mice. We generate a proteomic atlas of hepatocyte, myocyte, pericyte, and myeloid cell secretomes by direct purification of biotinylated secreted proteins from blood plasma. Our secretome dataset validates known cell type-protein pairs, reveals secreted polypeptides that distinguish between cell types, and identifies new cellular sources for classical plasma proteins. Lastly, we uncover a dynamic and previously undescribed nutrient-dependent reprogramming of the hepatocyte secretome characterized by increased unconventional secretion of the cytosolic enzyme BHMT. This secretome profiling strategy enables dynamic and cell-type dissection of the plasma proteome and the secreted polypeptides that mediate intercellular signaling.

Spatiotemporally-resolved mapping of RNA binding proteins via functional proximity labeling reveals a mitochondrial mRNA anchor promoting stress recovery

“…Addition of a small molecule substrate to live cells results in catalytic generation of a reactive biotin species (such as biotin-phenoxyl radical for APEX or biotin-AMP for TurboID) from the active site of the promiscuous enzyme, which diffuses outward to covalently tag proximal endogenous proteins. PL methods have been used to map organelle proteomes (mitochondrion, ER, lipid droplets, stress granules) 6 , dynamic interactomes 7 , 8 , and in vivo secretomes 9 , 10 . Recently, PL was extended to spatial mapping of transcriptomes in living cells 11 – 13 .…”

Cell type-specific biotin labeling in vivo resolves regional neuronal proteomic differences in mouse brain

“…The labeling of secreted and vesicular proteins also suggests that simultaneous profiling of secreted or released proteins in tissues and biofluids may be feasible. Indeed, secretome profiling using in vivo TurboID via AAV delivery was successful in a non-brain context 30 . Our ongoing studies with Rosa26 TurboID mice, in brain and non-brain disease contexts, will determine whether cellular origin of secreted proteins can indeed be measured in biofluids as biomarkers of underlying cellular mechanisms of disease.…”

Cell-specific Bioorthogonal Tagging of Glycoproteins

“…While glycans are a means to an end in this respect, the large signal-to-noise ratio in our fluorescent labelling experiments paired with the absence of toxicity in MOE indicates that our BOCTAG is complementary to other techniques, including the use of unnatural amino acids and proximity biotinylation. 30,31 Second, directly incorporating glycans in the analysis will give insight into cell-type-specific glycosylation sites and glycan structures to add another dimension to cell-type-specific glycoproteome profiling. A metabolic AND gate was necessary to ensure minimal background labelling while being able to supply the tagged monosaccharide as an easy-to-synthesise MOE reagent, which is in marked difference to highly unstable caged sugar-1-phosphates used previously.…”