Reagent-free total protein quantification of intact extracellular vesicles by attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy

Veronika Szentirmai,Zoltán Varga,Diána Kitka,Csaba Németh,Károly Héberger,Judith Mihály,Anita Rácz,András Wacha

doi:10.1007/s00216-020-02711-8

Abstract

Extracellular vesicles (EVs) are lipid bilayer–bounded particles that are actively synthesized and released by cells. The main components of EVs are lipids, proteins, and nucleic acids and their composition is characteristic to their type and origin, and it reveals the physiological and pathological conditions of the parent cells. The concentration and protein composition of EVs closely relate to their functions; therefore, total protein determination can assist in EV-based diagnostics and disease prognosis. Here, we present a simple, reagent-free method based on attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy to quantify the protein content of EV samples without any further sample preparation. After calibration with bovine serum albumin, the protein concentration of red blood cell–derived EVs (REVs) were investigated by ATR-FTIR spectroscopy. The integrated area of the amide I band was calculated from the IR spectra of REVs, which was proportional to the protein quantity in the sample‚ regardless of its secondary structure. A spike test and a dilution test were performed to determine the ability to use ATR-FTIR spectroscopy for protein quantification in EV samples, which resulted in linearity with R2 values as high as 0.992 over the concentration range of 0.08 to 1 mg/mL. Additionally, multivariate calibration with the partial least squares (PLS) regression method was carried out on the bovine serum albumin and EV spectra. R2 values were 0.94 for the calibration and 0.91 for the validation set. The results indicate that ATR-FTIR measurements provide a reliable method for reagent-free protein quantification of EVs.Graphical abstract

Highlights

Extracellular vesicles (EVs) are heterogeneous lipid bilayer– bounded particles created and secreted by cells [1, 2]
The presence and quality of red blood cell–derived EVs (REVs) in the samples was investigated by freeze-fracture combined electron microscopy (FF-TEM) and microfluidic resistive pulse sensing (MRPS) measurements
IR spectroscopy, the attenuated total reflection (ATR)-FTIR technique is widely used for the analysis of biofluids including quantification of

Summary

Introduction

Extracellular vesicles (EVs) are heterogeneous lipid bilayer– bounded particles created and secreted by cells [1, 2]. Their common feature is their size range from 30 nm up to several. On appropriate dilutions of gravimetrically prepared 10 mg/mL solutions of bovine serum albumin (BSA), Biuret, Lowry and Bradford assays provided results of 9.7, 8.4 and 21.1 mg/mL protein concentrations, respectively [10]. Our research group was a pioneer in the application of ATR-FTIR spectroscopy as a reagent-free, non-invasive and simple measurement mode to study extracellular vesicles [17]. Based on selected amide and C–H stretching band intensity ratios, we proposed a “spectroscopic protein-to-lipid ratio” which provides a useful index for EV characterization [8, 17,18,19]

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