Research output: Contribution to Journal/Magazine › Journal article › peer-review
Research output: Contribution to Journal/Magazine › Journal article › peer-review
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TY - JOUR
T1 - Monitoring the glycosylation status of proteins using Raman spectroscopy
AU - Brewster, Victoria L.
AU - Ashton, Lorna
AU - Goodacre, Royston
PY - 2011/8/1
Y1 - 2011/8/1
N2 - Protein-based biopharmaceuticals are becoming increasingly widely used as therapeutic agents, and the characterization of these biopharmaceuticals poses a significant analytical challenge. In particular, monitoring posttranslational modifications (PTMs), such as glycosylation, is an important aspect of this characterization because these glycans can strongly affect the stability, immunogenicity, and pharmacolcinetics of these biotherapeutic drugs. Raman spectroscopy is a powerful tool, with many emerging applications in the bioprocessing arena. Although the technique has a relatively rich history in protein science, only recently has Raman spectroscopy been investigated for assessing posttranslational modifications, including phosphorylation, acetylation, trimethylation, and ubiquitination. In this investigation, we develop for the first time Raman spectroscopy combined with multivariate data analyses, including principal components analysis and partial least-squares regression, for the determination of the glycosylation status of proteins and quantifying the relative concentrations of the native ribonuclease (RNase) A protein and RNase B glycoprotein within mixtures.
AB - Protein-based biopharmaceuticals are becoming increasingly widely used as therapeutic agents, and the characterization of these biopharmaceuticals poses a significant analytical challenge. In particular, monitoring posttranslational modifications (PTMs), such as glycosylation, is an important aspect of this characterization because these glycans can strongly affect the stability, immunogenicity, and pharmacolcinetics of these biotherapeutic drugs. Raman spectroscopy is a powerful tool, with many emerging applications in the bioprocessing arena. Although the technique has a relatively rich history in protein science, only recently has Raman spectroscopy been investigated for assessing posttranslational modifications, including phosphorylation, acetylation, trimethylation, and ubiquitination. In this investigation, we develop for the first time Raman spectroscopy combined with multivariate data analyses, including principal components analysis and partial least-squares regression, for the determination of the glycosylation status of proteins and quantifying the relative concentrations of the native ribonuclease (RNase) A protein and RNase B glycoprotein within mixtures.
KW - OPTICAL-ACTIVITY
KW - TRIFLUOROMETHANESULFONIC ACID
KW - 2-DIMENSIONAL RAMAN
KW - LINKED GLYCANS
KW - DEGLYCOSYLATION
KW - GLYCOPROTEINS
KW - QUANTIFICATION
KW - IDENTIFICATION
KW - CONFORMATION
KW - POLYPEPTIDE
U2 - 10.1021/ac2012009
DO - 10.1021/ac2012009
M3 - Journal article
VL - 83
SP - 6074
EP - 6081
JO - Analytical Chemistry
JF - Analytical Chemistry
SN - 0003-2700
IS - 15
ER -