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Glycosylation profiling of selected proteins in cerebrospinal fluid from Alzheimer's disease and healthy subjects
Stockholm University, Faculty of Science, Department of Environmental Science and Analytical Chemistry.ORCID iD: 0000-0002-3167-3772
Stockholm University, Faculty of Science, Department of Environmental Science and Analytical Chemistry.
Stockholm University, Faculty of Science, Department of Environmental Science and Analytical Chemistry.
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Number of Authors: 62019 (English)In: Analytical Methods, ISSN 1759-9660, E-ISSN 1759-9679, Vol. 11, no 26, p. 3331-3340Article in journal (Refereed) Published
Abstract [en]

Alteration of glycosylation has been observed in several diseases, such as cancer and neurodegenerative disorders. The study of changes in glycosylation could lead to a better understanding of mechanisms underlying these diseases and to the identification of new biomarkers. In this work the N-linked glycosylation of five target proteins in cerebrospinal fluid (CSF) from Alzheimer's disease (AD) patients and healthy controls have been analyzed for the first time. The investigated proteins, transferrin (TFN), alpha-1-antitrypsin (AAT), C1-inhibitor, immunoglobulin G (IgG), and alpha-1-acid glycoprotein (AGP), were selected based on the availability of VHH antibody fragments and their potential involvement in neurodegenerative and inflammation diseases. AD patients showed alterations in the glycosylation of low abundance proteins, such as C1-inhibitor and alpha-1-acid glycoprotein. These alterations would not have been detected if the glycosylation profile of the total CSF had been analyzed, due to the masking effect of the dominant profiles of high abundance glycoproteins, such as IgG. Information obtained from single proteins was not sufficient to correctly classify the two sample groups; however, by using an advanced multivariate technique a total non-error rate of 72 +/- 3% was obtained. In fact, the corresponding model was able to correctly classify 71 +/- 4% of the healthy subjects and 74 +/- 7% of the AD patients. Even if the results were not conclusive for AD, this approach could be extremely useful for diseases in which glycosylation changes are reported to be more extensive, such as several types of cancer and autoimmune diseases.

Place, publisher, year, edition, pages
2019. Vol. 11, no 26, p. 3331-3340
National Category
Chemical Sciences
Research subject
Analytical Chemistry
Identifiers
URN: urn:nbn:se:su:diva-170770DOI: 10.1039/c9ay00381aISI: 000474140100007OAI: oai:DiVA.org:su-170770DiVA, id: diva2:1338712
Available from: 2019-07-24 Created: 2019-07-24 Last updated: 2019-08-26Bibliographically approved
In thesis
1. Development and application of alternative methods for profiling proteins N-glycosylation
Open this publication in new window or tab >>Development and application of alternative methods for profiling proteins N-glycosylation
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Glycosylation is a post-translational modification (PTM) that exerts profound structural and functional effects on the modified protein. Glycan synthesis and conjugation to proteins are regulated by a myriad of factors, both genetic and environmental, and are also influenced by external stressors. Glycosylation patterns are known to vary in correlation to a large number of diseases; therefore, it is possible to study such alterations to identify reliable biomarkers and help elucidate mechanisms underlying the disease. For these reasons, the development of analytical methods able to investigate the glycosylation of proteins in complex samples and to measure and characterize disease-related alterations is of great importance.

In this thesis, the development and application of rapid and small-scale methods for the analysis of the glycosylation pattern on specific proteins in biological fluids, with a high degree of automation and potential for parallel sample treatment, is presented.

Paper I illustrates a profiling method based on a microfluidic compact disc (CD) and its application to humans serum samples. The workflow integrated all the sample preparation steps, allowing a high degree of automation and sample treatment parallelization, significantly reducing the required processing time. In Paper II, a bead-based procedure for the immunoaffinity extraction of selected proteins from complex biological matrices was developed. This procedure improved and extended the applicability of the microfluidic CD method, increasing the flexibility and maintaining a good potential for automation. Paper III included a derivatization procedure in the bead-based methodology, to stabilize sialic acids for matrix-assisted lased desorption/ionization (MALDI) and to discriminate between connectivity isomers. Additionally, the method was applied to different biological fluids in order to highlight interpersonal variations of glycosylation. To increase the sample throughput, the method was scaled to a multi-wells format in Paper IV and subsequently applied to the investigation of alterations in the glycosylation pattern correlated to Alzheimer’s disease.

Papers V and VI focus on applications based on electrospray ionization (ESI). In Paper V, a source for paper spray ionization (PSI) was modified to create a new set-up to extend the applicability of this mass spectrometry (MS) technique to large biomolecules. It was possible to measure intact proteins, identifying many glycoforms together with other PTMs, as well as to characterize released glycans, performing structural analysis by tandem mass spectrometry (MS/MS). In Paper VI ESI-MS and the bead-based sample preparation method developed in Papers II, III, and IV were used for quantification of various glycoforms of intact proteins. Additionally, a travelling wave ion mobility spectrometry (TWIMS) MS/MS method was developed to structurally characterize the related N-glycans after enzymatic release.

The methods proposed in this thesis show valid approaches, which could be applied to investigate alterations of glycosylation at different levels, with potential implementation for biomarker investigation and development.

Place, publisher, year, edition, pages
Stockholm: Department of Environmental Science and Analytical Chemistry, Stockholm University, 2019. p. 98
Keywords
N-glycosylation, Glycomics, Glycosylation Biomarkers, Intact Glycoproteins, Glycoform Quantification, Mass Spectrometry, Ion Mobility Spectrometry, MALDI-MS, Paper Spray Ionization, Microfluidics, Magnetic Beads, Immunoaffinity Purification, Nanobodies
National Category
Analytical Chemistry
Research subject
Analytical Chemistry
Identifiers
urn:nbn:se:su:diva-171844 (URN)978-91-7797-783-4 (ISBN)978-91-7797-784-1 (ISBN)
Public defence
2019-10-04, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16B, Stockholm, 10:00 (English)
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Supervisors
Note

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 5: Manuscript. Paper 6: Manuscript.

Available from: 2019-09-11 Created: 2019-08-21 Last updated: 2019-09-03Bibliographically approved

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