Stolz, Alexander
[Author]
;
Scriba, Gerhard
[Degree supervisor];
Neusüß, Christian
[Degree supervisor];
Tholey, Andreas
[Degree supervisor]Friedrich-Schiller-Universität Jena
Capillary electrophoresis-mass spectrometry for top-down protein analysis
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Media type:
E-Book;
Thesis
Title:
Capillary electrophoresis-mass spectrometry for top-down protein analysis
:
development of one- and two-dimensional separation methods and their application in the clinical context
University thesis:
Dissertation, Friedrich-Schiller-Universität Jena, 2023
Footnote:
Kumulative Dissertation, enthält Zeitschriftenaufsätze
Tag der Verteidigung: 13.01.2023
Zusammenfassungen in deutscher und englischer Sprache
Description:
Due to the high sensitivity, selectivity, and the possibility for detailed molecular characterisation, mass spectrometry (MS) is the analytical method of choice for top-down protein biomarker discovery. Due to the low sample consumption, high separation efficiency, and the unique and complementary selectivity, capillary zone electrophoresis (CZE)-MS represents an interesting alternative to the traditionally used liquid chromatography (LC)-MS platforms. In this thesis, instrumental and methodological concepts were developed to increase the potential of CZE-MS for intact proteins. The first part of the thesis describes the development of a nanoflow sheath liquid interface for the efficient coupling of CZE and MS. The interface was developed with a focus on fast setup, easy handling and analytical robustness and has been used for most applications in this thesis. Furthermore, a CZE-MS screening platform for the identification and characterisation of known and unknown Hb variants from DBS samples was developed. The application of SMIL coatings enables efficient separation of closely-related proteoforms and even positional isomers of glycated Hb on the intact level. In the last part of the thesis, nanoLC and CZE-MS were coupled in a heart-cut approach using a polymer nanoliter valve. The platform was used for the glycosylation profiling of heterogeneous alpha-1 acid glycoprotein (AGP). This approach enables the assignment of notably more glycoforms from a lower concentrated AGP sample, compared to CZE-MS alone. In a proof-of-concept study, the platform was further extended to operate in the selective comprehensive mode. With a single injection, 19% more glycoforms were assigned compared to the heart-cut approach with 3 injections. The here presented instrumental and methodological concepts show the great potential of CZE-MS in the context of clinical protein analysis. Especially the combination of LC and CZE in multidimensional separation platforms shows great potential.