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

Tuesday, October 25, 2011 — Poster Session II
Noon – 2:00 p.m.	Natcher Conference Center	NIAID	PROTEOM-5

Authors

• NP Manes
• E An
• V Sjoelund
• M Ishii
• M Meier-Schellersheim
• RN Germain
• A Nita-Lazar

Abstract

Osteoclasts are monocyte-derived multinuclear cells that attach to the bone matrix and are responsible for bone resorption. Osteoclast misregulation has been implicated in numerous skeletal diseases including osteoporosis and osteoarthritis. Our laboratory has previously 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 (S1PR1 and S1PR2) which antagonize each other in an S1P-concentration dependent manner. In this investigation, targeted proteomics is being applied to map the S1P signaling pathway, and to discover and validate potential therapeutic targets in close collaboration with an ongoing shotgun proteomics + phosphoproteomics study. Triple quadrupole mass spectrometry (nanoLC-QqQ-SRM) is being used to perform extremely sensitive (100 amol), highly reproducible (CV<10%) absolute quantitation measurements of 409 tryptic peptides derived from 171 targeted proteins within cultured cell lysates. SRM assay development is ongoing and includes in silico selection of proteotypic peptides, SPOT synthesis of peptide standards, and nanoLC-QqQ-SRM optimization. Absolute quantitation of phosphopeptides originating from the 171 proteins will be employed to build a model of S1P-mediated chemotaxis in osteoclast precursors.

back to top