BackgroundMolecular components in blood, such as proteins, are used as biomarkers to detect or predict disease states, guide clinical interventions and aid in the development of therapies. While... Show moreBackgroundMolecular components in blood, such as proteins, are used as biomarkers to detect or predict disease states, guide clinical interventions and aid in the development of therapies. While multiplexing proteomics methods promote discovery of such biomarkers, their translation to clinical use is difficult due to the lack of substantial evidence regarding their reliability as quantifiable indicators of disease state or outcome. To overcome this challenge, a novel orthogonal strategy was developed and used to assess the reliability of biomarkers and analytically corroborate already identified serum biomarkers for Duchenne muscular dystrophy (DMD). DMD is a monogenic incurable disease characterized by progressive muscle damage that currently lacks reliable and specific disease monitoring tools.MethodsTwo technological platforms are used to detect and quantify the biomarkers in 72 longitudinally collected serum samples from DMD patients at 3 to 5 timepoints. Quantification of the biomarkers is achieved by detection of the same biomarker fragment either through interaction with validated antibodies in immuno-assays or through quantification of peptides by Parallel Reaction Monitoring Mass Spectrometry assay (PRM-MS).ResultsFive, out of ten biomarkers previously identified by affinity-based proteomics methods, were confirmed to be associated with DMD using the mass spectrometry-based method. Two biomarkers, carbonic anhydrase III and lactate dehydrogenase B, were quantified with two independent methods, sandwich immunoassays and PRM-MS, with Pearson correlations of 0.92 and 0.946 respectively. The median concentrations of CA3 and LDHB in DMD patients was elevated in comparison to those in healthy individuals by 35- and 3-fold, respectively. Levels of CA3 vary between 10.26 and 0.36 ng/ml in DMD patients whereas those of LDHB vary between 15.1 and 0.8 ng/ml.ConclusionsThese results demonstrate that orthogonal assays can be used to assess the analytical reliability of biomarker quantification assays, providing a means to facilitate the translation of biomarkers to clinical practice. This strategy also warrants the development of the most relevant biomarkers, markers that can be reliably quantified with different proteomics methods. Show less
Protein glycosylation has profound implications in a wide range of molecular and biological processes occurring in cancer, where specific changes in the glycan structures have shown to be... Show moreProtein glycosylation has profound implications in a wide range of molecular and biological processes occurring in cancer, where specific changes in the glycan structures have shown to be associated with the development and progression of the disease paving the way for the development of new clinical biomarkers as well as providing specific targets for therapeutic intervention, patient stratification and personalized medicine. Protein glycosylation is also critical for the development of biopharmaceuticals, as even minor shifts in manufacturing procedures can substantially impact the bioactivity, safety, and efficacy of therapeutic proteins. Although a variety of mass spectrometric and chromatographic methods are available for the identification and characterization of glycans from complex sample mixtures, the lack of standardized protocols across platforms often results in inconsistent results, making data integration and comparison challenging. Furthermore, most of the current technology for the study of intact glycans would not be suitable for the rapid analysis of large sample sets, mainly due to limitations in sample throughput. The scope of this thesis is to establish standardized, high-throughput glycomics technologies for the quantitative analysis of protein N- and O-glycosylation and improve current methodologies in order to facilitate the characterization of intact oligosaccharides from in vitro established model systems. Show less
Novel food proteases (Maxipro A, Maxipro B, Maxipro C, and Maxipro D) with broad specificity and different effective pH range (from 2.5 to 9.0) were evaluated to produce hypocholesterolemic... Show moreNovel food proteases (Maxipro A, Maxipro B, Maxipro C, and Maxipro D) with broad specificity and different effective pH range (from 2.5 to 9.0) were evaluated to produce hypocholesterolemic peptides from olive seeds proteins. Box-Behnken experimental design was employed to evaluate the effect of the hydrolysis time, pH, temperature, and enzyme:substrate ratio on the amount of released peptides. The production of peptides, under optimal conditions, was higher with Maxipro C and lower with Maxipro B. The capability of extracts to reduce the absorption of exogenous cholesterol and the production of endogenous cholesterol was also evaluated. All hydrolysates showed a higher capacity to bind bile acids and inhibit cholesterol esterase enzyme than the obtained with usual Alcalase enzyme. Proteomic analysis using high resolution RP-HPLC-ESI-QTOF with database searching and de novo identification was employed to identify peptides in hydrolysates. Some sequences with hypocholesterolemic features were detected among the 45 peptides identified in Maxipro A hydrolysate, the 56 peptides in that of Maxipro C, and the 50 in the Maxipro B one. This work demonstrates that Maxipro A enzyme can hydrolyze olive seeds proteins, under acidic pH, releasing peptides with multifunctional and higher hypo-cholesterolemic capacity than the obtained with Alcalase, under alkaline conditions. Show less
