TY - JOUR
T1 - Reagent-free total protein quantification of intact extracellular vesicles by attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy
AU - Szentirmai, Veronika
AU - Wacha, András
AU - Németh, Csaba
AU - Kitka, Diána
AU - Rácz, Anita
AU - Héberger, Károly
AU - Mihály, Judith
AU - Varga, Zoltán
N1 - Publisher Copyright:
© 2020, The Author(s).
PY - 2020/7/1
Y1 - 2020/7/1
N2 - 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.].
AB - 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.].
KW - ATR-FTIR
KW - Chemometrics
KW - Extracellular vesicle (EV)
KW - Infrared spectroscopy
KW - Protein quantification
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U2 - 10.1007/s00216-020-02711-8
DO - 10.1007/s00216-020-02711-8
M3 - Article
C2 - 32472144
AN - SCOPUS:85085743964
SN - 1618-2642
VL - 412
SP - 4619
EP - 4628
JO - Analytical and Bioanalytical Chemistry
JF - Analytical and Bioanalytical Chemistry
IS - 19
ER -