# In vivo discovery of the osteocyte protein secretome: identification of novel factors and functions > **NIH NIH R33** · INDIANA UNIVERSITY INDIANAPOLIS · 2020 · $396,250 ## Abstract The osteocyte—a dynamic and metabolically active cell—regulates numerous and diverse physiologic functions (e.g., kidney, immune system, bone turnover, and others). Discoveries regarding the osteocyte’s regulatory roles are largely fortuitous, i.e., they were identified not by a systematic screen of the osteocyte’s products/functions, but rather, by identifying a particular messenger molecule that has important effects on physiologic function, then tracing its source back to the osteocyte. This approach has yielded numerous important factors, several of which have proven to be attractive drug targets for skeletal and non-skeletal therapies. However, it is very likely that the osteocyte produces many more medically important factors than are currently known, and a systematic approach to identifying the totality of osteocyte-derived factors, in vivo (where cell culture conditions are not a factor) is long overdue. The problem with a systematic approach to quantifying the osteocyte’s in vivo proteome, both the intracellular protein pool and secreted proteins, has been the lack of biological/biochemical research tools and technical proteomics tools to successfully attempt such an endeavor, until now. We now have a genetically engineered mouse model that facilitates metabolic labeling of proteins selectively within osteocytes, using an azide-tagged synthetic amino acid— Azidonorleucine (Anl)—that substitutes for Methionine in synthesizing peptide chains, only in osteocytes. Anl is bio- orthogonal, i.e., it does not perturb the biology of proteins into which it incorporates. The physical properties of Anl exclude it from interacting with the wild-type enzyme (MetRS) that attaches (“charges”) Methionine to tRNA carriers, but expression of a mutant MetRS (MetRSL274G) promotes Anl charging to tRNA. Therefore, expression of the MetRSL274G allele selectively in osteocytes, plus dietary supplementation with Anl, allows in vivo metabolic labeling of proteins made by osteocytes. Osteocyte-generated proteins can subsequently be captured from bone tissue or serum using a “click” chemistry reaction to efficiently select for the functional azide group, and captured proteins can be identified/quantified using state-of-the-art mass spectrometry-based proteomics. The proteomics approach proposed will facilitate unprecedented sensitivity, depth, and control for very low abundance proteins. We capitalize on these advances to, for the first time, identify and quantify the entirety of the osteocyte proteome in vivo, including the secreted portion of the osteocyte proteome—the protein secretome. In Aim 1, procedural optimization for protein labeling and capture from bone tissue samples will be accomplished. In Aim 2, special techniques will be employed to capture and reveal circulating factors secreted into the serum by osteocytes, including the development of novel biomarker assays. Aim 3 (the R33 phase) will follow up on the novel protein leads generated by Aims 1 ... ## Key facts - **NIH application ID:** 10197344 - **Project number:** 4R33AR073551-03 - **Recipient organization:** INDIANA UNIVERSITY INDIANAPOLIS - **Principal Investigator:** ALEXANDER G ROBLING - **Activity code:** R33 (R01, R21, SBIR, etc.) - **Funding institute:** NIH - **Fiscal year:** 2020 - **Award amount:** $396,250 - **Award type:** 4N - **Project period:** 2018-03-01 → 2022-07-31 ## Primary source NIH RePORTER: https://reporter.nih.gov/project-details/10197344 ## Citation > US National Institutes of Health, RePORTER application 10197344, In vivo discovery of the osteocyte protein secretome: identification of novel factors and functions (4R33AR073551-03). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10197344. Licensed CC0. --- *[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*