Sphingosine-1-phosphate mediated chemotaxis of osteoclast precursors investigated using targeted proteomics via mass spectrometry

Thursday, October 11, 2012 — Poster Session IV
2:00 p.m. – 4:00 p.m.	Natcher Conference Center, Building 45	NIAID	PROTEOM-2

Authors

• N.P. Manes
• E. An
• V. Sjoelund
• J. Sun
• M. Ishii
• M. Meier-Schellersheim
• R.N. Germain
• A. Nita-Lazar

Abstract

Osteoclasts are monocyte-derived multinuclear cells that attach to the bone matrix and are responsible for bone resorption. Misregulation of osteoclasts has been implicated in numerous skeletal diseases including osteoporosis and osteoarthritis. Our laboratory recently reported that the phosphosphingolipid sphingosine-1-phosphate (S1P) regulates bone resorption in mice by mediating both chemotaxis and chemorepulsion of osteoclast precursors through two G-protein coupled receptors, which antagonize each other in an S1P-concentration dependent manner. In this investigation, transcriptomics, shotgun proteomics, and targeted proteomics are being employed to enable quantitative simulation of the S1P-chemotaxis signaling pathway within osteoclast precursors (RAW 264.7 cells). RNA-Seq was used to identify proteins expressed in RAW cells, and proteotypic peptides were identified using shotgun mass spectrometry. SPOT synthesis was used to prepare 409 crude, unlabeled, semi-quantitated peptide standards against 171 target proteins. Targeted mass spectrometry of these peptides was used to develop SRM assays. SRM of RAW cell samples resulted in the identification and semi-quantitation of 208 of the 409 peptide targets. Robust absolute quantitation using heavy-labeled, quantitated internal peptide standards is underway. Additionally, a supplementary set of crude, unlabeled peptides is being prepared to target the missed proteins. Quantitative pathway simulation will be performed using Simmune Modeler.

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