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N-glycosylation profiling of selected intact proteins by high-resolution mass spectrometry (MS) and glycan analysis using ion mobility-MS/MS
Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för miljövetenskap och analytisk kemi.ORCID-id: 0000-0002-3167-3772
Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för miljövetenskap och analytisk kemi.
Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för miljövetenskap och analytisk kemi.
Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för miljövetenskap och analytisk kemi.
Visa övriga samt affilieringar
(Engelska)Manuskript (preprint) (Övrigt vetenskapligt)
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.

Nyckelord [en]
N-Glycosylation, Intact Glycoproteins, Ion Mobility Spectrometry, Mass Spectrometry, Glycomics, Quantitative Analysis
Nationell ämneskategori
Analytisk kemi
Forskningsämne
analytisk kemi
Identifikatorer
URN: urn:nbn:se:su:diva-172111OAI: oai:DiVA.org:su-172111DiVA, id: diva2:1344610
Tillgänglig från: 2019-08-21 Skapad: 2019-08-21 Senast uppdaterad: 2019-08-26Bibliografiskt granskad
Ingår i avhandling
1. Development and application of alternative methods for profiling proteins N-glycosylation
Öppna denna publikation i ny flik eller fönster >>Development and application of alternative methods for profiling proteins N-glycosylation
2019 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
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.

Ort, förlag, år, upplaga, sidor
Stockholm: Department of Environmental Science and Analytical Chemistry, Stockholm University, 2019. s. 98
Nyckelord
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
Nationell ämneskategori
Analytisk kemi
Forskningsämne
analytisk kemi
Identifikatorer
urn:nbn:se:su:diva-171844 (URN)978-91-7797-783-4 (ISBN)978-91-7797-784-1 (ISBN)
Disputation
2019-10-04, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16B, Stockholm, 10:00 (Engelska)
Opponent
Handledare
Anmärkning

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 5: Manuscript.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

Tillgänglig från: 2019-09-11 Skapad: 2019-08-21 Senast uppdaterad: 2019-09-03Bibliografiskt granskad

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Quaranta, AlessandroKarlsson, IsabellaNdreu, LorenaIlag, Leopold
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Institutionen för miljövetenskap och analytisk kemi
Analytisk kemi

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