The ideas underlying this thesis are that lipidomics may improve the diagnosis of liver metabolic diseases, and can provide further insights into the underlying pathophysiology of these diseases.... Show moreThe ideas underlying this thesis are that lipidomics may improve the diagnosis of liver metabolic diseases, and can provide further insights into the underlying pathophysiology of these diseases. However to achieve this, the measurement of lipids should be further improved by enabling comprehensive coverage, accurate identification and quantitation. Therefore, in this thesis, new analytical methodologies using LC-MS for the accurate detection and quantitation of lipids are developed. These methods are then applied to MCADD samples to identify lipid biomarkers, which are further correlated with gene expression analysis to understand the underlying biochemical pathways. Show less
Harish, P.; Malerba, A.; Kroon, R.H.M.J.M.; Shademan, M.; Engelan, B. van; Raz, V.; ... ; Snowden, S.G. 2023
The identification of metabolomic biomarkers relies on the analysis of large cohorts of patients compared to healthy controls followed by the validation of markers in an independent sample set.... Show moreThe identification of metabolomic biomarkers relies on the analysis of large cohorts of patients compared to healthy controls followed by the validation of markers in an independent sample set. Indeed, circulating biomarkers should be causally linked to pathology to ensure that changes in the marker precede changes in the disease. However, this approach becomes unfeasible in rare diseases due to the paucity of samples, necessitating the development of new methods for biomarker identification. The present study describes a novel approach that combines samples from both mouse models and human patients to identify biomarkers of OPMD. We initially identified a pathology-specific metabolic fingerprint in murine dystrophic muscle. This metabolic fingerprint was then translated into (paired) murine serum samples and then to human plasma samples. This study identified a panel of nine candidate biomarkers that could predict muscle pathology with a sensitivity of 74.3% and specificity of 100% in a random forest model. These findings demonstrate that the proposed approach can identify biomarkers with good predictive performance and a higher degree of confidence in their relevance to pathology than markers identified in a small cohort of human samples alone. Therefore, this approach has a high potential utility for identifying circulating biomarkers in rare diseases. Show less
Lentjes, E.G.W.M.; Bui, H.N.; Ruhaak, L.R.; Kema, I.P.; Coene, K.L.M.; Ouweland, J.M.W. van den 2023
Background: Over the past decade the use of LC-MS/MS has increased significantly in the hospital laboratories. Clinical laboratories have switched from immunoassays to LC-MS/MS methods due to the... Show moreBackground: Over the past decade the use of LC-MS/MS has increased significantly in the hospital laboratories. Clinical laboratories have switched from immunoassays to LC-MS/MS methods due to the promise of improve-ments in sensitivity and specificity, better standardization with often non-commutable international standards, and better between-laboratory comparison. However, it remains unclear whether routine performance of the LC-MS/MS methods have met these expectations. Method: This study examined the EQAS results, from the Dutch SKML, of serum cortisol, testosterone, 25OH-vita-minD and cortisol in urine and saliva over 9 surveys (2020 to first half of 2021). Results: The study found a significant increase in the number of compounds and in the number of results measured in the different matrices, with LC-MS/MS over a period of eleven years. In 2021, approximately 4000 LC-MS/MS results were submitted (serum: urine: saliva = 58:31:11%) compared to only 34 in 2010. When compared to the individual immunoassays, the LC-MS/MS based methods for serum cortisol, testosterone and 25OH-vitaminD showed comparable but also higher between-laboratory CVs in different samples of the surveys. For cortisol, testosterone and 25OH-vitaminD the median CV was 6.8%, 6.1% and 4.7% respectively for the LC-MS/MS compared to 3.9-8.0%,4.5-6.7%, and 7.5-18.3% for immunoassays. However, the bias and imprecision of the LC-MS/MS was better than that of the immunoassays. Conclusion: Despite the expectation that LC-MS/MS methods would result in smaller between-laboratory dif-ferences, as they are relatively matrix independent and better to standardize, the results of the SKML round robins do not reflect this for some analytes and may be in part explained by the fact that in most cases laboratory developed tests were used. Show less
