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

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

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25 Scopus citations


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. [Figure not available: see fulltext.].

Original languageEnglish (US)
Pages (from-to)4619-4628
Number of pages10
JournalAnalytical and Bioanalytical Chemistry
Issue number19
StatePublished - Jul 1 2020
Externally publishedYes

Bibliographical note

Funding Information:
Open access funding provided by Research Centre for Natural Sciences. This study was funded by the National Research, Development and Innovation Office NKFIH, Hungary under grant numbers NVKP_16-1-2016-0007, PD 121326 (Zoltán Varga), PD 124451 (András Wacha), K 131594 (Judith Mihály) and K 119269 (Anita Rácz and Károly Héberger). Zoltán Varga was supported by the János Bolyai Research Scholarship of the Hungarian Academy of Sciences. Diana Kitka was supported by the ÚNKP-19-3 New National Excellence Program of the Ministry for Innovation and Technology. Acknowledgments

Publisher Copyright:
© 2020, The Author(s).


  • Chemometrics
  • Extracellular vesicle (EV)
  • Infrared spectroscopy
  • Protein quantification


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