Session: Session 5

Metabolomic Profiling of Osteocyte Extracellular Vesicles and Matrix Bound Vesicles using a Prototype Benchtop Multi Reflecting Time-of-Flight (MRT) Mass Spectrometer

Introduction

Extracellular vesicles (EVs) are lipid delimited nanoparticles that function in development and intercellular signaling events throughout the body. Within bone there exists a subset of EVs, known as matrix binding vesicles (MBVs), which have been long proposed to associate with the underlying collagenous extracellular matrix to drive early mineralization events during bone development. However, the precise relationship between EVs and MBVs and their differential roles in bone development and metabolism remains a point of contention.



This study uses a prototype benchtop MRT to construct a comparative metabolite profile for EVs and MBVs obtained from MC3T3 pre-osteoblasts under osteogenic culture conditions.



Preliminary data 

EVs and MBV’s were extracted from MC3T3 osteocyte cells and analysed by both HILIC and Reversed-Phase chromatographic methods - to ensure maximal compound coverage – on a prototype benchtop MRT mass spectrometer. Chromatographic separation was investigated using ‘conventional’ scale (10 minute gradient on a 2.1x100mm column) and also a higher throughput methodology which utilized a 3.5 minute gradient, using a 1x50mm column. Data were acquired using a data independent analysis (DIA) strategy, across the mass range 50-1200 Da, and the instrument consistently produced a mass spectral resolution in the region of 100,000 FWHM.



This feasibility dataset has putatively identified several significant biologically relevant polar metabolites, including amino acids, carboxylic acids, fatty acids, phenols, pyridines and indoles being present within both the EV and MBV extracts. LC-MS data were processed using MARS (Mass Analytica). The data were peak picked, normalized and putative compound identifications were gained with database searches conducted using a library consisting of the human metabolome (HMDB) databas