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Quantification of a Peptide Standard Using the Intrinsic Fluorescence of Tyrosine

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Quantification of a Peptide Standard Using the Intrinsic Fluorescence of Tyrosine. / Preston, George W.; Phillips, David H.

In: ANALYTICAL AND BIOANALYTICAL CHEMISTRY, Vol. 408, 03.2016, p. 2187–2193.

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Harvard

Preston, GW & Phillips, DH 2016, 'Quantification of a Peptide Standard Using the Intrinsic Fluorescence of Tyrosine', ANALYTICAL AND BIOANALYTICAL CHEMISTRY, vol. 408, pp. 2187–2193. https://doi.org/10.1007/s00216-016-9334-1

APA

Preston, G. W., & Phillips, D. H. (2016). Quantification of a Peptide Standard Using the Intrinsic Fluorescence of Tyrosine. ANALYTICAL AND BIOANALYTICAL CHEMISTRY, 408, 2187–2193. https://doi.org/10.1007/s00216-016-9334-1

Vancouver

Preston GW, Phillips DH. Quantification of a Peptide Standard Using the Intrinsic Fluorescence of Tyrosine. ANALYTICAL AND BIOANALYTICAL CHEMISTRY. 2016 Mar;408:2187–2193. https://doi.org/10.1007/s00216-016-9334-1

Author

Preston, George W. ; Phillips, David H. / Quantification of a Peptide Standard Using the Intrinsic Fluorescence of Tyrosine. In: ANALYTICAL AND BIOANALYTICAL CHEMISTRY. 2016 ; Vol. 408. pp. 2187–2193.

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@article{4edebc659c3c438daf3cc1419b61c0a0,
title = "Quantification of a Peptide Standard Using the Intrinsic Fluorescence of Tyrosine",
abstract = "Absolute quantification of peptides is typically achieved using amino acid analysis, elemental analysis or derivatisation chemistry. Impurities, if present, may be accounted for using analytical high-performance liquid chromatography (HPLC) with detection of the peptide bond ultraviolet (UV) absorbance. To do this, peak areas from a UV chromatogram are used to estimate percentage purity on a mass basis, and this purity value is used as a correction. However, because the approach assumes that UV absorbance is uniformly proportional to mass, the result may be only semi-quantitative. Here, an alternative approach involving HPLC with detection of intrinsic tyrosine fluorescence is described. The fluorescence properties of a 21-residue synthetic peptide corresponding to an S-carbamidomethylated tryptic fragment of human serum albumin were characterised, and a method involving quantification relative to a non-peptidic calibrant, N-acetyl-l-tyrosine ethyl ester, was established. The method was used to quantify the thiol form of the peptide, and the results were compared with a parallel analysis involving derivatisation of the same material with Ellman’s reagent. When differences in fluorescence response (analyte versus calibrant) were accounted for, the measurements obtained via the two methods were in good agreement. Contributions from peptidic impurities were also considered, and their influence on the validity of the conclusions was evaluated. Despite some ambiguities introduced by the impurities, and the identification of some other potential sources of error, the results demonstrate that use of Tyr fluorescence is a promising solution to the challenging problem of absolute peptide quantification.",
author = "Preston, {George W.} and Phillips, {David H.}",
year = "2016",
month = "3",
doi = "10.1007/s00216-016-9334-1",
language = "English",
volume = "408",
pages = "2187–2193",
journal = "ANALYTICAL AND BIOANALYTICAL CHEMISTRY",
issn = "1618-2642",
publisher = "Springer Verlag",

}

RIS (suitable for import to EndNote) Download

TY - JOUR

T1 - Quantification of a Peptide Standard Using the Intrinsic Fluorescence of Tyrosine

AU - Preston, George W.

AU - Phillips, David H.

PY - 2016/3

Y1 - 2016/3

N2 - Absolute quantification of peptides is typically achieved using amino acid analysis, elemental analysis or derivatisation chemistry. Impurities, if present, may be accounted for using analytical high-performance liquid chromatography (HPLC) with detection of the peptide bond ultraviolet (UV) absorbance. To do this, peak areas from a UV chromatogram are used to estimate percentage purity on a mass basis, and this purity value is used as a correction. However, because the approach assumes that UV absorbance is uniformly proportional to mass, the result may be only semi-quantitative. Here, an alternative approach involving HPLC with detection of intrinsic tyrosine fluorescence is described. The fluorescence properties of a 21-residue synthetic peptide corresponding to an S-carbamidomethylated tryptic fragment of human serum albumin were characterised, and a method involving quantification relative to a non-peptidic calibrant, N-acetyl-l-tyrosine ethyl ester, was established. The method was used to quantify the thiol form of the peptide, and the results were compared with a parallel analysis involving derivatisation of the same material with Ellman’s reagent. When differences in fluorescence response (analyte versus calibrant) were accounted for, the measurements obtained via the two methods were in good agreement. Contributions from peptidic impurities were also considered, and their influence on the validity of the conclusions was evaluated. Despite some ambiguities introduced by the impurities, and the identification of some other potential sources of error, the results demonstrate that use of Tyr fluorescence is a promising solution to the challenging problem of absolute peptide quantification.

AB - Absolute quantification of peptides is typically achieved using amino acid analysis, elemental analysis or derivatisation chemistry. Impurities, if present, may be accounted for using analytical high-performance liquid chromatography (HPLC) with detection of the peptide bond ultraviolet (UV) absorbance. To do this, peak areas from a UV chromatogram are used to estimate percentage purity on a mass basis, and this purity value is used as a correction. However, because the approach assumes that UV absorbance is uniformly proportional to mass, the result may be only semi-quantitative. Here, an alternative approach involving HPLC with detection of intrinsic tyrosine fluorescence is described. The fluorescence properties of a 21-residue synthetic peptide corresponding to an S-carbamidomethylated tryptic fragment of human serum albumin were characterised, and a method involving quantification relative to a non-peptidic calibrant, N-acetyl-l-tyrosine ethyl ester, was established. The method was used to quantify the thiol form of the peptide, and the results were compared with a parallel analysis involving derivatisation of the same material with Ellman’s reagent. When differences in fluorescence response (analyte versus calibrant) were accounted for, the measurements obtained via the two methods were in good agreement. Contributions from peptidic impurities were also considered, and their influence on the validity of the conclusions was evaluated. Despite some ambiguities introduced by the impurities, and the identification of some other potential sources of error, the results demonstrate that use of Tyr fluorescence is a promising solution to the challenging problem of absolute peptide quantification.

U2 - 10.1007/s00216-016-9334-1

DO - 10.1007/s00216-016-9334-1

M3 - Article

VL - 408

SP - 2187

EP - 2193

JO - ANALYTICAL AND BIOANALYTICAL CHEMISTRY

JF - ANALYTICAL AND BIOANALYTICAL CHEMISTRY

SN - 1618-2642

ER -

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