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  • 1.
    Karlsson, Isabella
    et al.
    Stockholm University, Faculty of Science, Department of Environmental Science and Analytical Chemistry.
    Ndreu, Lorena
    Stockholm University, Faculty of Science, Department of Environmental Science and Analytical Chemistry.
    Quaranta, Alessandro
    Stockholm University, Faculty of Science, Department of Environmental Science and Analytical Chemistry.
    Thorsén, Gunnar
    Stockholm University, Faculty of Science, Department of Environmental Science and Analytical Chemistry.
    Glycosylation patterns of selected proteins in individual serum and cerebrospinal fluid samples2017In: Journal of Pharmaceutical and Biomedical Analysis, ISSN 0731-7085, E-ISSN 1873-264X, Vol. 145, p. 431-439Article in journal (Refereed)
    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.

  • 2.
    Quaranta, Alessandro
    Stockholm University, Faculty of Science, Department of Environmental Science and Analytical Chemistry.
    Development and application of alternative methods for profiling proteins N-glycosylation2019Doctoral 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.

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  • 3.
    Quaranta, Alessandro
    et al.
    Stockholm University, Faculty of Science, Department of Environmental Science and Analytical Chemistry.
    Karlsson, Isabella
    Stockholm University, Faculty of Science, Department of Environmental Science and Analytical Chemistry.
    Ndreu, Lorena
    Stockholm University, Faculty of Science, Department of Environmental Science and Analytical Chemistry.
    Marini, Federico
    Ingelsson, Martin
    Thorsén, Gunnar
    Stockholm University, Faculty of Science, Department of Environmental Science and Analytical Chemistry.
    Glycosylation profiling of selected proteins in cerebrospinal fluid from Alzheimer's disease and healthy subjects2019In: Analytical Methods, ISSN 1759-9660, E-ISSN 1759-9679, Vol. 11, no 26, p. 3331-3340Article in journal (Refereed)
    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.

  • 4.
    Quaranta, Alessandro
    et al.
    Stockholm University, Faculty of Science, Department of Environmental Science and Analytical Chemistry.
    Spasova, Maya
    Stockholm University, Faculty of Science, Department of Environmental Science and Analytical Chemistry.
    Passarini, Elena
    Stockholm University, Faculty of Science, Department of Environmental Science and Analytical Chemistry.
    Karlsson, Isabella
    Stockholm University, Faculty of Science, Department of Environmental Science and Analytical Chemistry.
    Ndreu, Lorena
    Stockholm University, Faculty of Science, Department of Environmental Science and Analytical Chemistry.
    Thorsén, Gunnar
    Ilag, Leopold
    Stockholm University, Faculty of Science, Department of Environmental Science and Analytical Chemistry.
    N-glycosylation profiling of selected intact proteins by high-resolution mass spectrometry (MS) and glycan analysis using ion mobility-MS/MSManuscript (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.

  • 5.
    Quaranta, Alessandro
    et al.
    Stockholm University, Faculty of Science, Department of Environmental Science and Analytical Chemistry.
    Spasova, Maya
    Stockholm University, Faculty of Science, Department of Environmental Science and Analytical Chemistry.
    Passarini, Elena
    Stockholm University, Faculty of Science, Department of Environmental Science and Analytical Chemistry.
    Karlsson, Isabella
    Stockholm University, Faculty of Science, Department of Environmental Science and Analytical Chemistry.
    Ndreu, Lorena
    Stockholm University, Faculty of Science, Department of Environmental Science and Analytical Chemistry.
    Thorsén, Gunnar
    Ilag, Leopold L.
    Stockholm University, Faculty of Science, Department of Environmental Science and Analytical Chemistry.
    N-Glycosylation profiling of intact target proteins by high-resolution mass spectrometry (MS) and glycan analysis using ion mobility-MS/MS2020In: The Analyst, ISSN 0003-2654, E-ISSN 1364-5528, Vol. 145, no 5, p. 1737-1748Article in journal (Refereed)
    Abstract [en]

    Glycosylation influences the structure and functionality of glycoproteins, and is regulated by genetic and environmental factors. The types and abundance of glycans on glycoproteins can vary due to diseases such as cancer, inflammation, autoimmune and neurodegenerative disorders. Due to the crucial role glycans play in modulating protein function, glycosylation analysis could lead to the discovery of novel biomarkers and is of prime importance in controlling the quality of glycoprotein biopharmaceuticals. Here, we present a method for the 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 the biopharmaceutical trastuzumab. The accuracy of the method, expressed as the relative error (RE), ranged from 2.1 (at high concentrations) to 7.9% (at low concentrations), and intra- and inter-day precision, expressed as relative standard deviation (RSD), was 3.2 and 8.2%, respectively. The sensitivity and linearity of the method were suitable for serum analysis and the LOQ was calculated to be 3.1 and 4.4 mu g mL(-1) for transferrin (TFN) and trastuzumab (TRA), respectively. Its application to transferrin from five healthy human serum samples yielded concentrations between 1.61 and 3.17 mg mL(-1), which are in agreement with blood reference levels. In parallel, the structure of the identified glycans was determined by ion mobility spectrometry coupled with tandem mass spectrometry. No chromatographic separation was required and sample preparation was performed in a semi-automatic manner, facilitating the handling of up to 12 samples at a time. This method should be useful for clinical laboratories and for the quality control of large batches of biopharmaceuticals.

