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Next-Generation Dried Blood Spot Samplers for Protein Analysis: Describing Trypsin-Modified Smart Sampling Paper

Separations, 2021-05, Vol.8 (5), p.66 [Peer Reviewed Journal]

2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License. ;info:eu-repo/semantics/openAccess ;ISSN: 2297-8739 ;EISSN: 2297-8739 ;DOI: 10.3390/separations8050066

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  • Title:
    Next-Generation Dried Blood Spot Samplers for Protein Analysis: Describing Trypsin-Modified Smart Sampling Paper
  • Author: Pizzi, Eleonora ; Halvorsen, Trine Grønhaug ; Koehler, Christian J. ; Reubsaet, Léon
  • Subjects: Binding ; Biological properties ; bottom-up analysis ; Cellulose ; Cytochrome ; Cytochromes ; dried blood spot ; Enzyme activity ; immobilized trypsin ; Laboratories ; Medical screening ; Polyhydroxyethyl methacrylate ; Polymerization ; Potassium ; protein determination ; Proteins ; Samplers ; Sampling ; smart sampler ; Spectrometry ; Substrates ; Trypsin
  • Is Part Of: Separations, 2021-05, Vol.8 (5), p.66
  • Description: This paper describes smart sampling paper to be used for bottom-up protein analysis. Four different manners to immobilize trypsin on cellulose were evaluated. Untreated paper, potassium-periodate-functionalized paper (with and without post-immobilization reduction) and 2-hydroxyethyl methacrylate (HEMA)/2-vinyl-4,4-dimethylazlactone (VDM)-functionalized paper were all used to immobilize trypsin. For the evaluation, Coomassie Brilliant Blue staining of proteins on paper and the BAEE trypsin activity assay needed to be modified. These methods allowed, together with data from mass spectrometric analysis of cytochrome C digestions, us to acquire fundamental insight into protein binding, and trypsin action and activity on paper. All functionalized discs bind more protein than the untreated discs. Protein binding to functionalized discs is based on both adsorption and covalent binding. Trypsin immobilized on potassium-periodate-functionalized discs exhibits the highest trypsin activity when using cytochrome C as substrate. It is proven that it is trypsin attached to paper (and not desorbed trypsin) which is responsible for the enzyme activity. The use of discs on complex biological samples shows that all functionalized discs are able to digest diluted serum; for the best-performing disc, HEMA-VDM functionalized, up to 200 high-confidence proteins are qualified, showing its potential.
  • Publisher: Basel: MDPI AG
  • Language: English;Norwegian
  • Identifier: ISSN: 2297-8739
    EISSN: 2297-8739
    DOI: 10.3390/separations8050066
  • Source: AUTh Library subscriptions: ProQuest Central
    NORA Norwegian Open Research Archives
    ROAD: Directory of Open Access Scholarly Resources
    DOAJ Directory of Open Access Journals
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