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
This thesis describes the development and evaluation of a new laboratory tool to improve early kidney injury diagnosis. In the first part, we define the unmet clinical needs in the clinical care... Show moreThis thesis describes the development and evaluation of a new laboratory tool to improve early kidney injury diagnosis. In the first part, we define the unmet clinical needs in the clinical care pathways in kidney injury, by a questionnaire and s literature review to summarize current state-of-the-art diagnostics. The second part of this thesis comprises the analytical phase of test development. Liquid-chromatography (LC) coupled to tandem MS was chosen as analytical platform for protein-based kidney injury biomarker quantitation from human urine specimens. The third part of this thesis covers aspects of the postanalytical phase of medical test development, which included the establishment of reference intervals and clinical observational cohort studies. Elevated levels of the studied kidney injury biomarkers, and their combined signatures, were associated with acute kidney injury induced by ischemia-reperfusion injury after kidney transplantation, but not with stable chronic kidney disease. In a general perspective, this thesis highlights the role of quantitative protein mass spectrometry for biomarker translation from research towards the clinical laboratory. Show less
The research described in this thesis aims at the development of ubiquitin-based research tools to study the enzymes of the ubiquitination pathway, the ligase enzymes and the deubiquitinating... Show moreThe research described in this thesis aims at the development of ubiquitin-based research tools to study the enzymes of the ubiquitination pathway, the ligase enzymes and the deubiquitinating enzymes. These enzymes are responsible for the conjugation and the removal of the post-translational modifier ubiquitin. This small protein is involved in almost all cellular processes, and when conjugated onto a substrate protein it can signal for degradation and influence the localization, interaction, stability and activity of the protein. Therefore, the dysregulation of these processes can have detrimental effects of cell organization and survival which in turn has implications in numerous processes related to diseases. Hence, it is important to fully understand the ubiquitination pathway and how to interact with it. The ubiquitin-based research tools described in this thesis aim to shine light on parts of this pathway. Ranging from the selectivity and specificity of DUBs for specific Ub linkages in competition and the catalytic efficiency of these proteolytic cleavage processes to the selectivity and activity of ligases and the activity of DUBs in cells. All ubiquitin research tools are based on synthetic ubiquitin modified with unnatural amino acids, neutron-encoded amino acids, point mutations and/or fluorescent labels, in order to study the characteristics of the enzymes in vitro. Show less
Glycosylation is a widely occurring and complex modification found on lipids and proteins and is involved in the recognition, signaling and interaction events within the cell and between cells.... Show moreGlycosylation is a widely occurring and complex modification found on lipids and proteins and is involved in the recognition, signaling and interaction events within the cell and between cells. These events based on glycan structures result in adhesion, cell-matrix interaction and immune recognition. Alterations in the glycomic profile are considered a hallmark of various diseases, including cancer where it contributes to the development and progression of cancer, affecting cell-cell communication, cell-matrix interactions, tumor angiogenesis, invasion and metastasis. These functions are governed by different glycans and their terminal structures. In order to further explore these structures with regard to their potential as biomarkers and specific targets for diagnostic applications and therapeutical strategies for various diseases, in-depth glycomic analysis is needed. It is further noted that aberrant glycosylation not only results from the altered expression of glycosyltransferases (GTs) but also from the changed activity of GTs and glycosidases as well as the availability and abundance of sugar nucleotide donors. The aim of the research described in this thesis was to explore the glycomic signatures of colorectal cancer (CRC) in cell lines and tissues as well as of acute myeloid leukemia (AML) cell lines. Show less
