High-throughput mass spectrometric N-glycomics

Glycosylation is an important way in which proteins, the functional agents of our body, can be modified to alter and expand their functional repertoire. Glycans consist of monosaccharides that are linked in a chained and branching fashion, often to form specific epitopes that are of clinical and biopharmaceutical interest. In order to study glycosylation, there is a need for high-throughput analysis methodology. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) is a prominent example of this, as it can rapidly provide information on the monosaccharide compositions of glycans. However, it is challenging for the method to yield information on the structural aspects of glycosylation, as well as on glycans carrying sialic acids. These sialylated glycans are prone to in-source and metastable decay, and tend to require chemical derivatization to allow their analysis. The aim of this thesis is the development and application of new methodology for MALDI-MS N-glycomics, and, with a focus on metabolic syndrome and rheumatoid arthritis, to increase our understanding of the role of N-glycosylation in health and disease. A principal outcome of the work is a sialic acid derivatization protocol that allows the mass-based discrimination of alpha-2,3- and alpha-2,6-linked sialic acids, facilitating their study in a high-throughput setting. LUMC / Geneeskund

Advances in neuroproteomics for neurotrauma: unraveling insights for personalized medicine and future prospects

Neuroproteomics, an emerging field at the intersection of neuroscience and proteomics, has garnered significant attention in the context of neurotrauma research. Neuroproteomics involves the quantitative and qualitative analysis of nervous system components, essential for understanding the dynamic events involved in the vast areas of neuroscience, including, but not limited to, neuropsychiatric disorders, neurodegenerative disorders, mental illness, traumatic brain injury, chronic traumatic encephalopathy, and other neurodegenerative diseases. With advancements in mass spectrometry coupled with bioinformatics and systems biology, neuroproteomics has led to the development of innovative techniques such as microproteomics, single-cell proteomics, and imaging mass spectrometry, which have significantly impacted neuronal biomarker research. By analyzing the complex protein interactions and alterations that occur in the injured brain, neuroproteomics provides valuable insights into the pathophysiological mechanisms underlying neurotrauma. This review explores how such insights can be harnessed to advance personalized medicine (PM) approaches, tailoring treatments based on individual patient profiles. Additionally, we highlight the potential future prospects of neuroproteomics, such as identifying novel biomarkers and developing targeted therapies by employing artificial intelligence (AI) and machine learning (ML). By shedding light on neurotrauma’s current state and future directions, this review aims to stimulate further research and collaboration in this promising and transformative field

Human plasma N-glycosylation as analyzed by MALDI-FTICR-MS associates with markers of inflammation and metabolic health

Glycosylation is an abundant co- and post-translational protein modification of importance to protein processing and activity. While not template-defined, glycosylation does reflect the biological state of an organism and is a high-potential biomarker for disease and patient stratification. However, to interpret a complex but informative sample like the total plasma N-glycome (TPNG), it is important to establish its baseline association with plasma protein levels and systemic processes. Thus far, large scale studies (n > 200) of the TPNG have been performed with methods of chromatographic and electrophoretic separation, which, while being informative, are limited in resolving the structural complexity of plasma N-glycans. Mass spectrometry (MS) has the opportunity to contribute additional information on, among others, antennarity, sialylation, and the identity of high-mannose type species. Here, we have used matrix-assisted laser desorption/ionization (MALDI)-Fourier transform ion cyclotron resonance (FTICR)- MS to study the TPNGs of 2,144 healthy middle-aged individuals from the Leiden Longevity Study, to allow association analysis with markers of metabolic health and inflammation. To achieve this, N-glycans were enzymatically released from their protein backbones, labeled at the reducing end with 2-aminobenzoic acid, and following purification analyzed by negative ion mode intermediate pressure MALDI-FTICR-MS. In doing so, we achieved the relative quantification of 61 glycan compositions, ranging from Hex4HexNAc2 to Hex7HexNAc6dHex1Neu5Ac4, as well as that of 39 glycosylation traits derived thereof. Next to confirming known associations of glycosylation with age and sex by MALDI-FTICR-MS, we report novel associations with C-reactive protein (CRP), interleukin 6 (IL-6), body mass index (BMI), leptin, adiponectin, HDL cholesterol, triglycerides (TG), insulin, gamma-glutamyl transferase (GGT), alanine aminotransferase (ALT) and smoking. Overall, the bisection, galactosylation and sialylation of diantennary species, the sialylation of tetraantennary species, and the size of high-mannose species proved to be important plasma characteristics associated with inflammation and metabolic health