  • 6.
    Quaranta, Alessandro
    et al.
    Stockholm University, Faculty of Science, Department of Environmental Science and Analytical Chemistry.
    Sroka-Bartnicka, Anna
    Stockholm University, Faculty of Science, Department of Environmental Science and Analytical Chemistry. Marie Curie Sklodowska University, Poland.
    Tengstrand, Erik
    Stockholm University, Faculty of Science, Department of Environmental Science and Analytical Chemistry.
    Thorsén, Gunnar
    Stockholm University, Faculty of Science, Department of Environmental Science and Analytical Chemistry.
    N-Glycan profile analysis of transferrin using a microfluidic compact disc and MALDI-MS2016In: Analusis, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 408, no 17, p. 4765-4776Article in journal (Refereed)
    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.

  • 7.
    Riboni, Nicolò
    et al.
    Stockholm University, Faculty of Science, Department of Environmental Science and Analytical Chemistry. University of Parma, Italy.
    Quaranta, Alessandro
    Stockholm University, Faculty of Science, Department of Environmental Science and Analytical Chemistry.
    Motwani, Hitesh
    Stockholm University, Faculty of Science, Department of Environmental Science and Analytical Chemistry.
    Österlund, Nicklas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Bianchi, Federica
    Ilag, Leopold
    Stockholm University, Faculty of Science, Department of Environmental Science and Analytical Chemistry.
    Solvent-Assisted Paper Spray Ionization (SAPSI) for the Analysis of Biomolecules and BiofluidsManuscript (preprint) (Other academic)
  • 8.
    Riboni, Nicoló
    et al.
    Stockholm University, Faculty of Science, Department of Environmental Science and Analytical Chemistry. University of Parma, Italy.
    Quaranta, Alessandro
    Stockholm University, Faculty of Science, Department of Environmental Science and Analytical Chemistry.
    Motwani, Hitesh
    Stockholm University, Faculty of Science, Department of Environmental Science and Analytical Chemistry.
    Österlund, Nickles
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Bianchi, Federica
    Ilag, Leopold L.
    Stockholm University, Faculty of Science, Department of Environmental Science and Analytical Chemistry.
    Solvent-Assisted Paper Spray Ionization Mass Spectrometry (SAPSI-MS) for the Analysis of Biomolecules and Biofluids2019In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 9, article id 10296Article in journal (Refereed)
    Abstract [en]

    Paper Spray Ionization (PSI) is commonly applied for the analysis of small molecules, including drugs, metabolites, and pesticides in biological fluids, due to its high versatility, simplicity, and low costs. In this study, a new setup called Solvent Assisted Paper Spray Ionization (SAPSI), able to increase data acquisition time, signal stability, and repeatability, is proposed to overcome common PSI drawbacks. The setup relies on an integrated solution to provide ionization potential and constant solvent flow to the paper tip. Specifically, the ion source was connected to the instrument fluidics along with the voltage supply systems, ensuring a close control over the ionization conditions. SAPSI was successfully applied for the analysis of different classes of biomolecules: amyloidogenic peptides, proteins, and N-glycans. The prolonged analysis time allowed real-time monitoring of processes taking places on the paper tip, such as amyloid peptides aggregation and disaggregation phenomena. The enhanced signal stability allowed to discriminate protein species characterized by different post translational modifications and adducts with electrophilic compounds, both in aqueous solutions and in biofluids, such as serum and cerebrospinal fluid, without any sample pretreatment. In the next future, application to clinical relevant modifications, could lead to the development of quick and cost-effective diagnostic tools.

  • 9.
    Sroka-Bartnicka, Anna
    et al.
    Stockholm University, Faculty of Science, Department of Environmental Science and Analytical Chemistry.
    Karlsson, Isabella
    Stockholm University, Faculty of Science, Department of Environmental Science and Analytical Chemistry.
    Ndreu, Lorena
    Stockholm University, Faculty of Science, Department of Environmental Science and Analytical Chemistry.
    Quaranta, Alessandro
    Stockholm University, Faculty of Science, Department of Environmental Science and Analytical Chemistry.
    Pijnappel, Matthijs
    Stockholm University, Faculty of Science, Department of Environmental Science and Analytical Chemistry.
    Thorsén, Gunnar
    Stockholm University, Faculty of Science, Department of Environmental Science and Analytical Chemistry.
    Particle-based N-linked glycan analysis of selected proteins from biological samples using nonglycosylated binders2017In: Journal of Pharmaceutical and Biomedical Analysis, ISSN 0731-7085, E-ISSN 1873-264X, Vol. 132, p. 125-132Article in journal (Refereed)
    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.

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