Analysis of Oligosaccharides via Ion Mobility-Mass Spectrometry

Complex carbohydrates, also referred to as glycans, are ubiquitous in nature and represent one of the major classes of biopolymers. In contrast to peptides and oligonucleotides whose structure is directly related to specific genes, glycans are not directly encoded in the DNA. Instead, their structure is the result of a dynamic biosynthetic pathway that is heavily affected by environmental factors. Consequently, glycans are extremely diverse and exhibit branched sites, as well as a complex regio- and stereochemistry. A common way to address this complexity is liquid chromatography (LC) in combination with spectroscopic and mass spectrometric (MS) detection. It allows for high throughput measurements and can identify the general building block composition of a glycan, but it often fails to unambiguously assign the linkage between building blocks. The type of linkage, however, is a key factor for the three-dimensional structure of the glycan and therefore its biological functions. A promising alternative, which is sensitive to the globular structure of a glycan, is ion mobility-mass spectrometry (IM-MS). In addition to the separation based on the mass-to-charge (m/z) ratio, IM-MS allows to distinguish ions based on their size, shape, and charge. LC-MS and IM-MS both showed enormous potential for the analysis of glycans as standalone techniques, each providing a different level of information. However, there are very few examples of combining both approaches into a consistent LC-IM-MS workflow. In this thesis, it was investigated if IM-MS can be hyphenated to classical LC-MS workflows to enable a comprehensive structural elucidation of complex N-glycans. As sample preparation for LC-MS usually includes the modification of the reducing end of the glycan, the first step was to study the effect of these derivatizations on the quality of IMS separation. The problem was addressed by the systematic study of a set of six isomeric fucose-containing blood group antigens that are derivatized with the most common fluorescent tags using IMS. The quality of the separation was evaluated by comparing the CCS values of all species in positive and negative ion mode as well as with adduct ions. Afterwards, the application of LC-IMS-MS for the investigation of complex N-glycans was demonstrated. For this purpose, the glycans from human alpha-1-acid glycoprotein (hAGP) were investigated to identify the sialylation (α2,3- vs α2,6-linked residues) and fucosylation (core vs terminal) patterns. It was shown that IMS enables the structural elucidation of even highly sialylated glycans up to tetraantennary species without changing the sample preparation and within a single LC run. The main parameters that are obtained from LC-IMS-MS measurements are retention times, mass-to-charge-ratios, and drift times, which represent a powerful dataset to identify glycan isomers. However, retention times in LC and drift times in IMS depend significantly on instrumental parameters and are difficult to compare between experiments. We therefore introduced an internal calibration method for the conversion of retention times into universal glucose units (GU) and drift times into collisional cross-sections (CCS). It was shown that the internal calibration approach enabled a faster, more accurate analysis for both LC and IMS without loss in calibration accuracy or informational content. Overall, the addition of IMS into classical LC-MS workflows is straightforward and only requires minor adaptions in sample preparation and data treatment. It enables a fast and accurate identification of structural motifs and complements the informational content of LC-MS experiments. LC-IM-MS therefore represents a powerful combinational approach for the comprehensive analysis of complex N-glycans.Komplexe Kohlenhydrate, auch als Glykane bezeichnet, sind in der Natur allgegenwärtig und stellen eine der Hauptklassen von Biopolymeren dar. Im Gegensatz zu Peptiden und Oligonukleotiden, deren Struktur in direktem Zusammenhang mit bestimmten Genen steht, sind Glykane nicht direkt in der DNA kodiert. Stattdessen ist ihre Struktur das Ergebnis einer dynamischen und komplexen Biosynthese, der stark von Umweltfaktoren beeinflusst wird. Folglich sind Glykane äußerst vielfältig und weisen verzweigte Stellen sowie eine komplexe Regio- und Stereochemie auf. Ein gängiger Weg, um dieser Komplexität zu begegnen, ist die Flüssigchromatographie (LC) in Kombination mit spektroskopischer und massenspektrometrischer (MS) Detektion. Es ermöglicht Messungen mit hohem Durchsatz und kann die allgemeine Zusammensetzung eines Glykans identifizieren, kann jedoch häufig die Verknüpfung zwischen Bausteinen nicht eindeutig zuordnen. Die Art der Verknüpfung ist jedoch ein Schlüsselfaktor für die dreidimensionale Struktur eines Glykans und damit mitverantwortlich für seine biologischen Funktionen. Eine vielversprechende Alternative, die sensitiv für die globuläre Struktur eines Glykans ist, ist die Ionenmobilitäts-Massenspektrometrie (IM-MS). Zusätzlich zur Trennung auf Basis des Masse-zu-Ladungsverhältnis (m/z) ermöglicht IM-MS die Unterscheidung von Ionen basierend auf ihrer Größe, Form und Ladung. LC-MS und IM-MS zeigten beide großes Potenzial für die Analyse von Glykanen als eigenständige Techniken, die jeweils eine andere Informationsebene liefern. Es gibt jedoch nur sehr wenige Beispiele für die Kombination beider Ansätze zu einem konsistenten LC-IM-MS Arbeitsablauf. In dieser Arbeit wurde untersucht, ob IM-MS mit klassischen LC-MS Arbeitsabläufen verknüpft werden kann, um eine umfassende Strukturaufklärung komplexer N-Glykane zu ermöglichen. Da die Probenvorbereitung für LC-MS normalerweise die Modifikation des reduzierenden Endes am Glykan beinhaltet, bestand der erste Schritt darin, die Auswirkung dieser Derivatisierungen auf die Qualität der IMS-Trennung zu untersuchen. Das Problem wurde durch die systematische Untersuchung eines Satzes von sechs isomeren Blutgruppen Antigenen angegangen, die mit den gängigsten Fluoreszenzmarkern derivatisiert wurden und anschließend mittels IMS gemessen wurden. Die Qualität der Trennung wurde durch den Vergleich der CCS-Werte aller Spezies im positiven und negativen Ionenmodus sowie mit Addukt-Ionen bewertet. Anschließend wurde die Anwendung von LC-IMS-MS zur Untersuchung komplexer N-Glykane demonstriert. Zu diesem Zweck wurden die Glykane aus menschlichem alpha-1-acid Glykoprotein (hAGP) untersucht, um das Sialylierungs- (α2,3- vs. α2,6-Verknüpfung) und Fucosylierungsmuster (am reduzierenden Ende vs. terminal) zu identifizieren. Es konnte gezeigt werden, dass IMS die Strukturaufklärung selbst hochsialylierter Glykane bis hin zu tetraantennären Spezies ohne Änderung der Probenvorbereitung und innerhalb eines einzigen LC-Laufs ermöglicht. Die Hauptparameter, die aus LC-IMS-MS-Messungen erhalten werden, sind Retentionszeiten, Masse-zu-Ladungsverhältnisse und Driftzeiten, die eine hohe Informationsdichte zur Identifizierung von Glykanisomeren darstellen. Retentionszeiten in LC und Driftzeiten in IMS hängen jedoch erheblich von instrumentellen Parametern ab und sind zwischen Experimenten nur schwer zu vergleichen. Wir haben daher ein internes Kalibrierverfahren eingeführt, dass die Umrechnung von Retentionszeiten in universelle Glucoseeinheiten (GU) und von Driftzeiten in Kollisionsquerschnitte (CCS) ermöglicht. Es wurde gezeigt, dass dies eine schnellere und genauere Analyse sowohl für LC als auch für IMS ohne Verlust der Kalibrierungsgenauigkeit oder des Informationsgehalts ermöglicht. Insgesamt ist die Integration von IMS in klassische LC-MS-Workflows unkompliziert und erfordert nur geringfügige Anpassungen bei der Probenvorbereitung und Datenverarbeitung. Es ermöglicht eine schnelle und genaue Identifizierung von Strukturmotiven und ergänzt den Informationsgehalt von LC-MS-Experimenten. LC-IM-MS stellt daher einen leistungsstarken kombinatorischen Ansatz für die umfassende Analyse komplexer N-Glykane dar

