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@article{4e491bfa885648dab1120478f78c62de,
title = "Quantification of Intrinsically Disordered Proteins: A Problem Not Fully Appreciated",
abstract = "Protein quantification is essential in a great variety of biochemical assays, yet the inherent systematic errors associated with the concentration determination of intrinsically disordered proteins (IDPs) using classical methods are hardly appreciated. Routinely used assays for protein quantification, such as the Bradford assay or ultraviolet absorbance at 280 nm, usually seriously misestimate the concentrations of IDPs due to their distinct and variable amino acid composition. Therefore, dependable method(s) have to be worked out/adopted for this task. By comparison to elemental analysis as the gold standard, we show through the example of four globular proteins and nine IDPs that the ninhydrin assay and the commercial QubitTM Protein Assay provide reliable data on IDP quantity. However, as IDPs can show extreme variation in amino acid composition and physical features not necessarily covered by our examples, even these techniques should only be used for IDPs following standardization. The far-reaching implications of these simple observations are demonstrated through two examples: (i) circular dichroism spectrum deconvolution, and (ii) receptor-ligand affinity determination. These actual comparative examples illustrate the potential errors that can be incorporated into the biophysical parameters of IDPs, due to systematic misestimation of their concentration. This leads to inaccurate description of IDP functions.",
keywords = "Circular dichroism, Coomassie brilliant blue, Elemental analysis, Error propagation, Nanoorange, Ninhydrin, Protein concentration, UV absorbance",
author = "{Contreras Martos}, Sara and Nguyen, {Huy Hung} and {Nguyen Nhu}, Phuong and Hristozova, {Nevena Ivanova} and {Macossay Castillo}, Mauricio and Kovacs, {Sandor Denes} and Angela Bekesi and Oemig, {Jesper Skottegaard} and Dominique Maes and Kris Pauwels and Peter Tompa and Pierre Lebrun",
year = "2018",
month = "9",
day = "4",
doi = "10.3389/fmolb.2018.00083",
language = "English",
volume = "5",
pages = "83--83",
journal = "Frontiers in Molecular Biosciences",
issn = "2296-889X",
publisher = "Frontiers Media",
number = "SEP",

}

RIS

TY - JOUR

T1 - Quantification of Intrinsically Disordered Proteins: A Problem Not Fully Appreciated

AU - Contreras Martos, Sara

AU - Nguyen, Huy Hung

AU - Nguyen Nhu, Phuong

AU - Hristozova, Nevena Ivanova

AU - Macossay Castillo, Mauricio

AU - Kovacs, Sandor Denes

AU - Bekesi, Angela

AU - Oemig, Jesper Skottegaard

AU - Maes, Dominique

AU - Pauwels, Kris

AU - Tompa, Peter

AU - Lebrun, Pierre

PY - 2018/9/4

Y1 - 2018/9/4

N2 - Protein quantification is essential in a great variety of biochemical assays, yet the inherent systematic errors associated with the concentration determination of intrinsically disordered proteins (IDPs) using classical methods are hardly appreciated. Routinely used assays for protein quantification, such as the Bradford assay or ultraviolet absorbance at 280 nm, usually seriously misestimate the concentrations of IDPs due to their distinct and variable amino acid composition. Therefore, dependable method(s) have to be worked out/adopted for this task. By comparison to elemental analysis as the gold standard, we show through the example of four globular proteins and nine IDPs that the ninhydrin assay and the commercial QubitTM Protein Assay provide reliable data on IDP quantity. However, as IDPs can show extreme variation in amino acid composition and physical features not necessarily covered by our examples, even these techniques should only be used for IDPs following standardization. The far-reaching implications of these simple observations are demonstrated through two examples: (i) circular dichroism spectrum deconvolution, and (ii) receptor-ligand affinity determination. These actual comparative examples illustrate the potential errors that can be incorporated into the biophysical parameters of IDPs, due to systematic misestimation of their concentration. This leads to inaccurate description of IDP functions.

AB - Protein quantification is essential in a great variety of biochemical assays, yet the inherent systematic errors associated with the concentration determination of intrinsically disordered proteins (IDPs) using classical methods are hardly appreciated. Routinely used assays for protein quantification, such as the Bradford assay or ultraviolet absorbance at 280 nm, usually seriously misestimate the concentrations of IDPs due to their distinct and variable amino acid composition. Therefore, dependable method(s) have to be worked out/adopted for this task. By comparison to elemental analysis as the gold standard, we show through the example of four globular proteins and nine IDPs that the ninhydrin assay and the commercial QubitTM Protein Assay provide reliable data on IDP quantity. However, as IDPs can show extreme variation in amino acid composition and physical features not necessarily covered by our examples, even these techniques should only be used for IDPs following standardization. The far-reaching implications of these simple observations are demonstrated through two examples: (i) circular dichroism spectrum deconvolution, and (ii) receptor-ligand affinity determination. These actual comparative examples illustrate the potential errors that can be incorporated into the biophysical parameters of IDPs, due to systematic misestimation of their concentration. This leads to inaccurate description of IDP functions.

KW - Circular dichroism

KW - Coomassie brilliant blue

KW - Elemental analysis

KW - Error propagation

KW - Nanoorange

KW - Ninhydrin

KW - Protein concentration

KW - UV absorbance

UR - http://www.scopus.com/inward/record.url?scp=85054204626&partnerID=8YFLogxK

U2 - 10.3389/fmolb.2018.00083

DO - 10.3389/fmolb.2018.00083

M3 - Article

VL - 5

SP - 83

EP - 83

JO - Frontiers in Molecular Biosciences

JF - Frontiers in Molecular Biosciences

SN - 2296-889X

IS - SEP

M1 - 83

ER -

ID: 39823154