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Medulloblastomas (MBs) are malignant pediatric brain tumors that are molecularly and clinically heterogenous. The application of omics technologies—mainly studying nucleic acids—has significantly improved MB classification and stratification, but treatment options are still unsatisfactory. The proteome and their N-glycans hold the potential to discover clinically relevant phenotypes and targetable pathways. We compile a harmonized proteome dataset of 167 MBs and integrate findings with DNA methylome, transcriptome and N-glycome data. We show six proteome MB subtypes, that can be assigned to two main molecular programs: transcription/translation (pSHHt, pWNT and pG3myc), and synapses/immunological processes (pSHHs, pG3 and pG4). Multiomic analysis reveals different conservation levels of proteome features across MB subtypes at the DNA methylome level. Aggressive pGroup3myc MBs and favorable pWNT MBs are most similar in cluster hierarchies concerning overall proteome patterns but show different protein abundances of the vincristine resistance-associated multiprotein complex TriC/CCT and of N-glycan turnover-associated factors. The N-glycome reflects proteome subtypes and complex-bisecting N-glycans characterize pGroup3myc tumors. Our results shed light on targetable alterations in MB and set a foundation for potential immunotherapies targeting glycan structures. Medulloblastomas (MBs) are highly heterogeneous paediatric brain tumours that remain challenging to treat. Here, the authors integrate proteomics, epigenomics, transcriptomics and post-translational modification analyses to find molecular subgroups and potential therapeutic targets in MB tumours.

The glomerular filtration barrier (GFB) produces primary urine and is composed of a fenestrated endothelium, a glomerular basement membrane (GBM), podocytes, and a slit diaphragm. Impairment of the GFB leads to albuminuria and microhematuria. The GBM is generated via secreted proteins from both endothelial cells and podocytes and is supposed to majorly contribute to filtration selectivity. While genetic mutations or variations of GBM components have been recently proposed to be a common cause of glomerular diseases, pathways modifying and stabilizing the GBM remain incompletely understood. Here, we identified prolyl 3-hydroxylase 2 (P3H2) as a regulator of the GBM in an a cohort of patients with albuminuria. P3H2 hydroxylates the 3' of prolines in collagen IV subchains in the endoplasmic reticulum. Characterization of a P3h2ΔPod mouse line revealed that the absence of P3H2 protein in podocytes induced a thin basement membrane nephropathy (TBMN) phenotype with a thinner GBM than that in WT mice and the development of microhematuria and microalbuminuria over time. Mechanistically, differential quantitative proteomics of the GBM identified a significant decrease in the abundance of collagen IV subchains and their interaction partners in P3h2ΔPod mice. To our knowledge, P3H2 protein is the first identified GBM modifier, and loss or mutation of P3H2 causes TBMN and focal segmental glomerulosclerosis in mice and humans.

In recent years, there has been a significant increase in the number of mass spectrometry-based spatial omics studies. Considering the spatial resolution allows for a deeper understanding of the cellular organization and tissue interactions, whereby physiological and pathological processes can be investigated more comprehensively. Despite the growing demand, there is currently a lack of suitable workflows in which both the spatial information in the tissue is retained during sampling and comprehensive coverage is achieved in the subsequent proteome and lipidome analysis. A novel approach utilizes short pulse midinfrared laser ablation for the simultaneous sampling and homogenization of tissues. Biomolecules are released from their cellular tissue compartments via a gentle and cold vaporization process. Moreover, this technology preserves the spatial information of the sample location and provides highly precise sampling by maintaining the integrity of the surrounding tissue area. In this dissertation, the methodology of nanosecond infrared laser (NIRL)-based tissue sampling and subsequent processing for low-input mass spectrometric omics was further developed, paving the way for an application in three-dimensional spatial omics. Given that common sample processing workflows for bottom-up proteomics and shotgun lipidomics necessitate the use of tissue amounts of several milligrams for robust processing, workflows were developed enabling the robust processing of miniaturized tissue volumes of 500 nL, corresponding to low input amounts of 500 µg. The implementation of a novel aerosol collection method, as described in the study performed by Hahn & Moritz et al., enabled the successful bottom-up proteome analysis of miniaturized tissue volumes of murine spleen and colon tissue. This resulted in the identification of 1,889 proteins with quantitative information. In comparison to a previous PIRL-based study conducted by our research group, in which a 50-fold larger tissue volume was utilized, a comparable number of proteins was identified. The results of differential quantitative proteome analysis revealed significantly different protein abundances in the tissues displaying expected proteomes of spleen and colon tissue. Accordingly, the applicability of NIRL ablation for soft and muscle-rich tissue types was confirmed. In a further methodological development, NIRL-based tissue sampling was combined with quantitative shotgun lipidome analysis, for the first time. In the study of Stadlhofer & Moritz et al., biopsies from human squamous cell carcinomas of the oropharynx (OPSCC) and surrounding mucosa tissue were ablated by NIRL. In total, 755 lipid species from 13 lipid classes were quantified. The data demonstrated not only intra- but also interpatient alterations in lipid composition. The findings confirm similarities in the lipid profiles of the OPSCC samples from different tumor locations. For instance, the lipid classes phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were found to be present in higher concentrations in the OPSCC samples than in the respective non-tumorous oropharyngeal tissue. In contrast, mucosa samples from the base of tongue and tonsil exhibit distinct lipid compositions. Since comprehensive analysis with high coverage was achieved for both bottom-up proteomics and shotgun lipidomics, the suitability of the developed processing workflows for the sampled tissue volumes was verified and provided the foundation for further development towards spatial omics. In the study of Voß & Moritz et al., NIRL ablation was utilized for the tissue sampling of three different colon parts (ascending, transverse, descending) and for layer-by-layer sampling of eight consecutive intestinal tissue layers.