Development of ultrahigh resolution FTICR mass spectrometry methods for clinical proteomics

Fourier transform ion cyclotron resonance (FTICR) MS provides (a) ultrahigh mass resolving power that allows the analysis of large proteins and complex mixtures; (b) ultrahigh mass measurement accuracy and precision that allow a more reliable identification of the detected species; (c) a wide dynamic range that is favourable for the detection of low abundant components. Despite these excellent characteristics, MALDI-FTICR MS has been rarely used for peptide and protein profiling of large cohorts of samples. In this thesis, the development and application of novel MALDI-FTICR-MS methods for high-throughput analysis of human serum peptides and proteins is described. Due to the large biological variability of human serum, the analysis of a large cohort is needed to obtain reliable statistical results.UBL - phd migration 201

Incorporating standardised drift-tube ion mobility to enhance non-targeted assessment of the wine metabolome (LC×IM-MS)

Liquid chromatography with drift-tube ion mobility spectrometry-mass spectrometry (LCxIM-MS) is emerging as a powerful addition to existing LC-MS workflows for addressing a diverse range of metabolomics-related questions [1,2]. Importantly, excellent precision under repeatability and reproducibility conditions of drift-tube IM separations [3] supports the development of non-targeted approaches for complex metabolome assessment such as wine characterisation [4]. In this work, fundamentals of this new analytical metabolomics approach are introduced and application to the analysis of 90 authentic red and white wine samples originating from Macedonia is presented. Following measurements, intersample alignment of metabolites using non-targeted extraction and three-dimensional alignment of molecular features (retention time, collision cross section, and high-resolution mass spectra) provides confidence for metabolite identity confirmation. Applying a fingerprinting metabolomics workflow allows statistical assessment of the influence of geographic region, variety, and age. This approach is a state-of-the-art tool to assess wine chemodiversity and is particularly beneficial for the discovery of wine biomarkers and establishing product authenticity based on development of fingerprint libraries

Practical Applications of NMR to Solve Real-World Problems

Nuclear magnetic resonance spectroscopy (NMR) has developed from primarily a method of academic study into a recognized technology that has advanced measurement capabilities within many different industrial sectors. These sectors include areas such as national security, energy, forensics, life sciences, pharmaceuticals, etc. Despite this diversity, these applications have many shared technical challenges and regulatory burdens, yet interdisciplinary cross-talk is often limited. To facilitate the sharing of knowledge, this Special Issue presents technical articles from four different areas, including the oil industry, nanostructured systems and materials, metabolomics, and biologics. These areas use NMR or magnetic resonance imaging (MRI) technologies that range from low-field relaxometry to magnetic fields as high as 700 MHz. Each article represents a practical application of NMR. A few articles are focused on basic research concepts, which will likely have the cross-cutting effect of advancing multiple disciplinary areas

High-throughput glycopeptide profiling of prostate-specific antigen from seminal plasma by MALDI-MS

An altered total seminal plasma glycosylation has been associated with male infertility, and the highly abundant seminal plasma glycoprotein prostate-specific antigen (PSA) plays an important role in fertilization. However, the exact role of PSA glycosylation in male fertility is not clear. To understand the involvement of PSA glycosylation in the fertilization process, analytical methods are required to study the glycosylation of PSA from seminal plasma with a high glycoform resolution and in a protein-specific manner. In this study, we developed a novel, high-throughput PSA glycopeptide workflow, based on matrix-assisted laser desorption/ionization-mass spectrometry, allowing the discrimination of sialic acid linkage isomers via the derivatization of glycopeptides. The method was successfully applied on a cohort consisting of seminal plasma from infertile and fertile men (N = 102). Forty-four glycopeptides were quantified in all samples, showing mainly complex-type glycans with high levels of fucosylation and sialylation. In addition, N,N-diacetyllactosamine (LacdiNAc) motives were found as well as hybrid-type and high mannose-type structures. Our method showed a high intra- and interday repeatability and revealed no difference in PSA glycosylation between fertile and infertile men. Next to seminal plasma, the method is also expected to be of use for studying PSA glycopeptides derived from other biofluids and/or in other disease contexts.Proteomic