Varunjikar, M.S.; Bohn, T.; Sanden, M.; Belghit, I.; Pineda-Pampliega, J.; Palmblad, M.; ... ; Rasinger, J.D. 2023
The present study compared genetically modified (GM) crops with crops from different farming practices using high-resolution tandem mass spectrometry (HR-MS) and proteomics bioinformatics tools. In... Show moreThe present study compared genetically modified (GM) crops with crops from different farming practices using high-resolution tandem mass spectrometry (HR-MS) and proteomics bioinformatics tools. In a previously pub-lished study, a number of significant differences regarding nutritional and elemental composition between a selection of GM, non-GM conventionally farmed, and organic soybeans have been found. In the present study, the proteome-level equivalence of the same samples was assessed using HR-MS. Direct comparison of tandem mass spectra and bottom-up proteomics bioinformatics indicated that proteomes of all samples investigated were very similar overall, with only a few distinct protein expression clusters obtained for GM and organic samples. Standard bottom-up proteome analyses identified 1025 soy proteins; of these 39 were found to be differentially expressed (p < 0.01) between GM, non-GM conventionally farmed, and organically farmed soybeans. Subsequent bioinformatics analyses of these proteins highlighted several potentially affected biochemical pathways that could contribute to the compositional differences reported earlier. In addition, protein markers separating conventionally, and organically farmed soybean seeds were found and peptide markers for the detection of GM soy in food and feed samples are described. Taken together, the data presented here shows that HR-MS based proteomics approaches can be used for the detection of transgenic events in food and feed grade soy, the dif-ferentiation of organically and conventionally farmed plants, and provide mechanistic explanations of effects observed on the phenotypic level of GM plants. HR-MS and proteomic bioinformatics thus should be considered key tools when developing molecular panel approaches for detection and safety assessments of novel crop va-rieties destined for use in feed and food. Show less
Despite being a relatively new addition to the Omics' land-scape, lipidomics is increasingly being recognized as an important tool for the identification of druggable targets and biochemical... Show moreDespite being a relatively new addition to the Omics' land-scape, lipidomics is increasingly being recognized as an important tool for the identification of druggable targets and biochemical markers. In this review we present recent ad-vances of lipid analysis in drug discovery and development. We cover current state of the art technologies which are constantly evolving to meet demands in terms of sensitivity and selectivity. A careful selection of important examples is then provided, illustrating the versatility of lipidomics analysis in the drug discovery and development process. Integration of lipi-domics with other omics', stem-cell technologies, and meta-bolic flux analysis will open new avenues for deciphering pathophysiological mechanisms and the discovery of novel targets and biomarkers. Show less
Ubiquitin and SUMO modify thousands of substrates to regulate most cellular processes. System-wide identifi-cation of ubiquitin and SUMO substrates provides global understanding of their cellular... Show moreUbiquitin and SUMO modify thousands of substrates to regulate most cellular processes. System-wide identifi-cation of ubiquitin and SUMO substrates provides global understanding of their cellular functions. In this review, we discuss the biological importance of site-specific modifications by ubiquitin and SUMO regulating the DNA damage response, protein quality control and cell cycle progression. Furthermore we discuss the machinery responsible for these modifications and methods to purify and identify ubiquitin and SUMO modified sites by mass spectrometry. We provide a framework to aid in the selection of appropriate purification, digestion and acquisition strategies suited to answer different biological questions. We highlight opportunities in the field for employing innovative technologies, as well as discuss challenges and long-standing questions in the field that are difficult to address with the currently available tools, emphasizing the need for further innovation. 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
