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Bioanalysis using capillary electrophoresis and mass spectrometry: Applied on proteins, protein nanofibrils and polyvinyl alcohol microbubbles
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH).
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The sequencing of the genome of various species, including the human species, have led to increased understanding about how a protein structure is generated, and how specific structures are related to the proteins’ functionality. In paper I and II of this thesis, the folding of proteins in vitro to form hierarchical nanostructures, which in vivo often have a pathological effect, have been studied. Protein isolates from soybean and potato, that are byproducts from oil and starch production, respectively, were used as a starting material for protein nanofibril (PNF) formation, and mass spectrometry was used to identify the building blocks that are included in the formed PNF. The five peptides identified in soybean PNF and the six peptides identified in potato PNF originated from the major seed storage proteins for the respective crop.

The use of ionic liquids has increased for improvement of the performance of different separation techniques due to their adjustable properties, and good solvating ability. In paper III, an ionic liquid and water mixture was used as background electrolyte in capillary electrophoresis for protein separation. The system showed high reproducibility at basic conditions, and could potentially be used for routine control analysis.

Many diseases and injuries require clinical diagnosis techniques e.g. ultrasound imaging, to be detected, and for the physician to be able to decide the correct therapy. To increase the resolution of such imaging techniques, contrast agents can be used. In paper IV-VI, a newly developed contrast agent consisting of air-filled microbubbles stabilized with a shell of polyvinyl alcohol (PVA-MBs) was studied. Development of a capillary electrophoretic method for analysis of the PVA-MBs with the intentions to be used for clinical diagnosis is performed, where different detectors such as a UV detector, a UV area imaging detector and an in-house constructed microscope are used to increase the sensitivity of detection for the PVA-MBs. The developed method could be used for quantification of the contrast agent, since individual PVA-MBs were visible using the imaging detectors. Findings regarding the mobility of the PVA-MBs in human blood plasma and in water implies that a protein corona was formed around the MBs.

Abstract [sv]

Sekvensering av genomet för många arter, inklusive den mänskliga, har lett till en ökad förståelse för hur proteiners strukturer är genererade, och hur specifika proteinstrukturer är relaterade till proteinernas funktioner. I artikel I och II i denna avhandling studeras proteinveckning till hierarkiska nanostrukturer in vitro, vilka vanligtvis har en patologisk effekt in vivo. Proteinisolat från sojabönor och potatis, d.v.s. biprodukter från respektive olje- och stärkelseproduktion, användes som startmaterial för tillverkningen av protein-nanofibriller (PNF). Masspektrometri användes för att identifiera byggstenarna i dessa nanofibriller. De fem identifierade peptiderna från sojabönans PNF och de sex peptiderna från potatis PNF har sitt ursprung i de större lagringsproteinerna från respektive gröda.

Användningen av jonvätskor har ökat i avseende att förbättra prestandan för olika separationstekniker på grund av jonvätskors justerbara egenskaper och effektivitet vid användning som lösningsmedel. I artikel III används en utspädd jonvätska som bakgrundselektrolyt i kapillärelektrofores för separation av proteiner. Systemet uppvisade så god reproducerbarhet vid alkaliska förhållanden att det potentiellt skulle kunna användas vid rutinanalyser.

Många sjukdomar och skador kräver kliniska diagnostiska tekniker såsom ultraljudsavbildning (sonografi), för att rätt terapi ska kunna tillämpas. För att förbättra upplösningen för ultraljudsavbildning kan kontrastmedel användas. I artikel IV-VI studerades nyligen utvecklade mikrobubblor av luft med ett hårt och stabiliserande skal av polyvinylalkohol (PVA-MBs) som kontrastmedel. En kapillärelektroforetisk metod har tagits fram för analys av dessa PVA-MBs som i avsikt att använda dessa i klinisk diagnostik. Tre olika detektorer har utvärderats för att uppnå känslig detektion av detta kontrastmedel: en UV-detektor, en ytavbildande UV-detektor och en egen-konstruerad mikroskopdetektor. Den utvecklade kapillärelektroforetiska metoden kan användas för kvantifiering av PVA-MBs då känsligheten för de avbildande detektorerna var så hög att individuella MB var synliga. Den elektroforetiska mobiliteten för PVA-MB varierade när PVA-MBs suspenderats i vatten med suspension i humant blodplasma, vilket indikerar att en proteinkorona kring PVA-MB bildades.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2019. , p. 43
Series
TRITA-CBH-FOU ; 2019:49
Keywords [en]
: amyloid, capillary electrophoresis, contrast agents, detection, human blood plasma, ionic liquids, mass spectrometry, microbubbles, polyvinyl alcohol microbubbles, protein nanofibrils, proteins
National Category
Analytical Chemistry
Research subject
Chemistry
Identifiers
URN: urn:nbn:se:kth:diva-257962ISBN: 978-91-7873-311-8 (print)OAI: oai:DiVA.org:kth-257962DiVA, id: diva2:1352339
Public defence
2019-10-24, sal F3, Lindstedtsvägen 26, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

QC 2019-09-20

Available from: 2019-09-20 Created: 2019-09-18 Last updated: 2019-09-20Bibliographically approved
List of papers
1. Structural basis for the formation of soy protein nanofibrils
Open this publication in new window or tab >>Structural basis for the formation of soy protein nanofibrils
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2019 (English)In: RSC Advances, ISSN 2046-2069, E-ISSN 2046-2069, Vol. 9, no 11, p. 6310-6319Article in journal (Refereed) Published
Abstract [en]

