Metabolomics is a powerful tool that can provide a comprehensive insight into the complexity of human biology and the pathophysiology of diseases. By analyzing the metabolome, which refers to the... Show moreMetabolomics is a powerful tool that can provide a comprehensive insight into the complexity of human biology and the pathophysiology of diseases. By analyzing the metabolome, which refers to the complete set of small (endogenous) molecules (with a mass <1500 Da) present within a given biological system, a direct functional read-out of an organism's physiological status can be obtained. In particular, metabolomics has emerged as a promising approach for neuroscience research, as it helps to shed light on the molecular mechanisms underlying neurodegenerative and neuropsychiatric diseases. One of the main challenges in (brain) metabolomics studies is the reliable and sensitive analysis of volume-limited biological samples. Therefore, in this thesis, innovative microscale analytical workflows based on capillary electrophoresis (CE) coupled to mass spectrometry (MS) are developed in order to generate metabolic profiles in volume-restricted samples. The ultimate aim of this thesis is to showcase that the technical reproducibility, detection sensitivity, and metabolic coverage of CE-MS are sufficient to allow use for different material-limited matrices of interest for neuroscience research. Show less
In this thesis, we developed new ways to use a technique called electroextraction (EE) to extract charged molecules from one liquid into another using an electric field. We wanted to make EE more... Show moreIn this thesis, we developed new ways to use a technique called electroextraction (EE) to extract charged molecules from one liquid into another using an electric field. We wanted to make EE more practical and easy to use for extracting different types of molecules. We tackled three main challenges: making EE work seamlessly with other techniques, avoiding the need to dilute small samples, and making EE more robust and user-friendly. To do this, we used automation and made use of existing analysis systems. This allowed us to fully take advantage of EE without needing manual processing or dilution. As a result, we were able to expand the range of compounds and types of molecules that EE can extract, and even extract both positively and negatively charged molecules at the same time. Show less
Capillary electrophoresis has matured into a highly sensitive and widely applied analytical method over the last forty years. Here we combine text mining and computational chemistry to paint, with... Show moreCapillary electrophoresis has matured into a highly sensitive and widely applied analytical method over the last forty years. Here we combine text mining and computational chemistry to paint, with very broad strokes, the applicability and trends in the scientific literature on capillary electrophoresis, simulta-neously demonstrating that this is not only possible, but reveal both expected and unexpected details of this history. All software and data are freely available on GitHub (https://github.com/ReinV/SCOPE) and OSF (https://osf.io/e56zt/). (c) 2023 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Show less
We have explored the history of the field of capillary electrophoresis using bibliometric methods. The analysis shows that 416 prolific researchers are connected in a single, large, co-authorship... Show moreWe have explored the history of the field of capillary electrophoresis using bibliometric methods. The analysis shows that 416 prolific researchers are connected in a single, large, co-authorship network based on publications on capillary electrophoresis between 1980 and 2021, with a few pioneers having remained active throughout much of this time period. Looking at research topics revealed electro-chemistry, sensors, nanotechnology and metabolomics as 'hot' topics, with fundamental method development being more 'mature', and reveal that capillary electrophoresis technology have matured over a 30-year time period, with research efforts moving from separations to quantitative measurements to biomedical applications. The citation patterns showed the strongest coupling between journals of similar scope. Interactive versions of the bibliometric network visualizations are available on-line at https://tinyurl.com/2z7q7wcx (researcher co-authorship network), https://tinyurl.com/2jmhsgxx (research topic network) and https://tinyurl.com/2lnfzzgn (journal bibliographic coupling citation network).(c) 2022 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Show less
Analytical assay development, particularly pertaining to glycomics, is an exciting amalgam of biology, chemistry and engineering. Besides academic research in natural and medical sciences,... Show moreAnalytical assay development, particularly pertaining to glycomics, is an exciting amalgam of biology, chemistry and engineering. Besides academic research in natural and medical sciences, glycomics assays have immense importance in industrial applications such as in quality control and quality assurance of glycoproteins. An up-coming industrial and clinical application is the high-throughput glycan profiling of clinical samples, such as plasma, for identifying disease associations. These glycomics assays are often based on chromatographic and mass spectrometric instrumentation. Thus, they create a requirement of instrumentation infrastructure as well as technical skills which are both not always readily available. This creates a demand in industry for the development of glycomics assays that have a low infrastructure cost as well as minimal training requirements and that are user-friendly. With these objectives in focus, this thesis develops novel exoglycosidase-based high-throughput glycomics assays for use in industrial glycan profiling. In doing so, this thesis also contributes to the development of potential products, such as glycomics kits. Show less
Benschop, C.C.G.; Gaag, K.J. van der; Vreede, J. de; Backx, A.J.; Leeuw, R.H. de; Zuniga, S.; ... ; Sijen, T. 2021
