ATR-FTIR spectroscopy for the routine quality control of exosome isolations

Abstract

Exosomes are nanosized vesicles containing specific cargos of DNA, RNA, proteins, metabolites, and intracellular and membrane lipids. Exosome isolation needs to be optimized carefully depending on the type of biofluid and tissue and the retrieved exosomes need to be characterized. The main objective of this study was to determine the feasibility of a multimodal analysis of Attenuated Total Reflectance – Fourier Transform Infrared (ATR-FTIR) spectroscopy and UPLC–QqTOF-MSMS for the development of a routine quality control tool of isolated exosomes and the rapid characterization of their lipid profiles and total protein content. Using human milk as model example, exosomes were isolated by multi-stage ultracentrifugation. After single-phase extraction, lipidomic analysis was carried out by UPLC–QqTOF-MSMS with automated MSMS-based annotation using HMDB, METLIN, LipidBlast and MSDIAL databases. The classes with the largest number of annotated features were glycerophospholipids, sphingolipids, and glycerolipids. Then, dry films of 2 ​μL exosomes were directly analyzed by ATR-FTIR. Multivariate analysis showed significant associations between ATR-FTIR specific regions and the concentrations of different lipid classes. Principal component analysis and Hierarchical Cluster Analysis of IR and lipidomic data showed that ATR−FTIR renders valuable qualitative descriptors of the lipid content of isolated exosomes. Total LC-MS lipid and total protein contents could also be quantified by using the area of CHs and CO stretching bands as well as the amide I band. As a conclusion, results obtained show that multimodal analysis of ATR-FTIR and UPLC-MS data is a useful tool for the development of spectroscopic methods. ATR-FTIR provided both, qualitative and quantitative chemical descriptors of isolated exosomes, enabling a fast and direct quantification of total protein and lipid contents.