Amyloid-like protein nanofibrils (PNFs) can assemble from a range of different proteins including disease-associated proteins, functional amyloid proteins and several proteins for which the PNFs are neither related to disease nor function. We here examined the core building blocks of PNFs formed by soy proteins. Fibril formation at pH 2 and 90 degrees C is coupled to peptide hydrolysis which allows isolation of the PNF-forming peptides and identification of them by mass spectrometry. We found five peptides that constitute the main building blocks in soy PNFs, three of them from the protein b-conglycinin and two from the protein glycinin. The abilities of these peptides to form PNFs were addressed by amyloid prediction software and by PNF formation of the corresponding synthetic peptides. Analysis of the structural context in the native soy proteins revealed two structural motifs for the PNF-forming peptides: (i) so-called b-arches and (ii) helical segments involved in quaternary structure contacts. However, the results suggest that neither the native structural motifs nor the protein of origin defines the morphology of the PNFs formed from soy protein isolate.

Place, publisher, year, edition, pages
ROYAL SOC CHEMISTRY, 2019
National Category
Physical Chemistry
Identifiers
urn:nbn:se:kth:diva-246262 (URN)10.1039/c8ra10610j (DOI)000459507800054 ()2-s2.0-85062456643 (Scopus ID)
Note

QC 20190326

Available from: 2019-03-26 Created: 2019-03-26 Last updated: 2019-09-18Bibliographically approved
2. Protein nanofibrils from potato protein isolate - preparation and characterization
Open this publication in new window or tab >>Protein nanofibrils from potato protein isolate - preparation and characterization
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(English)Manuscript (preprint) (Other academic)
National Category
Materials Chemistry
Research subject
Chemistry
Identifiers
urn:nbn:se:kth:diva-257961 (URN)
Available from: 2019-09-14 Created: 2019-09-14 Last updated: 2019-09-18
3. Evaluation of ethylammonium nitrate as background electrolyte in capillary electrophoresis
Open this publication in new window or tab >>Evaluation of ethylammonium nitrate as background electrolyte in capillary electrophoresis
(English)Manuscript (preprint) (Other academic)
National Category
Analytical Chemistry
Research subject
Chemistry
Identifiers
urn:nbn:se:kth:diva-215628 (URN)
Note

QC 20171013

Available from: 2017-10-11 Created: 2017-10-11 Last updated: 2019-09-18
4. Analysis of polyvinyl alcohol microbubbles in human blood plasma using capillary electrophoresis
Open this publication in new window or tab >>Analysis of polyvinyl alcohol microbubbles in human blood plasma using capillary electrophoresis
2016 (English)In: Journal of Separation Science, ISSN 1615-9306, E-ISSN 1615-9314, Vol. 39, no 8, p. 1551-1558Article in journal (Refereed) Published
Abstract [en]

Recently, a new type of ultrasound contrast agent that consists of air-filled microbubbles stabilized with a shell of polyvinyl alcohol was developed. When superparamagnetic nanoparticles of iron oxide are incorporated in the polymer shell, a multimodal contrast agent can be obtained. The biodistribution and elimination pathways of the polyvinyl alcohol microbubbles are essential to investigate, which is limited with today's techniques. The aim of the present study was, therefore, to develop a method for qualitative and quantitative analysis of microbubbles in biological samples using capillary electrophoresis with ultraviolet detection. The analysis parameters were optimized to a wavelength at 260 nm and pH of the background electrolyte ranging between 11.9 and 12. Studies with high-intensity ultrasonication degraded microbubbles in water showed that degraded products and intact microbubbles could be distinguished, thus it was possible to quantify the intact microbubbles solely. Analysis of human blood plasma spiked with either plain microbubbles or microbubbles with nanoparticles demonstrated that it is possible to separate them from biological components like proteins in these kinds of samples.

Place, publisher, year, edition, pages
Wiley-VCH Verlagsgesellschaft, 2016
Keywords
Capillary electrophoresis, Contrast agents, Human blood plasma, Polyvinyl alcohol microbubbles
National Category
Analytical Chemistry
Identifiers
urn:nbn:se:kth:diva-187204 (URN)10.1002/jssc.201501342 (DOI)000374773000018 ()2-s2.0-84963701455 (Scopus ID)
Funder
EU, FP7, Seventh Framework Programme, 245572
Note

QC 20160518

Available from: 2016-05-18 Created: 2016-05-18 Last updated: 2019-09-18Bibliographically approved
5. Implementation of a UV area imaging detector for analysis of polyvinyl alcohol microbubbles in capillary electrophoresis
Open this publication in new window or tab >>Implementation of a UV area imaging detector for analysis of polyvinyl alcohol microbubbles in capillary electrophoresis
(English)Manuscript (preprint) (Other academic)
National Category
Analytical Chemistry
Research subject
Chemistry
Identifiers
urn:nbn:se:kth:diva-257959 (URN)
Note

QC 20191011

Available from: 2019-09-14 Created: 2019-09-14 Last updated: 2019-10-11Bibliographically approved
6. Imaging of polyvinyl alcohol microbubbles in a capillary using a homebuilt microscope
Open this publication in new window or tab >>Imaging of polyvinyl alcohol microbubbles in a capillary using a homebuilt microscope
(English)Manuscript (preprint) (Other (popular science, discussion, etc.))
National Category
Analytical Chemistry
Research subject
Chemistry
Identifiers
urn:nbn:se:kth:diva-257960 (URN)
Note

QC 20191011

Available from: 2019-09-14 Created: 2019-09-14 Last updated: 2019-10-11Bibliographically approved

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