A detection and quantification label-free tool to speed up downstream processing of model mucins

Sofia B. Carvalho, Ana Sofia Moreira, Joana Gomes, Manuel J.T. Carrondo, David J. Thornton, Paula M. Alves, Julia Costa, Cristina Peixoto

Research output: Contribution to journalArticle

3 Citations (Scopus)
8 Downloads (Pure)

Abstract

Mucins are high-molecular weight glycoproteins (0.25–20 MDa) containing one or more domains that are heavily O-glycosylated. Their implications as targets for cancer treatment have increased the interest in these glycoproteins, mainly in the fields of vaccines and antibodies. However, mucins present high heterogeneity, posing challenges that affect purification processes and quality control analysis. In that sense, it is necessary to develop and improve downstream processes and analytical methods to characterize these products. Here a tool based on biolayer interferometry analysis to improve mucin’s detection and quantification in a fast, simple and label free-way is presented. Taking advantage of lectin recognition of mucins’ carbohydrate structures, several lectins were evaluated and immobilized on streptavidin biosensors. Different assay conditions were optimized and the most suitable lectin, Aleuria aurantia lectin (AAL), was selected. Bovine Submaxillary Gland and human MUC5B mucins were used as proof of concept and were successfully detected and quantified at different stages of purification. High sensitivity levels were achieved with LOD and LOQ of 3.8 μg mL-1 and 11.7 μg mL-1 for BSM, and 0.2 μg mL-1 and 0.6 μg mL-1 for MUC5B. AAL binding specificity was also confirmed with fucose competition assays. Our method represents an advance on mucins detection and quantification since the existing methods present several disadvantages for process development. Hereafter, it can be applied to the optimization of new or already established downstream processes for mucins’ purification.

Original languageEnglish
Article numbere0190974
JournalPLoS ONE
Volume13
Issue number1
DOIs
Publication statusPublished - 1 Jan 2018

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mucins
Mucins
Labels
lectins
Processing
Aleuria
Lectins
Purification
Citrus
glycoproteins
Assays
Glycoproteins
carbohydrate structure
Interferometry
streptavidin
purification methods
Oncology
Fucose
fucose
Streptavidin

Cite this

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title = "A detection and quantification label-free tool to speed up downstream processing of model mucins",
abstract = "Mucins are high-molecular weight glycoproteins (0.25–20 MDa) containing one or more domains that are heavily O-glycosylated. Their implications as targets for cancer treatment have increased the interest in these glycoproteins, mainly in the fields of vaccines and antibodies. However, mucins present high heterogeneity, posing challenges that affect purification processes and quality control analysis. In that sense, it is necessary to develop and improve downstream processes and analytical methods to characterize these products. Here a tool based on biolayer interferometry analysis to improve mucin’s detection and quantification in a fast, simple and label free-way is presented. Taking advantage of lectin recognition of mucins’ carbohydrate structures, several lectins were evaluated and immobilized on streptavidin biosensors. Different assay conditions were optimized and the most suitable lectin, Aleuria aurantia lectin (AAL), was selected. Bovine Submaxillary Gland and human MUC5B mucins were used as proof of concept and were successfully detected and quantified at different stages of purification. High sensitivity levels were achieved with LOD and LOQ of 3.8 μg mL-1 and 11.7 μg mL-1 for BSM, and 0.2 μg mL-1 and 0.6 μg mL-1 for MUC5B. AAL binding specificity was also confirmed with fucose competition assays. Our method represents an advance on mucins detection and quantification since the existing methods present several disadvantages for process development. Hereafter, it can be applied to the optimization of new or already established downstream processes for mucins’ purification.",
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A detection and quantification label-free tool to speed up downstream processing of model mucins. / Carvalho, Sofia B.; Moreira, Ana Sofia; Gomes, Joana; Carrondo, Manuel J.T.; Thornton, David J.; Alves, Paula M.; Costa, Julia; Peixoto, Cristina.

In: PLoS ONE, Vol. 13, No. 1, e0190974, 01.01.2018.

Research output: Contribution to journalArticle

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T1 - A detection and quantification label-free tool to speed up downstream processing of model mucins

AU - Carvalho, Sofia B.

AU - Moreira, Ana Sofia

AU - Gomes, Joana

AU - Carrondo, Manuel J.T.

AU - Thornton, David J.

AU - Alves, Paula M.

AU - Costa, Julia

AU - Peixoto, Cristina

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Y1 - 2018/1/1

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