Nowadays, therapeutic antibodies are the major and fastest growing class of biotherapeuticals. Since their invention, they are continuously developed to improve structural and functional... Show moreNowadays, therapeutic antibodies are the major and fastest growing class of biotherapeuticals. Since their invention, they are continuously developed to improve structural and functional characteristics. The high complexity of recently generated antibody derivatives, with various modifications induced during the manufacturing process itself leads to many proteoform variants of the desired product. These proteoforms can potentially exhibit altered activity. Therefore, an adequate characterization of the proteoforms, the assessment of their impact and careful monitoring of critical species is indispensable in order to guarantee effective and safe biopharmaceuticals. As the landscape of next-generation Ab formats continuously evolves, it is likewise of great importance to further develop appropriate analytical methods for their thorough attribute analysis. Hence, the focus of the research performed in this thesis is the development of multi-level approaches for the in-depth, primarily MS-based characterization of biopharmaceuticals to overcome the present restrictions and challenges arising e.g. by the implementation of complex Ab formats. Show less
Alarcon-Barrera, J.C.; Kostidis, S.; Ondo-Mendez, A.; Giera, M. 2022
The pharmaceutical industry adapted proteomics and other 'omics technologies for drug research early following their initial introduction. Although metabolomics lacked behind in this development,... Show moreThe pharmaceutical industry adapted proteomics and other 'omics technologies for drug research early following their initial introduction. Although metabolomics lacked behind in this development, it has now become an accepted and widely applied approach in early drug development. Over the past few decades, metabolomics has evolved from a pure exploratory tool to a more mature and quantitative biochemical technology. Several metabolomics-based platforms are now applied during the early phases of drug discovery. Metabolomics analysis assists in the definition of the physiological response and target engagement (TE) markers as well as elucidation of the mode of action (MoA) of drug candidates under investigation. In this review, we highlight recent examples and novel developments of metabolomics analyses applied during early drug development. Show less
Chinello, C.; Haan, N. de; Capitoli, G.; Trezzi, B.; Radice, A.; Pagani, L.; ... ; Magni, F. 2022
The podocyte injury, and consequent proteinuria, that characterize the pathology of idiopathic membranous nephropathy (IMN) is mediated by an autoimmune reaction against podocyte antigens. In... Show moreThe podocyte injury, and consequent proteinuria, that characterize the pathology of idiopathic membranous nephropathy (IMN) is mediated by an autoimmune reaction against podocyte antigens. In particular, the activation of pathways leading to abundant renal deposits of complement is likely to involve the binding of mannose-binding lectin (MBL) to aberrant glycans on immunoglobulins. To obtain a landscape of circulatory IgG Fc glycosylation characterizing this disease, we conducted a systematic N-glycan profiling study of IgG1, 2, and 4 by mass spectrometry. The cohort included 57 IMN patients, a pathological control group with nephrotic syndrome (PN) (n = 20), and 88 healthy control subjects. The effect of sex and age was assessed in all groups and controlled by rigorous matching. Several IgG Fc glycan traits were found to be associated with IMN. Interestingly, among them, only IgG4-related results were specific for IMN and not for PN. Hypo-galactosylation of IgG4, already shown for IMN, was observed to occur in the absence of core fucose, in line with a probable increase of pro-inflammatory IgG. In addition, elevated levels of fucosylated IgG4, along with low levels of hybrid-type glycans, were detected. Some of these IgG4 alterations are likely to be more pronounced in high PLA2R (phospholipase A2 receptor) patients. IgG Fc glycosylation patterns associated with IMN warrant further studies of their role in disease mechanisms and may eventually enrich the diagnostic spectrum regarding patient stratification. Show less
