Infrared spectroscopy used to evaluate glycosylation of proteins

Khajehpour, Mazdak; Dashnau, Jennifer L.; Vanderkooi, Jane M.

doi:10.1016/j.ab.2005.10.009

Cited by 114 publications

(97 citation statements)

References 34 publications

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“…Specifically, increasing the pH value from 3 to 7 leads to 266 a decrease in the intensity of the carbonyl band (ν(C=O)) near 1720 cm -1 , which is attributed to 267 the COOH groups of the mucin's sialic acid. 46 Together with this, an increase in the absorption 268 near 1556 and 1400 cm -1 corresponding, respectively, to the antisymmetric ν a (COO -) and 269 symmetric ν s (COO -) stretching vibrations of carboxylate groups is observed with increasing 270 pH. 271…”

Section: Introductionmentioning

confidence: 97%

Spectroscopic Ellipsometry of Mucin Layers on an Amphiphilic Diblock Copolymer Surface

Nikonenko

Bushnak²,

Keddie³

2009

Appl Spectrosc

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10 11Both visible and infrared (IR) spectroscopic ellipsometry have been employed to study the 12 structure of thin layers of bovine submaxillary mucin (BSM) adsorbed on poly(acrylic acid-13 block-methyl methacrylate) (PAA-b-PMMA) copolymer and poly(methyl methacrylate) 14 surfaces at three pH values (3, 7 and 10). The adsorbed mucin layer on the copolymer surface 15 had the greatest thickness (17 nm) when adsorbed from a mucin solution at a pH of 3. For the 16 first time, IR ellipsometry was used to identify adhesive interactions and conformational 17 changes in mucin/polymer double layers. After applying the regularized method of 18 deconvolution in the analysis, the formation of hydrogen bonds between the carboxyl groups of 19 the BSM and PAA-b-PMMA copolymer in double layers has been found. The IR ellipsometry 20 data, in agreement with the visible ellipsometry analysis, indicate the pH dependence of 21 adhesion of mucin to the copolymer surface. There is an increase in the amount of hydrogen-22 bonded carboxyl groups in mucin deposited at a pH of 3. There is no evidence that the mucin's 23 amide groups participate in this bonding. At the lower pH, the IR ellipsometry spectra after 24 deconvolution reveal an increase in the proportion of β-sheets in the BSM upon adsorption on 25 the copolymer surface, indicating a more unfolded, aggregated structure. The IR ellipsometry 26 data also indicated some changes in the conformational states of the side groups in the 27 copolymer induced by entanglements and bonding interactions with the mucin 28 macromolecules. Deconvolution provides an unprecedented level of information from the IR 29 ellipsometry spectra and yields important insights. 30 31

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Section: Introductionmentioning

confidence: 97%

Spectroscopic Ellipsometry of Mucin Layers on an Amphiphilic Diblock Copolymer Surface

Nikonenko

Bushnak²,

Keddie³

2009

Appl Spectrosc

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“…It is possible to note that the heat treatment does not lead to changes in the protein secondary structure and no obvious changes in glycosylation to ovomucoid (Figure 1), indicating the heat stability of the protein. Moreover, the shape of peaks in the region 900-1200 cm-1 resembles those of N-acetylglucosamine (GlcNAc) as indicated by Khajehpour et al [2].…”

Section: Ftir Analysismentioning

confidence: 88%

“…Glycosylation is a common posttranscriptional modification of proteins. Covalently bound sugar residues stabilize proteins, act as recognition sites, and provide loci for immuno response [2]. Carbohydrate modifications of proteins fall into three general categories: N-linked modification of asparagine (Asn), O-linked modification of serine or threonine and glycosylphosphatidyl inositol derivatization of the C-terminus carboxyl group [3].…”

Section: Introductionmentioning

confidence: 99%

Optimization of In Vitro N-Deglycosylation of Ovomucoid Protein

Giosafto¹

2016

MOJFPT

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Glycosylation is a common post-translational modification that confers important properties to proteins. Glycans are implicated in a wide range of intracellular, cell-cell and cell-matrix recognition events and, therefore, are of great biological interest. In this paper we have taken an enzymatic approach by using peptide N-glycosidase F to release N-linked glycans from ovomucoid. Our study revealed that the protein in its native conformation was susceptible to this enzyme. Nevertheless, denaturation by means of both heat (at 100 °C) and reducing agent treatment, before the enzymatic exposure, greatly enhanced the extent of deglycosylation.SDS-PAGE and Western blot analyses revealed that the intact protein migrates as a diffuse band that was attributed to glycosylation heterogeneity. The complete deglycosylation of the protein resulted in a shift of the protein migration and in the formation of a sharp protein band and it was confirmed by gel filtration, FTIR and MALDI-TOF analyses. Importantly, LC-MS/MS revealed for the first time the presence of eight potential deglycosylation sites on the protein.

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“…The absorption features in the carbohydrate region in protein spectra have been used to probe protein glycosylation and to identify types of sugars attached to proteins [44,45]. Therefore, our findings suggest that Type-IV pili of M. bovis, as occurs with many Type-IV pili of other Gram-negative bacteria, could be post-translationally modified by glycosylation.…”

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confidence: 84%

“…In addition, N-acetyl glycosidic functional groups have their typical absorption bands in the W 2 region, in particular bands at 1410 and 1380 cm À1 [45,46]. Therefore, based on these band assignments and on the fact that pili belonging to Type-IV pili family share structural and chemical properties [47] we consider that the features involved in the W 2 window as Type-IV pili spectroscopic markers, and suggest that they could be attributed mainly to N-acetil glycosylations of M. bovis pilin protein.…”

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confidence: 99%

Type‐IV pili spectroscopic markers: Applications in the quantification of piliation levels inMoraxella boviscells by a FT‐IR ANN‐based model

Bosch

Prieto

Serra

et al. 2010

Journal of Biophotonics

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Type‐IV pili are cell surface organelles found in a wide variety of Gram‐negative bacteria. They have traditionally been detected by electron microscopy and ELISA techniques. However, these methodologies are not appropriate for the rapid discrimination and quantification of piliated and nonpiliated cells in industrial or field conditions. Here, the analysis of FT‐IR spectra of piliated, nonpiliated and sheared Moraxella bovis cells, together with purified pili suspensions spectra, allowed the identification of 3 IR regions associated to spectroscopic markers of Type‐IV pili: 1750–1600, 1450–1350 and 1280–950 cm–1. Such IR‐specific markers were found for piliated cells grown in different culture systems (liquid or solid media), independently of the strain or pili serotype. They were also sensitive to pili expression levels. Therefore, on the bases of these specific spectral features, an FT‐IR ANN‐based model was developed to classify piliation levels in 5 distinct groups. An overall classification rate of almost 90% demonstrates the strong potential of the ANN system developed to monitor M. bovis cultures in vaccine production. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

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Infrared spectroscopy used to evaluate glycosylation of proteins

Cited by 114 publications

References 34 publications

Spectroscopic Ellipsometry of Mucin Layers on an Amphiphilic Diblock Copolymer Surface

Spectroscopic Ellipsometry of Mucin Layers on an Amphiphilic Diblock Copolymer Surface

Optimization of In Vitro N-Deglycosylation of Ovomucoid Protein

Type‐IV pili spectroscopic markers: Applications in the quantification of piliation levels inMoraxella boviscells by a FT‐IR ANN‐based model

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