Immunoglobulin G (IgG) antibodies can exert their functions via both Fab-mediated neutralization and Fc-mediated effector functions, both of which are crucial for protective immunity in COVID-19.... Show moreImmunoglobulin G (IgG) antibodies can exert their functions via both Fab-mediated neutralization and Fc-mediated effector functions, both of which are crucial for protective immunity in COVID-19. Importantly, effector functions and resulting inflammatory responses are impacted by the structure of N-glycans linked to the Fc-tail of IgG. Studying antibody glycosylation in emerging infectious diseases such as SARS-CoV-2 allows to gain insight into specific glycan signatures at the early stages of infection, and to investigate whether these reflect how the disease would progress. For example, low fucosylation is a common glyco-phenotypic signature of IgG1 produced against the spike (S) protein of severely ill SARS-CoV-2 infected patients early on in their disease course, but has likewise been described in other disease settings, where the antigen is presented in the context of host-cell membranes (Chapter 2). In this thesis, antibody glycomics signatures of SARS-CoV-2 infection and vaccination have been explored using an established liquid chromatography – mass spectrometry-based method relying on affinity-isolation and proteolytic digestion of both total and anti-S IgG. In Chapter 3, the glycosylation of SARS-CoV-2 anti-S IgG antibodies were found to be vastly skewed relative to total IgG and to change in a highly dynamic fashion. Moreover, IgG glycosylation was shown to be an early severity marker and showed patient stratification potential, with predicting power for intensive care admission within a hospitalized patient population. Early detection of a pro-inflammatory glycosylation pattern may provide a broader intervention window and decrease the number of ICU-admissions. Furthermore, anti-S IgG1 glycosylation levels obtained with LC-MS show promise to supplement clinical parameters and biomarkers of inflammation, that have together been used for the severity score calculation of hospitalized COVID-19 patients. Similarly to SARS-CoV-2 infection, antibodies generated against the spike protein upon BNT162b2 mRNA vaccination also induced a transient afucosylated anti-S IgG1 response in antigen naïve individuals, albeit to a lower extent than in severely ill patients, exemplifying the influence of the type of immunization on antibody glycosylation (Chapter 4). Upon vaccination, the observed initial, mild afucosylated response was additionally accompanied by low fucosyltransferase (FUT8) expression in antigen-specific plasma cells. Furthermore, the observed initial anti-S IgG afucosylation signature may aided mounting a stronger immune response, as indicated by its correlation with antibody amounts following the second vaccination dose. Given the impact of glycosylation on antibody function, deciphering theunderlying regulatory mechanisms influencing IgG glycosylation will be of great importance to better understand the inflammatory potential, vaccine efficacy and protective capacity of vaccine- or pathogen-induced IgG in both body fluids and tissues in the future.In Chapter 5 and 6, the reaction steps of a previously developed linkage-specific sialic acid derivatization workflow were studied in more detail. Key players in such reactions are catalyst, of which novel types with different physico-chemical properties were introduced in Chapter 5. In Chapter 6, prior lactone formation was found to be a prerequisite for subsequent amidation of α2,3-linked sialic acids, which proceeds via direct aminolysis of the C2 lactone. Together, these new insights will be beneficial for the rational optimization of high-throughput (MALDI-)MS-based glycomics and glycoproteomics workflows relying on linkage-specific sialic acid derivatization. Show less
Duijl, T.T. van; Ruhaak, R.; Hoogeveen, E.; Mutsert, R. de; Rosendaal, F.; Cessie, S. le; ... ; Cobbaert, C. 2022
Background and aims: There is an ongoing need to recognize early kidney injury and its progression in structural chronic pathologies. The proteins NGAL, IGFBP7, TIMP2, KIM-1, CXCL9, TGF-beta 1,... Show moreBackground and aims: There is an ongoing need to recognize early kidney injury and its progression in structural chronic pathologies. The proteins NGAL, IGFBP7, TIMP2, KIM-1, CXCL9, TGF-beta 1, SLC22A2, nephrin, cubilin and uromodulin have been proposed as early kidney injury biomarkers. To guide clinical interpretation, their urinary concentrations should be accompanied by reference intervals, which we here establish in a representative Dutch middle-aged population. Materials and Methods: The 24-h urine samples from 1443 Caucasian middle-aged men and women, were analyzed for the biomarkers by quantitative LC-MS/MS. Biomarker excretion per 24-h were calculated, and urine creatinine and osmolality were measured for dilution normalization. This population was characterized by demographic and anthropometric parameters, comorbid conditions, and conventional kidney function measures. Results: NGAL, IGFBP7, TIMP2, KIM-1 and uromodulin could be quantified in this population, whereas