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Separation and characterization of aggregated species of amyloid-beta peptides
KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry.
KTH, School of Chemical Science and Engineering (CHE), Chemistry, Analytical Chemistry.
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2010 (English)In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 397, no 6, p. 2357-2366Article in journal (Refereed) Published
Abstract [en]

We have investigated the use of isoelectric focusing and immunodetection for the separation of low molecular weight species of amyloid-beta (A beta) peptides from their aggregates. From solutions of A beta(1-40) or A beta(1-42) monomeric peptides, low molecular weight material appeared at a pI value of ca. 5, while the presence of aggregates was detected as bands, observed at a pI of 6-6.5. The formation of A beta aggregates (protofibrils) was verified by a sandwich ELISA, employing the protofibril conformation-selective antibody mAb158. In order to study the aggregation behavior when using a mixture of the monomers, we utilized the IEF separation combined with Western blot using two polyclonal antisera, selective for A beta(1-40) and A beta(1-42), respectively. We conclude that both monomers were incorporated in the aggregates. In a further study of the mixed aggregates, we used the protofibril conformation-selective antibody mAb158 for immunoprecipitation, followed by nanoelectrospray mass spectrometry (IP-MS). This showed that the A beta(1-42) peptide is incorporated in the aggregate in a significantly larger proportion than its relative presence in the original monomer composition. IP-MS with mAb158 was also performed, and compared to IP-MS with the A beta-selective antibody mAb1C3, where a monomeric A beta(1-16) peptide was added to the protofibril preparation. A beta(1-16) is known for its poor aggregation propensity, and acted therefore as a selectivity marker. The results obtained confirmed the protofibril conformation selectivity of mAb158.

Place, publisher, year, edition, pages
2010. Vol. 397, no 6, p. 2357-2366
Keywords [en]
Alzheimer's disease, Protofibrils, Amyloid-beta (A beta), Isoelectric focusing (IEF)., Mass spectrometry (MS), Bioanalytical methods, Capillary electrophoresis / Electrophoresis
National Category
Analytical Chemistry
Identifiers
URN: urn:nbn:se:kth:diva-26073DOI: 10.1007/s00216-010-3839-9ISI: 000279453000038Scopus ID: 2-s2.0-77955981824OAI: oai:DiVA.org:kth-26073DiVA, id: diva2:369695
Conference
6th International Conference on Instrumental Methods of Analysis Athens, GREECE, OCT 04-08, 2009
Note
QC 20110214Available from: 2010-11-11 Created: 2010-11-11 Last updated: 2017-12-12Bibliographically approved
In thesis
1. New methods for sensitive analysis with nanoelectrospray ionization mass spectrometry
Open this publication in new window or tab >>New methods for sensitive analysis with nanoelectrospray ionization mass spectrometry
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

In this thesis, new methods that address some current limitations in nanoelectrospray mass spectrometry (nESI-MS) analysis are presented. One of the major objectives is the potential gain in sensitivity that can be obtained when employing the proposed techniques.

In the first part of this thesis, a new emitter, based on the generation of electrospray from a spray orifice with variable size, is presented. Electrospray is generated from an open gap between the edges of two individually mounted, pointed tips. The fabrication and evaluation of two different types of such emitters is presented; an ESI emitter fabricated from polyethylene terephtalate (Paper I), and a high-precision silicon device (Paper II). Both emitters were surface-treated in a selective way for an improved wetting of the gap and to confine the sample solution into the gap.

In the second part of this thesis, different methods for improved sensitivity of nESI-MS analysis have been developed. In Paper III, a method for nESI-MS analysis from discrete sample volumes down to 1.5 nL is presented, using commercially available nESI needles. When analyzing attomole amounts of analyte in such a small volume of sample, an increased sensitivity was obtained, compared to when analyzing equal amounts in conventional nESI-MS analysis. To be able to analyze smaller sample volumes, needles with a narrower orifice and a higher flow resistance were needed. This triggered the development of a new method for fabrication of fused silica nESI needles (Paper IV). The fabrication is based on melting of a fused silica capillary by means of a rotating plasma, prior to pulling the capillary into a fine tip. Using the described technique, needles with sub-micrometer orifices could be fabricated. Such needles enabled the analysis of sample volumes down to 275 pL, and a further improvement of the sensitivity was obtained. In a final project (Paper V), nESI-MS was used to study the aggregation behavior of Aβ peptides, related to Alzheimer’s disease. An immunoprecipitation followed by nESI-MS was employed. This technique was also utilized to study the selectivity of the antibodies utilized.

Place, publisher, year, edition, pages
Stockholm: KTH, 2010. p. vii, 70
Series
Trita-CHE-Report, ISSN 1654-1081 ; 2010:41
Keywords
mass spectrometry, electrospray ionization, nanoelectrospray ionization, adjustable gap, emitter, miniaturization, improved sensitivity, nanoliter/picoliter sample volumes, Alzheimer’s disease, amyloid-beta (Aβ)
National Category
Analytical Chemistry
Identifiers
urn:nbn:se:kth:diva-25917 (URN)978-91-7415-751-2 (ISBN)
Public defence
2010-11-26, K1, Teknikringen 56, Stockholm, 10:00 (English)
Opponent
Supervisors
Note
QC 20101112Available from: 2010-11-12 Created: 2010-11-04 Last updated: 2010-11-12Bibliographically approved
2. Analytical Approaches to Neurodegenerative Disease Protein Aggregation
Open this publication in new window or tab >>Analytical Approaches to Neurodegenerative Disease Protein Aggregation
2011 (English)Licentiate thesis, comprehensive summary (Other academic)
Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2011. p. vi, 38
Series
Trita-CHE-Report, ISSN 1654-1081 ; 2011:42
Keywords
neurodegenerative diseases, protein misfolding, protein aggregation, gel electrophoresis, isoelectric focusing, immunodetection, ELISA, immunoprecipitation, mass spectrometry, nanoelectrospray, liquid chromatography, neurodegenerativa sjukdomar, felveckade proteiner, proteinaggregering, geleektrofores, isoelektrisk fokusering, immundetektion, ELISA, immunoprecipitering, masspektrometri, nanoelektrospray, vätskekromatografi
National Category
Analytical Chemistry
Identifiers
urn:nbn:se:kth:diva-34027 (URN)978-91-7501-023-6 (ISBN)
Presentation
2011-06-15, Hörsal D2, KTH, Lindstedtsvägen 5, Stockholm, 13:00 (English)
Opponent
Supervisors
Note
QC 20110615Available from: 2011-06-15 Created: 2011-05-23 Last updated: 2011-06-16Bibliographically approved

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