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Development and application of alternative methods for profiling proteins N-glycosylation
Stockholm University, Faculty of Science, Department of Environmental Science and Analytical Chemistry.ORCID iD: 0000-0002-3167-3772
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 [en]
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: urn:nbn:se:su:diva-171844ISBN: 978-91-7797-783-4 (print)ISBN: 978-91-7797-784-1 (electronic)OAI: oai:DiVA.org:su-171844DiVA, id: diva2:1344642
Public defence
2019-10-04, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16B, Stockholm, 10:00 (English)
Opponent
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
List of papers
1. N-Glycan profile analysis of transferrin using a microfluidic compact disc and MALDI-MS
Open this publication in new window or tab >>N-Glycan profile analysis of transferrin using a microfluidic compact disc and MALDI-MS
2016 (English)In: Analusis, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 408, no 17, p. 4765-4776Article in journal (Refereed) Published
Abstract [en]

It has been known for a long time that diseases can be associated with changes to the glycosylation of specific proteins. This has been shown for cancer, immunological disorders, and neurodegenerative diseases. The possibility of using the glycosylation patterns of proteins as biomarkers for disease would be a great asset for clinical research or diagnosis. There is at present a lack of rapid, automated, and cost-efficient analytical techniques for the determination of the glycosylation of specific serum proteins. We have developed a method for determining the glycosylation pattern of proteins based on the affinity capture of a specific serum protein, the enzymatic release of the N-linked glycans, and the analysis of the glycan pattern using MALDI-MS. All sample preparation is performed in a disposable centrifugal microfluidic disc. The sample preparation is miniaturized, requiring only 1 mu L of sample per determination, and automated with the possibility of processing 54 samples in parallel in 3.5 h. We have developed a method for the glycosylation pattern analysis of transferrin. The method has been tested on serum samples from chronic alcohol abusers and a control group. Also, a SIMCA model was created and evaluated to discriminate between the two groups.

Keywords
Glycan pattern, Glycomics, N-linked oligosaccharide, Transferrin, Microfluidic, MALDI-MS
National Category
Chemical Sciences
Research subject
Analytical Chemistry
Identifiers
urn:nbn:se:su:diva-132558 (URN)10.1007/s00216-016-9570-4 (DOI)000378725200025 ()27137515 (PubMedID)
Available from: 2016-08-18 Created: 2016-08-15 Last updated: 2019-08-26Bibliographically approved
2. Particle-based N-linked glycan analysis of selected proteins from biological samples using nonglycosylated binders
Open this publication in new window or tab >>Particle-based N-linked glycan analysis of selected proteins from biological samples using nonglycosylated binders
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2017 (English)In: Journal of Pharmaceutical and Biomedical Analysis, ISSN 0731-7085, E-ISSN 1873-264X, Vol. 132, p. 125-132Article in journal (Refereed) Published
Abstract [en]

Glycosylation is one of the most common and important post-translational modifications, influencing both the chemical and the biological properties of proteins. Studying the glycosylation of the entire protein population of a sample can be challenging because variations in the concentrations of certain proteins can enhance or obscure changes in glycosylation. Furthermore, alterations in the glycosylation pattern of individual proteins, exhibiting larger variability in disease states, have been suggested as biomarkers for different types of cancer, as well as inflammatory and neurodegenerative diseases. In this paper, we present a rapid and efficient method for glycosylation analysis of individual proteins focusing on changes in the degree of fucosylation or other alterations to the core structure of the glycans, such as the presence of bisecting N-acetylglucosamines and a modified degree of branching. Streptavidin-coated magnetic beads are used in combination with genetically engineered immunoaffinity binders, called VHH antibody fragments. A major advantage of the VHHs is that they are nonglycosylated; thus, enzymatic release of glycans from the targeted protein can be performed directly on the beads. After deglycosylation, the glycans are analyzed by MALDI-TOF-MS. The developed method was evaluated concerning its specificity, and thereafter implemented for studying the glycosylation pattern of two different proteins, alpha-1-antitrypsin and transferrin, in human serum and cerebrospinal fluid. To our knowledge, this is the first example of a protein array-type experiment that employs bead-based immunoaffinity purification in combination with mass spectrometry analysis for fast and efficient glycan analysis of individual proteins in biological fluid.