Developments in mass spectrometry (MS)-based analyses of glycoproteins have been important to study changes in glycosylation related to disease. Recently, the characteristic pattern of oxonium ions... Show moreDevelopments in mass spectrometry (MS)-based analyses of glycoproteins have been important to study changes in glycosylation related to disease. Recently, the characteristic pattern of oxonium ions in glycopeptide fragmentation spectra had been used to assign different sets of glycopeptides. In particular, this was helpful to discriminate between O-GalNAc and O-GlcNAc. Here, we thought to investigate how such information can be used to examine quantitative proteomics data. For this purpose, we used tandem mass tag (TMT)-labeled samples from total cell lysates and secreted proteins from three different colorectal cancer cell lines. Following automated glycopeptide assignment (Byonic) and evaluation of the presence and relative intensity of oxonium ions, we observed that, in particular, the ratio of the ions at m/z 144.066 and 138.055, respectively, could be used to discriminate between O-GlcNAcylated and O-GalNAcylated peptides, with concomitant relative quantification between the different cell lines. Among the O-GalNAcylated proteins, we also observed anterior gradient protein 2 (AGR2), a protein which glycosylation site and status was hitherto not well documented. Using a combination of multiple fragmentation methods, we then not only assigned the site of modification, but also showed different glycosylation between intracellular (ER-resident) and secreted AGR2. Overall, our study shows the potential of broad application of the use of the relative intensities of oxonium ions for the confident assignment of glycopeptides, even in complex proteomics datasets. Show less
Lindstrom, M.; Schinkelshoek, M.; Tienari, P.J.; Kukkonen, J.P.; Renkonen, R.; Fronczek, R.; ... ; Itkonen, O. 2021
Background: Orexin-A and-B are neuropeptides involved in sleep-wake regulation. In human narcolepsy type 1, this cycle is disrupted due to loss of orexin-producing neurons in the hypothalamus.... Show moreBackground: Orexin-A and-B are neuropeptides involved in sleep-wake regulation. In human narcolepsy type 1, this cycle is disrupted due to loss of orexin-producing neurons in the hypothalamus. Cerebrospinal fluid (CSF) orexin-A measurement is used in the diagnosis of narcolepsy type 1. Currently available immunoassays may lack specificity for accurate orexin quantification. We developed and validated a liquid chromatography mass spectrometry assay (LC-MS/MS) for CSF orexin-A and B.Methods: We used CSF samples from narcolepsy type 1 (n = 22) and type 2 (n = 6) and non-narcoleptic controls (n = 44). Stable isotope-labeled orexin-A and-B internal standards were added to samples before solid-phase extraction and quantification by LC-MS/MS. The samples were also assayed by commercial radioimmunoassay (RIA, n = 42) and enzymatic immunoassay (EIA, n = 72) kits. Stability of orexins in CSF was studied for 12 months.Results: Our assay has a good sensitivity (10 pmol/L = 35 pg/mL) and a wide linear range (35-3500 pg/mL). Added orexin-A and-B were stable in CSF for 12 and 3 months, respectively, when frozen. The median orexin-A concentration in CSF from narcolepsy type 1 patients was < 35 pg/mL (range < 35-131 pg/mL), which was lower than that in CSF from control individuals (98 pg/mL, range < 35-424 pg/mL). Orexin-A concentrations determined using our LC-MS/MS assay were five times lower than those measured with a commercial RIA. Orexin-B concentrations were undetectableConclusions: Orexin-A concentrations measured by our LC-MS/MS assay were lower in narcolepsy type 1 patients as compared to controls. RIA yielded on average higher concentrations than LC-MS/MS. Show less
Tissues and biofluids are important sources of information used for the detection of diseases and decisions on patient therapies. There are several accepted methods for preservation of tissues,... Show moreTissues and biofluids are important sources of information used for the detection of diseases and decisions on patient therapies. There are several accepted methods for preservation of tissues, among which the most popular are fresh-frozen and formalin-fixed paraffin embedded methods. Depending on the preservation method and the amount of sample available, various specific protocols are available for tissue processing for subsequent proteomic analysis. Protocols are tailored to answer various biological questions, and as such vary in lysis and digestion conditions, as well as duration. The existence of diverse tissue-sample protocols has led to confusion in how to choose the best protocol for a given tissue and