nephrin, SLC22A2 and CXCL9 were below their detection limits. Urine creatinine and osmolality ( r= -were correlated to urine volume (r = -0.71; -0.74) and to IGFBP7 (r = 0.73; 0.71) and TIMP2 (r = 0.71; 0.69). Crude and normalized biomarker concentrations were affected by sex, but not by age, BMI, smoking, kidney function or common comorbid conditions. The reference intervals (men; women) were 18-108; 21-131 pmol IGFBP7/mmol creatinine, 1- 63; 4-224 pmol NGAL/mmol creatinine, 7-48; 7- 59 pmol TIMP2/mmol creatinine, <1-9; <1-12 pmol KIM-1/mmol creatinine and 0.1-1.2; 0.1-1.7 mg uromodulin/mmol creatinine. Conclusion: We present dilution-normalized and sex-stratified urinary reference intervals of kidney injury biomarkers in a middle-aged Caucasian population. Show less
Piaggio, F.; Croce, M.; Reggiani, F.; Monti, P.; Bernardi, C.; Ambrosio, M.; ... ; Amaro, A. 2022
Background and aim of the study: Mutations in the G alpha-genes GNAQ and GNA11 are found in 85-90% of uveal melanomas (UM). Aim of the study is to understand whether the mutations in both genes... Show moreBackground and aim of the study: Mutations in the G alpha-genes GNAQ and GNA11 are found in 85-90% of uveal melanomas (UM). Aim of the study is to understand whether the mutations in both genes differentially affect tumor characteristics and outcome and if so, to identify potential mechanisms. Methods: We analyzed the association between GNAQ and GNA11 mutations with disease specific survival, gene expression profiles, and cytogenetic alterations in 219 UMs. We used tandem-affinity-purification, mass spectrometry and immunoprecipitation to identify protein interaction partners of the two G-proteins and analyzed their impact on DNA-methylation. Results: GNA11 mutation was associated with: i) an increased frequency of loss of BRCA1- associated protein 1 (BAP1) expression (p = 0.0005), ii) monosomy of chromosome 3 (p < 0.001), iii) amplification of chr8q (p = 0.038), iv) the combination of the latter two (p = 0.0002), and inversely with v) chr6p gain (p = 0.003). Our analysis also showed a shorter disease-specific survival of GNA11-mutated cases as compared to those carrying a GNAQ mutation (HR = 1.97 [95%CI 1.12-3.46], p = 0.02). GNAQ and GNA11 encoded G-proteins have different protein interaction partners. Specifically, the Tet Methylcytosine Dioxygenase 2 (TET2), a protein that is involved in DNA demethylation, physically interacts with the GNAQ protein but not with GNA11, as confirmed by immunoprecipitation analyses. High risk UM cases show a clearly different DNA-methylation pattern, suggesting that a different regulation of DNA methylation by the two G-proteins might convey a different risk of progression. Conclusions: GNA11 mutated uveal melanoma has worse prognosis and is associated with high risk cytogenetic, mutational and molecular tumor characteristics that might be determined at least in part by differential DNA-methylation. (C) 2022 Elsevier Ltd. All rights reserved. Show less
The surface of eukaryotic cells contains a very dense layer of oligosaccharides called glycans that are linked to protein and lipid carriers and play an important role in cell-cell and cell... Show moreThe surface of eukaryotic cells contains a very dense layer of oligosaccharides called glycans that are linked to protein and lipid carriers and play an important role in cell-cell and cell-extracellular matrix interactions. Cancer-induced changes in glycosylation have an impact on the function of major glycoproteins in the human colon, therefore studies focused on colorectal cancer (CRC)-specific glycosylation signatures can provide novel insights into onset and progression of this disease. The major focus of this thesis was to investigate mucin type O-glycosylation signatures of CRC. For this purpose, a protocol for in-depth analysis of N- and O-glycans obtained from cell lines was developed (Chapter 2) using nanoscale porous graphitized carbon liquid chromatography coupled to mass spectrometry (PGC-nano-LC-MS). In Chapter 3 additional conditions were optimized in the MS methodology by using polar protic dopant (methanol and isopropanol) enriched nitrogen gas to increase sensitivity on the MS and tandem MS level. In Chapter 4 we applied the methodology developed in Chapter 2 to the analysis of O-glycosylation signatures of 26 different CRC cell lines. This analysis resulted in the characterization of more than 150 O-glycan structures and increased our understanding of glycan expression in the analyzed cell lines. To gain further understanding in the mechanisms underlying glycomic changes with colon cell differentiation, we explored changes in the cell line glycome and proteome upon spontaneous and butyrate-stimulated differentiation in in vitro cell culture (Chapter 5). By performing an integrative approach, we generated hypotheses about glycosylation signatures of specific cell adhesion proteins, which may play an important role in cancer progression. The localization of glycans on the