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A flow-through nanoporous alumina trypsin bioreactor for mass spectrometry peptide fingerprinting
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Inorganic Chemistry.ORCID iD: 0000-0002-7018-9617
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2018 (English)In: Journal of Proteomics, ISSN 1874-3919, E-ISSN 1876-7737, Vol. 172, p. 165-172Article in journal (Refereed) Published
Abstract [en]

Mass spectrometry-based proteomics benefits from efficient digestion of protein samples. In this study, trypsinwas immobilized on nanoporous anodized alumina membranes to create an enzyme reactor suitable for peptidemassfingerprinting. The membranes were derivatized with 3-aminopropyltriethoxysilane and the amino groupswere activated with carbonyldiimidazole to allow coupling of porcine trypsin viaε-amino groups. The functionwas assessed using the artificial substrate Nα-Benzoyl-L-arginine 4-nitroanilide hydrochloride, bovine ribonu-clease A and a human plasma sample. A 10-membraneflow-through reactor was used for fragmentation and MSanalysis after a single pass of substrate both by collection of product and subsequent off-line analysis, and bycoupling on-line to the instrument. The peptide pattern allowed correct identification of the single target proteinin both cases, and of > 70 plasma proteins in single pass mode followed by LC-MS analysis. The reactor retained76% of the initial activity after 14 days of storage and repeated use at room temperature.

Significance:This manuscript describes the design of a stable enzyme reactor that allows efficient and fast di-gestion with negligible leakage of enzyme and enzyme fragments. The high stability facilitates the use in anonline-setup with MS detection since it allows the processing of multiple samples within an extended period of time without replacement.

Place, publisher, year, edition, pages
2018. Vol. 172, p. 165-172
Keyword [en]
nanoporous aluminum oxide, immobilization, trypsin, peptide mass fingerprinting, enzyme stability
National Category
Biochemistry and Molecular Biology Analytical Chemistry
Identifiers
URN: urn:nbn:se:uu:diva-210118DOI: 10.1016/j.jprot.2017.09.008ISI: 000423655300016PubMedID: 28942014OAI: oai:DiVA.org:uu-210118DiVA, id: diva2:661109
Funder
Swedish Research Council, 2005-3308Magnus Bergvall Foundation, 2015-01200Åke Wiberg Foundation, M14-0127Carl Tryggers foundation , CST 15:57Magnus Bergvall Foundation, 201601675
Available from: 2013-10-31 Created: 2013-10-31 Last updated: 2018-03-29Bibliographically approved
In thesis
1. Nanoporous Aluminum Oxide – A Promising Support for Modular Enzyme Reactors
Open this publication in new window or tab >>Nanoporous Aluminum Oxide – A Promising Support for Modular Enzyme Reactors
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Nanoporous alumina is a rather newly characterized material that so far has found limited use in the construction of bioreactors. The material has many advantages compared to conventional immobilization matrices. I have investigated its use in flow-through bioreactors. The rigidity and porous structure of the material makes it an excellent choice for multienzyme reactor construction. The total activity in a reactor is easily controlled by the number of membranes since the porosity makes the material less prone to increase flow system pressure. This bioreactor is suitable for characterization of new enzymes since the amount of immobilized enzyme is standardized and the enzyme may be reused many times.

We designed a simple stepwise technique for covalent immobilization on this matrix in a monolayer to minimize mass transfer effects in the reactor function. The kinetic parameters for ten different substrates were investigated for immobilized alcohol oxidase and, as a second step, a two-step reactor was also designed by addition of horseradish peroxidase. This bienzymatic reactor was, in turn, employed for measuring injected alcohol concentrations. The use of the matrix for substrate specificity screening was proven for two new epsilon-class glutathione transferases from Drosophila melanogaster. Immobilized trypsin showed a substantially prolonged lifetime and its potential use as an on-line digestion unit for peptide mass fingerprinting was also demonstrated. Finally, I investigated the immobilization of the model enzyme lactate dehydrogenase by adsorption mediated by metal ion chelation similar to IMAC. Regeneration was here possible multiple times without loss of capacity. In conclusion, immobilization of enzymes on nanoporous alumina is a convenient way to characterize, stabilize and reuse enzymes.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2013. p. 51
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1098
Keyword
nanoporous aluminum oxide, immobilized enzymes, bioreactor
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-210120 (URN)978-91-554-8807-9 (ISBN)
Public defence
2013-12-13, Sal: B42, BMC, Husargatan 3, Uppsala, 14:00 (English)
Opponent
Supervisors
Available from: 2013-11-21 Created: 2013-10-31 Last updated: 2014-01-23

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Kjellander, MarcusBillinger, ErikaBoman, MatsBergström Lind, SaraJohansson, Gunnar

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