Significant changes of glycan structures are observed in humans if diseases like cancer, arthritis or inflammation are present. Thus, interest in biomarkers based on glycan structures has rapidly emerged in recent years and monitoring disease specific changes of glycosylation and their quantification is of great interest. Mass spectrometry is most commonly used to characterize and quantify glycopeptides and glycans liberated from the glycoprotein of interest. However, ionization properties of glycopeptides can strongly depend on their composition and can therefore lead to intensities that do not reflect the actual proportions present in the intact glycoprotein. Here we show that an increase in the length of the peptide can lead to a more accurate determination and quantification of the glycans. The four glycosylation sites of human serum ceruloplasmin from 17 different individuals were analyzed using glycopeptides of varying peptide lengths, obtained by action of different proteases and by limited digestion. In most cases, highly sialylated compositions showed an increased relative abundance with increasing peptide length. We observed a relative increase of triantennary glycans of up to a factor of three and, even more, MS peaks corresponding to tetraantennary compositions on ceruloplasmin at glycosite 137N in all 17 samples, which we did not detect using a bottom up approach. The data presented here leads to the conclusion that a middle down - or when possible a top down - approach is favorable for qualitative and quantitative analysis of the glycosylation of glycoproteins.

It was recently shown that ultrashort pulse infrared (IR) lasers, operating at the wavelength of the OH vibration stretching band of water, are highly efficient for sampling and homogenizing biological tissue. In this study we utilized a tunable nanosecond infrared laser (NIRL) for tissue sampling and homogenization with subsequent liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis for mass spectrometric proteomics. For the first time, laser sampling was performed with murine spleen and colon tissue. An ablation volume of 1.1 × 1.1 × 0.4 mm³ (approximately 0.5 µL) was determined with optical coherence tomography (OCT). The results of bottom-up proteomics revealed proteins with significant abundance differences for both tissue types, which are in accordance with the corresponding data of the Human Protein Atlas. The results demonstrate that tissue sampling and homogenization of small tissue volumes less than 1 µL for subsequent mass spectrometric proteomics is feasible with a NIRL.

For investigating the molecular physiology and pathophysiology in organs, the most exact data should be obtained; if not, organ-specific cell lines are analyzed, or the whole organ is homogenized, followed by the analysis of its biomolecules. However, if the morphological organization of the organ can be addressed, then, in the best case, the composition of molecules in single cells of the target organ can be analyzed. Laser capture microdissection (LCM) is a technique which enables the selection of specific cells of a tissue for further analysis of their molecules. However, LCM is a time-consuming two-dimensional technique, and optimal results are only obtained if the tissue is fixed, e.g., by formalin. Especially for proteome analysis, formalin fixation reduced the number of identifiable proteins, and this is an additional drawback. Recently, it was demonstrated that sampling of fresh-frozen (non-fixed) tissue with an infrared-laser is giving higher yields with respect to the absolute protein amount and number of identifiable proteins than conventional mechanical homogenization of tissues. In this study, the applicability of the infrared laser tissue sampling for the proteome analysis of different cell layers of murine intestine was investigated, using LC–MS/MS-based differential quantitative bottom-up proteomics. By laser ablation, eight consecutive layers of colon tissue were obtained and analyzed. However, a clear distinguishability of protein profiles between ascending, descending, and transversal colon was made, and we identified the different intestinal-cell-layer proteins, which are cell-specific, as confirmed by data from the Human Protein Atlas. Thus, for the first time, sampling directly from intact fresh-frozen tissue with three-dimensional resolution is giving access to the different proteomes of different cell layers of colon tissue.