cell surface and their role in biological processes are important in cancer pathogenesis, making them potential candidates for glycan targeting immunotherapy. Therefore, we further optimized the methodology to enable comprehensive analysis of N- and O-glycans from specific regions of formalin-fixed, paraffin-embedded tissues using laser capture microdissections and applied it for the analysis of selected regions of CRC tissues and their patient-matched colon mucosa controls (Chapter 6). We identified specific tumor-associated carbohydrate antigens (TACAs) that show expression only in the tumor samples, with no or limited expression in the normal colon mucosa. Since TACAs are present in high abundance on the surface of cancer cells which are linked to many different proteins, these are very promising targets for the development of tumor-specific immunotherapy. Show less
Background: Many patients who are diagnosed with coronavirus disease 2019 (COVID-19) suffer from venous thromboembolic complications despite the use of stringent anticoagulant prophylaxis. Studies... Show moreBackground: Many patients who are diagnosed with coronavirus disease 2019 (COVID-19) suffer from venous thromboembolic complications despite the use of stringent anticoagulant prophylaxis. Studies on the exact mechanism(s) underlying thrombosis in COVID-19 are limited as animal models commonly used to study venous thrombosis pathophysiology (i.e. rats and mice) are naturally not susceptible to Severe Acute Respiratory Syn-drome Coronavirus 2 (SARS-CoV-2). Ferrets are susceptible to SARS-CoV-2 infection, successfully used to study virus transmission, and have been previously used to study activation of coagulation and thrombosis during influenza virus infection.Objectives: This study aimed to explore the use of (heat-inactivated) plasma and lung material from SARS-CoV-2-inoculated ferrets studying COVID-19-associated changes in coagulation and thrombosis. Material and methods: Histology and longitudinal plasma profiling using mass spectrometry-based proteomics approach was performed.Results: Lungs of ferrets inoculated intranasally with SARS-CoV-2 demonstrated alveolar septa that were mildly expanded by macrophages, and diffuse interstitial histiocytic pneumonia. However, no macroscopical or microscopical evidence of vascular thrombosis in the lungs of SARS-CoV-2-inoculated ferrets was found. Lon-gitudinal plasma profiling revealed minor differences in plasma protein profiles in SARS-CoV-2-inoculated ferrets up to 2 weeks post-infection. The majority of plasma coagulation factors were stable and demonstrated a low coefficient of variation.Conclusions: We conclude that while ferrets are an essential and well-suited animal model to study SARS-CoV-2 transmission, their use to study SARS-CoV-2-related changes relevant to thrombotic disease is limited. Show less
Kotronoulas, A.; Lomana, A.L.G. de; Karvelsson, S.T.; Heijink, M.; Stone, R.; Giera, M.; Rolfsson, O. 2021
The use of acellular fish skin grafts (FSG) for the treatment of burn wounds is becoming more common due to its beneficial wound healing properties. In our previous study we demonstarted that FSG... Show moreThe use of acellular fish skin grafts (FSG) for the treatment of burn wounds is becoming more common due to its beneficial wound healing properties. In our previous study we demonstarted that FSG is a scaffold biomaterial that is rich in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) conjugated to phosphatidylcholines. Here we investigated whether EPA and DHA derived lipid mediators are influenced during the healing of burn wounds treated with FSG. Deep partial and full thickness burn wounds (DPT and FT, respectively) were created on Yorkshire pigs (n = 4). DPT were treated with either FSG or fetal bovine dermis while FT were treated either with FSG or cadaver skin initially and followed by a split thickness skin graft. Punch biopsies were collected on days 7, 14, 21, 28 and 60 and analyzed in respect of changes to approximately 45 derivatives of EPA, DHA, arachidonic acid (AA), and linoleic acid (LA) employing UPLC-MS/MS methodology. Nine EPA and DHA lipid mediators, principally mono-hydroxylated derivatives such as 18-HEPE and 17-HDHA, were significantly higher on day 7 in the DPT when treated with FSG. A similar but non-significant trend was observed for the FT. The results suggest that the use of FSG in burn wound treatment can alter the formation of EPA and DHA mono hydroxylated lipid mediators in comparison to other grafts of mammalian origin. The differences observed during the first seven days after treatment indicates that FSG affects the early stages of wound healing. Show less
Pot, S.; Gstottner, C.; Heinrich, K.; Hoelterhoff, S.; Grunert, I.; Leiss, M.; ... ; Dominguez Vega, E. 2021