made it difficult to compare results across sample types. Here, we summarize procedures used for tissue processing for subsequent bottom-up proteomic analysis. Furthermore, we compare protocols for their variations in the composition of lysis buffers, digestion procedures, and purification steps. For example, reports have shown that lysis buffer composition plays an important role in the profile of extracted proteins: the most common are tris(hydroxymethyl)aminomethane, radioimmunoprecipitation assay, and ammonium bicarbonate buffers. Although, trypsin is the most commonly used enzyme for proteolysis, in some protocols it is supplemented with Lys-C and/or chymotrypsin, which will often lead to an increase in proteome coverage. Data show that the selection of the lysis procedure might need to be tissue-specific to produce distinct protocols for individual tissue types. Finally, selection of the procedures is also influenced by the amount of sample available, which range from biopsies or the size of a few dozen of mm(2) obtained with laser capture microdissection to much larger amounts that weight several milligrams. Show less
In this thesis, methods were developed, and antibody glycosylation was characterized in order to further the clinical application of antibody glycosylation analysis. New mass spectrometric... Show moreIn this thesis, methods were developed, and antibody glycosylation was characterized in order to further the clinical application of antibody glycosylation analysis. New mass spectrometric workflows were introduced in Chapters 2 and 8. Chapter 7 showed the differential characteristics of murine IgG glycosylation of different strains and highlighted the profound differences between humans and mice, with regard to IgG glycosylation. Chapters 4 and 8 showed the use of controlled human situations to study the regulatory mechanisms of IgG and IgA glycosylation. Finally, Chapters 3, 5 and 6, identified glycosidic differences with specified (patho)physiological conditions, which might be exploited for patient stratification in the future. Show less
Haan, N. de; Reiding, K.R.; Kristic, J.; Ederveen, A.L.H.; Lauc, G.; Wuhrer, M. 2017
Glycosylation of immunoglobulins is suspected to play a key role in the regulation of the immune system. In this thesis, mass spectrometry-based glycoproteomics methods were used to characterize... Show moreGlycosylation of immunoglobulins is suspected to play a key role in the regulation of the immune system. In this thesis, mass spectrometry-based glycoproteomics methods were used to characterize the glycosylation of various immunoglobulins. In Chapter 2 we describe the development of a glycoproteomics method to analyze IgE glycosylation. In Chapter 3 we reported partial O-glycosylation of IgG3. In addition to structural glycosylation research, we also analyzed antibody glycosylation in population cohorts. In Chapter 4 and Chapter 5, IgG Fc glycopeptide analysis was performed on blood samples using LC-MS(/MS). In a cohort of 76 ANCA vasculitis patients, low galactosylation and sialylation of IgG was associated with a higher chance of future relapse. Furthermore, in the approximately 1800 participants of the Leiden Longevity study (LLS), low galactosylation and sialylation of IgG, together with high fucosylation, showed association with markers of inflammation. We hope that the novel data presented in this thesis may contribute to the elucidation of the role of antibody glycosylation in the immune system, of which the understanding is currently still very limited. Show less
Urinary tract infection is a complex clinical entity a common infectious disease that encompasses a variety of clinical syndromes with a positive bacterial culture as common denominator. This... Show moreUrinary tract infection is a complex clinical entity a common infectious disease that encompasses a variety of clinical syndromes with a positive bacterial culture as common denominator. This thesis provides an exhaustive exploratory study of the metabolic pattern of patients affected by urinary tract infection and Here this complex clinical entity was investigated with a multiplatform approach. Each of the used platforms added a unique perspective to the further understanding of the infection process. The assessment of the bacterial growth (NMR), of the host response (LC__MS) and of the physiological status (GC-APCI-MS) could eventually be useful during the assessment of the disease severity and/or decision making Show less
Reusch, D.; Haberger, M.; Kailich, T.; Heidenreich, A.K.; Kampe, M.; Bulau, P.; Wuhrer, M. 2014
