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

Friday, November 08, 2013 — Poster Session IV
2:00 p.m. – 4:00 p.m.	FAES Academic Center (Upper-Level Terrace)	NIAID	STRUCTBIO-10

Authors

N.P. Manes
E. An
V.H. Sjoelund
J. Sun
B.R. Angermann
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 (OPs) through two G-protein coupled receptors (S1PR1 and S1PR2), which antagonize each other in an S1P-concentration dependent manner. In this investigation, transcriptomics and proteomics are being used to enable simulation of the S1P-chemotaxis signaling pathway in OPs. A set of 171 mouse chemotaxis pathway target proteins was constructed from a literature review. RNA-Seq was used to identify OP protein transcripts. Shotgun nanoLC-LTQ-Velos-Orbitrap mass spectrometry was used to identify proteotypic peptides. In silico selection of the target peptides used a wide variety of criteria (e.g., proteotypic qualities, sequence uniqueness, vulnerability to PTMs). SPOT synthesis was used to prepare 557 crude peptide standards against the 171 target proteins. LC-SRM assays were developed using shotgun and targeted nanoLC-TSQ-Vantage-QqQ mass spectrometry. Absolute quantitation experiments were performed using 65 heavy-labeled, quantitated, internal peptide standards. Pathway simulation was performed using Simmune.