Keywords
Glycosylation pattern, Biomarkers, Magnetic beads, VHH antibody fragments, Cerebrospinal fluid, MALDI-MS
National Category
Chemical Sciences
Research subject
Analytical Chemistry
Identifiers
urn:nbn:se:su:diva-137554 (URN)10.1016/j.jpba.2016.09.029 (DOI)000389015700017 ()27718394 (PubMedID)
Available from: 2017-01-13 Created: 2017-01-09 Last updated: 2019-08-26Bibliographically approved
3. Glycosylation patterns of selected proteins in individual serum and cerebrospinal fluid samples
Open this publication in new window or tab >>Glycosylation patterns of selected proteins in individual serum and cerebrospinal fluid samples
2017 (English)In: Journal of Pharmaceutical and Biomedical Analysis, ISSN 0731-7085, E-ISSN 1873-264X, Vol. 145, p. 431-439Article in journal (Refereed) Published
Abstract [en]

A method we previously developed has been applied to the determination of the glycosylation pattern of specific proteins in biological samples. Six proteins (alpha-1-anthrypsin, transferrin, haptoglobin, Cl inhibitor, alpha-1 acid glycoprotein, and immunoglobulin G) were studied in serum samples from five individuals and cerebrospinal fluid (CSF) samples from three individuals, to investigate the expected normal distribution of glycosylation patterns and to assess whether this methodology can be used to discriminate between samples from different individuals. For serum samples, the differences were shown to be small, while much larger differences were found for the CSF samples, with a greater number of glycoforms present. This can be linked to the occurrence of differential glycosylation in proteins expressed in the brain compared with proteins expressed elsewhere in the body. The developed method could distinguish differences in the glycosylation pattern of specific proteins in the individual samples, which was not reflected in the glycan content of total CSF. This is the first time that the glycoforms of several of these proteins have been investigated in CSF.

Keywords
Glycosylation biomarkers, Acute phase proteins, Magnetic beads, VHH antibody fragments, Cerebrospinal fluid, MALDI-MS
National Category
Chemical Sciences
Research subject
Analytical Chemistry
Identifiers
urn:nbn:se:su:diva-147839 (URN)10.1016/j.jpba.2017.04.040 (DOI)000410872200053 ()28734272 (PubMedID)
Available from: 2017-11-02 Created: 2017-11-02 Last updated: 2019-08-26Bibliographically approved
4. Glycosylation profiling of selected proteins in cerebrospinal fluid from Alzheimer's disease and healthy subjects
Open this publication in new window or tab >>Glycosylation profiling of selected proteins in cerebrospinal fluid from Alzheimer's disease and healthy subjects
Show others...
2019 (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.

National Category
Chemical Sciences
Research subject
Analytical Chemistry
Identifiers
urn:nbn:se:su:diva-170770 (URN)10.1039/c9ay00381a (DOI)000474140100007 ()
Available from: 2019-07-24 Created: 2019-07-24 Last updated: 2019-08-26Bibliographically approved
5. Solvent-Assisted Paper Spray Ionization (SAPSI) for the Analysis of Biomolecules and Biofluids
Open this publication in new window or tab >>Solvent-Assisted Paper Spray Ionization (SAPSI) for the Analysis of Biomolecules and Biofluids
Show others...
(English)Manuscript (preprint) (Other academic)
National Category
Analytical Chemistry
Research subject
Analytical Chemistry
Identifiers
urn:nbn:se:su:diva-162305 (URN)
Available from: 2018-11-22 Created: 2018-11-22 Last updated: 2019-08-21Bibliographically approved
6. N-glycosylation profiling of selected intact proteins by high-resolution mass spectrometry (MS) and glycan analysis using ion mobility-MS/MS
Open this publication in new window or tab >>N-glycosylation profiling of selected intact proteins by high-resolution mass spectrometry (MS) and glycan analysis using ion mobility-MS/MS
Show others...
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Glycosylation influences structure and functionality of glycoproteins, and is regulated by genetic and environmental factors. Types and abundances of glycans on glycoproteins can vary due to diseases like cancer, inflammation, autoimmune and neurodegenerative disorders. Due to the crucial role of glycans in modulating protein function, glycosylation analysis is of prime importance in glycoprotein biopharmaceuticals quality control. We present a method for identification and quantification of glycoforms directly on intact proteins after immunoaffinity purification from biological fluids. The method was validated and applied to serum transferrin and to the biopharmaceutical trastuzumab. The accuracy ranged from 2.1 to 7.9%, and intra- and inter-day precision were 3.1 and 8.2%. Sensitivity and linearity were suitable for serum analysis and LOQs were calculated to be 3.1 (transferrin) and 4.4 (trastuzumab) µg/mL. Application to transferrin from five healthy serum samples yielded concentrations in agreement with blood reference levels (1.95-3.11 mg/mL). The structures of the identified glycans were assigned by ion mobility spectrometry coupled to tandem mass spectrometry. No chromatographic separation was required, and sample preparation was performed in a semi-automatic way, reducing the analysis time to 1-3 minutes. Hence, this method could be suitable for clinical laboratories and for quality control on large batches of biopharmaceuticals.

Keywords
N-Glycosylation, Intact Glycoproteins, Ion Mobility Spectrometry, Mass Spectrometry, Glycomics, Quantitative Analysis
National Category
Analytical Chemistry
Research subject
Analytical Chemistry
Identifiers
urn:nbn:se:su:diva-172111 (URN)
Available from: 2019-08-21 Created: 2019-08-21 Last updated: 2019-08-26Bibliographically approved

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1011121314151613 of 21
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