Complete surgical resection is essential for oropharyngeal squamous cell carcinoma (OPSCC) therapy, underscoring the need for improved intraoperative margin assessment. To advance in vivo diagnostics for OPSCC, Nanosecond infrared laser (NIRL) tissue sampling combined with shotgun lipidomic analysis reveals lipidome differences between OPSCC tissue and adjacent healthy tissue. In this study, ablations were performed on tonsil squamous cell carcinoma in 28 samples from 11 patients using an established chamber setup, and a subset of six samples from three patients with a custom‐made laser fiber‐coupled applicator, designed for handheld use. Welch's t ‐test results ( p = 0.05, two‐fold change) revealed a similar OPSCC lipid profile in seven out of 11 patients. Potential tumor lipid markers were identified as consistently and significantly increased, despite the biological heterogeneity of the samples, underscoring their potential diagnostic value. Tissue ablation with fiber‐coupled applicator was successful and the lipidomic analysis was consistent with the chamber setup. While limited to off‐line analysis, our approach highlights the potential of fiber‐based laser sampling as a rapid and minimally invasive method to obtain tissue material for advanced molecular profiling in surgical and endoscopic settings.

Ultrashort pulse infrared lasers can simultaneously sample and homogenize biological tissue using desorption by impulsive vibrational excitation (DIVE). With growing attention on alterations in lipid metabolism in malignant disease, mass spectrometry (MS)-based lipidomic analysis has become an emerging topic in cancer research. In this pilot study, we investigated the feasibility of tissue sampling with a nanosecond infrared laser (NIRL) for the subsequent lipidomic analysis of oropharyngeal tissues, and its potential to discriminate oropharyngeal squamous cell carcinoma (OPSCC) from non-tumorous oropharyngeal tissue. Eleven fresh frozen oropharyngeal tissue samples were ablated. The produced aerosols were collected by a glass fiber filter, and the lipidomes were analyzed with mass spectrometry. Data was evaluated by principal component analysis and Welch’s t-tests. Lipid profiles comprised 13 lipid classes and up to 755 lipid species. We found significant inter- and intrapatient alterations in lipid profiles for tumor and non-tumor samples (p-value < 0.05, two-fold difference). Thus, NIRL tissue sampling with consecutive MS lipidomic analysis is a feasible and promising approach for the differentiation of OPSCC and non-tumorous oropharyngeal tissue and may provide new insights into lipid composition alterations in OPSCC.

Advanced glycation end products (AGEs) accumulate in various tissues, including bone, due to aging and conditions like diabetes mellitus. To investigate the effects of AGEs on bone material quality and biomechanical properties, an in vitro study utilizing human tibial cortex, sectioned into 90 beams, and randomly assigned to three mechanical test groups was performed. Each test group included ribose (c = 0.6 M) treatment at 7-, 14-, and 21-d, alongside control groups (n = 5 per group). Fluorescent AGE (fAGE) and carboxymethyl-lysine (CML) levels were assessed through fluorometric analysis and mass spectrometry, while bone matrix composition was characterized using Fourier-transform infrared and Raman spectroscopy. Mechanical properties were determined through nanoindentation and three-point bending tests on non-notched and notched specimens. The results showed significant increases in fAGEs levels at 7-, 14-, and 21-d compared to controls (119%, 311%, 404%; p = .008, p < .0001, p < .0001, respectively), CML levels also rose substantially compared to controls (383%, 503%, 647%, p < .0001, p < .0001, p < .0001, respectively). Analysis of bone matrix composition showed greater sugars/Amide I ratio at 21-d glycation compared to controls, 7-d, and 14-d (p = .001, .011, .006, respectively); and higher carbonate-to-phosphate ratios in the ribose treatment group compared with controls (p < .05) in the interstitial bone area. Mechanical testing of notched specimens exhibited a higher yield force, pre-yield toughness, and maximum force at 14-d glycation compared to controls and to both 7-d and 21-d glycation (p < .05). Nanoindentation showed that the hardness was lower at 7-d glycation compared to the controls and 21-d glycation (p < .05). In conclusion, the study found altered mechanical properties at 7 and 14 d of glycation, which then returned to control levels at 21 d, indicating a dynamic relationship between glycation duration and mechanical characteristics that deserves further exploration.

Analytes during their journey from their natural sources to their identification and quantification are prone to adsorption to surfaces before they enter an analytical instrument, causing false quantities. This problem is especially severe in diverse omics. Here, thousands of analytes with a broad range of chemical properties and thus different affinities to surfaces are quantified within a single analytical run. For quantifying adsorption effects caused by surfaces of sample handling tools, an assay was developed, applying LC-MS/MS-based differential bottom-up proteomics and as probe a reference mixture of thousands of tryptic peptides, covering a broad range of chemical properties. The assay was tested by investigating the adsorption properties of several vials composed of polypropylene, including low-protein-binding polypropylene vials, borosilicate glass vials and low-retention glass vials. In total 3531 different peptides were identified and quantified across all samples and therefore used as probes. A significant number of hydrophobic peptides adsorbed on polypropylene vials. In contrast, only very few peptides adsorbed to low-protein-binding polypropylene vials. The highest number of peptides adsorbed to glass vials, driven by electrostatic as well as hydrophobic interactions. Calculation of the impact of the adsorption of peptides on differential quantitative proteomics showed significant false results. In summary, the new assay is suitable to characterize adsorption properties of surfaces getting into contact with analytes during sample preparation, thereby giving the opportunity to find parameters for minimizing false quantities.