The interpretation of short tandem repeat (STR) profiles can be challenging when, for example, alleles are masked due to allele sharing among contributors and/or when they are subject to drop-out,... Show moreThe interpretation of short tandem repeat (STR) profiles can be challenging when, for example, alleles are masked due to allele sharing among contributors and/or when they are subject to drop-out, for instance from sample degradation. Mixture interpretation can be improved by increasing the number of STRs and/or loci with a higher discriminatory power. Both capillary electrophoresis (CE, 6-dye) and massively parallel sequencing (MPS) provide a platform for analysing relatively large numbers of autosomal STRs. In addition, MPS enables distinguishing between sequence variants, resulting in enlarged discriminatory power. Also, MPS allows for small amplicon sizes for all loci as spacing is not an issue, which is beneficial with degraded DNA. Altogether, MPS has the potential to increase the weights of evidence for true contributors to (complex) DNA profiles. In this study, likelihood ratio (LR) calculations were performed using STR profiles obtained with two different MPS systems and analysed using different settings: 1) MPS PowerSeqTM Auto System profiles analysed using FDSTools equipped with optimized settings such as noise correction, 2) ForenSeqTM DNA Signature Prep Kit profiles analysed using the default settings in the Universal Analysis Software (UAS), and 3) ForenSeqTM DNA Signature Prep Kit profiles analysed using FDSTools empirically adapted to cope with one-directional reads and provisional, basic settings. The LR calculations used genotyping data for two- to four-person mixtures varying for mixture proportion, level of drop-out and allele sharing and were generated with the continuous model EuroForMix. The LR results for the over 2000 sets of propositions were affected by the variation for the number of markers and analysis settings used in the three approaches. Nevertheless, trends for true and non-contributors, effects of replicates, assigned number of contributors, and model validation results were comparable for the three MPS approaches and alike the trends known for CE data. Based on this analogy, we regard the probabilistic interpretation of MPS STR data fit for forensic DNA casework. In addition, guidelines were derived on when to apply LR calculations to MPS autosomal STR data and report the corresponding results. Show less
Recent developments in the coupling of liquid phase separations and mass spectrometry have yielded some new source designs for electrospray ionization. The research in this thesis covers the... Show moreRecent developments in the coupling of liquid phase separations and mass spectrometry have yielded some new source designs for electrospray ionization. The research in this thesis covers the application of a newly developed electrospray ionization source design and it's application for the coupling of capillary electrophoresis to mass spectrometry. The initial chapters cover improvements in sensitivity and separation power in capillary electrophoresis mass spectrometry (CE-MS) by allowing electrospray at ultra low flow rates and therefore optimal ionization and separation conditions. We showed strong complementarity of this CE-MS system with reversed phase liquid chromatography in the identification of peptides and proteins from e.coli digests. Finally a sample preparation method for peptide and protein identification from laser micro-dissected tissue samples from human kidneys was developed. It was shown that a few hundred proteins could be identified from amounts of material representative of one full human glomerulus. On the whole great strides were made in development of strategies employing CE-MS for proteomics analysis. Show less
Pattky, M.; Nicolardi, S.; Santiago-Schubel, B.; Sydes, D.; Burgt, Y.E.M. van der; Klein, A.N.; ... ; Huhn, C. 2015
In thesis the power of electrophoretic on-line concentration procedures for peptidomics and metabolomics is demonstrated. Tow examples are given: isotachophoresis with spacer molecules and... Show moreIn thesis the power of electrophoretic on-line concentration procedures for peptidomics and metabolomics is demonstrated. Tow examples are given: isotachophoresis with spacer molecules and electroextraction. Electromigration is capable of enhancing selective analyte migration. Selective analyte migration can be used to increase the amount of analyte that is injected into an analytical system, while it leaves the sample volume with the contaminants behind. In consequence, the injection of contaminants and bulk components that may disturb sample pretreatment, separation and detection can be avoided and LOD values can be lowered without the problems associated with direct injection of larger sample volumes. The work that is presented in this thesis is focused on fast electrophoresis-based LOD improvement of the analysis of peptides and/or metabolites in plasma and urine. Show less
Huhn, C.; Ruhaak, L.R.; Mannhardt, J.; Wuhrer, M.; Neususs, C.; Deelder, A.M.; Meyer, H. 2012
The chapters that comprised this thesis cover a broad range of subjects from analytical method development to clinical application of metabolic profiling. They are united by the facts that all of... Show moreThe chapters that comprised this thesis cover a broad range of subjects from analytical method development to clinical application of metabolic profiling. They are united by the facts that all of these studies aimed at analysis of biological fluids and that the presented methods and approaches may ultimately become parts of a robust metabolomics workflow that might be used in a future personalized medicine. Show less
Proteomics is the systematic analysis of proteins and their expression patterns in biological systems. It involves the localization, isolation, identification and functional characterization of the... Show moreProteomics is the systematic analysis of proteins and their expression patterns in biological systems. It involves the localization, isolation, identification and functional characterization of the proteins in an organism. One particular technique used in these studies is the shotgun approach. This involves enzymatic digestion of the proteins, followed by separation and analysis by tandem mass spectrometry (MS/MS).In this research, the application of capillary isoelectric focusing (CIEF) for the shotgun approach is studied. During CIEF-analysis, amphoteric compounds are separated according to their pI-values, in a pH gradient formed under the influence of an electric field. Normally, carrier ampholytes are used to establish a linear pH gradient. Because of their involatile nature, they cause dramatic suppression of MS signal intensity of the analyte ions.In this research, CIEF analysis of digested proteins is based on autofocusing, using only limited amounts of carrier ampholytes as spacers. The efficacy and the limitations of this approach are studied. Complex protein mixtures are successfully analyzed by CIEF-MS/MS.Furthermore, proteolytic 18O labeling is successfully applied in this approach for differential analysis. Overall, CIEF-MS provides a valuable alternative to LC-MS analysis of complex peptide mixtures, both in a qualitative way as well as in a quantitative way. Show less