Characterization of post-translational modifications (PTMs) of therapeutic antibodies is commonly performed by bottom-up approaches, involving sample preparation and peptide analysis by liquid... Show moreCharacterization of post-translational modifications (PTMs) of therapeutic antibodies is commonly performed by bottom-up approaches, involving sample preparation and peptide analysis by liquid chromatography-mass spectrometry (LC-MS). Conventional sample preparation requires extensive hands-on time and can increase the risk of inducing artificial modifications as many off-line steps - denaturation, disulfide-reduction, alkylation and tryptic digestion - are performed. In this study, we developed an on-line multidimensional (mD)-LC-MS bottom-up approach for fast sample preparation and analysis of (formulated) monoclonal antibodies and antibody-derived therapeutics. This approach allows on-column reduction, tryptic digestion and subsequent peptide analysis by RP-MS. Optimization of the 1D -and 2D flow and temperature improved the trapping of small polar peptides during on-line peptide mapping analysis. These adaptations increased the sequence coverage (95-98% versus 86-94% for off-line approaches) and allowed identification of various PTMs (i.e. deamidation of asparagine, methionine oxidation and lysine glycation) within a single analysis. This workflow enables a fast (<2 h) characterization of antibody heterogeneities within a single run and a low amount of protein (10 mu g). Importantly, the new mD-LC-MS bottom-up method was able to detect the polar, fast-eluting peptides: Fc oxidation at Hc-Met-252 and the Fc N-glycosylation at Hc-Asn-297, which can be challenging using mD-LC-MS. Moreover, the method showed good comparability across the different measurements (RSD of retention time in the range of 0.2-1.8% for polar peptides). The LC system was controlled by only a standard commercial software package which makes implementation for fast characterization of quality attributes relatively easy. (C) 2021 The Author(s). Published by Elsevier B.V. Show less
Biomarker molecules are analyzed in clinical tests to diagnose a disease, but often these test lack sensitivity or specificity. Also, for many diseases there is not even a blood based test... Show moreBiomarker molecules are analyzed in clinical tests to diagnose a disease, but often these test lack sensitivity or specificity. Also, for many diseases there is not even a blood based test available, while blood collection is relatively low invasive. For breast- and pancreatic cancer, there are several proteins that could potentially serve as biomarkers in blood, but these are not yet specific enough to use for diagnostic testing. Further research on other types of biomarkers may therefore be a valuable addition to eventually be able to develop a blood test. Methods for glycosylation profiling from serum and dried bloodspots with mass spectrometry were developed and applied to pancreatic- and breast cancer biomarker studies. Differences were found between profiles of healthy and sick persons for pancreatic cancer, but no clear differences were seen for breast cancer. This is probably due to the many different forms of breast cancer which result in different profiles. In the future, combining different types of markers from serum might ensure that differences between healthy and sick, between different diseases and between types of disease can be identified. This could lead to the development of a blood test for the early detection of cancer and other diseases. Show less
The chemical and structural heterogeneity of toxoid vaccines makes their analysis challenging. However, detailed insights on a molecular level can be obtained by mass spectrometry. Our initial... Show moreThe chemical and structural heterogeneity of toxoid vaccines makes their analysis challenging. However, detailed insights on a molecular level can be obtained by mass spectrometry. Our initial focus was the identification of formaldehyde-induced modifications in diphtheria toxin, which is described in Chapter 2. Subsequently, the methods described in Chapter 2 were applied to study what effects formaldehyde-induced modifications on model proteins have on their susceptibility to enzymatic proteolysis (Chapter 3). During the analysis of these model proteins, unknown formaldehyde-induced modifications were observed. The structural elucidation of these modifications, the discovery of a new type of crosslinks and various other subsequent reaction products are described in Chapter 4. The degradomics analysis described in Chapter 3 was applied to tetanus toxoids to distinguish heat-denaturated toxoids from their original state (Chapter 5). In order to reduce the analysis time and further improve the degradomics approach, an optimized strategy using Tandem Mass Tag multiplexing for the relative quantification of peptides was developed for the analysis of diphtheria toxoids (Chapter 6). Finally, Chapter 7 provides a brief discussion on the results presented in this thesis and offers some perspectives on implementation of the findings for toxoid vaccine development, quality control and further research. Show less