Synchronization of Directory Services with the Event Propagation Framework

This case study introduces the most relevant directory service standard LDAP and the approach how to synchronize these directory services using the Event Propagation Framework (EPF) of iC Consult to establish a cooperate directory service as it is done at Daimler Chrysler

Towards Integrated Variant Management in Global Software Engineering: An Experience Report

In the automotive domain, customer demands and market constraints are progressively realized by electric/ electronic components and corresponding software. Variant traceability in SPL is crucial in the context of different tasks, like change impact analysis, especially in complex global software projects. In addition, traceability concepts must be extended by partly automated variant configuration mechanisms to handle restrictions and dependencies between variants. Such variant configuration mechanism helps to reduce complexity when configuring a valid variant and to establish an explicit documentation of dependencies between components. However, integrated variant management has not been sufficiently addressed so far. Especially, the increasing number of software variants requires an examination of traceable and configurable software variants over the software lifecycle. This paper emphasizes variant traceability achievements in a large global software engineering project, elaborates existing challenges, and evaluates an industrial usage of an integrated variant management based on experiences

An Approach to Detect the Origin and Distribution of Software Defects in an Evolving Cyber-Physical System

Cyber-Physical Systems (CPS) are usually developed by an incremental approach. A changing environment like demanding user requirements or legislation amendments lead often to multiple development paths in an evolving CPS. Hence, software variability plays an increasingly important role adapting the characteristics of such CPS to different contexts. This paper focuses on software variability realized through a Software Product Line (SPL) more specifically. Thereby, variability and evolution are usually managed in different tools. However with respect to software defects, a holistic handling of variability and evolution is necessary to ensure a reliable software defect removal. Particularly, detecting software defects in different evolution stages and derived variants is ordinary, but complex and error-prone. To close the gap between variability and evolution, this paper presents a systematic approach to combine both disciplines. In particular, we apply existing variant management techniques in combination with software configuration management methods to determine a software defect's origin and distribution in an evolving SPL. We apply our approach to a CPS from the automotive domain to show its industrial relevance and usefulness

The role of the mobile proton in fucose migration

Fucose migration reactions represent a substantial challenge in the analysis of fucosylated glycan structures by mass spectrometry. In addition to the well-established observation of transposed fucose residues in glycan-dissociation product ions, recent experiments show that the rearrangement can also occur in intact glycan ions. These results suggest a low-energy barrier for migration of the fucose residue and broaden the relevance of fucose migration to include other types of mass spectrometry experiments, including ion mobility-mass spectrometry and ion spectroscopy. In this work, we utilize cold-ion infrared spectroscopy to provide further insight into glycan scrambling in intact glycan ions. Our results show that the mobility of the proton is a prerequisite for the migration reaction. For the prototypical fucosylated glycans Lewis x and blood group antigen H-2, the formation of adduct ions or the addition of functional groups with variable proton affinity yields significant differences in the infrared spectra. These changes correlate well with the promotion or inhibition of fucose migration through the presence or absence of a mobile proton

Herausforderungen an ein durchgängiges Variantenmanagement in Software-Produktlinien und die daraus resultierende Entwicklungsprozessadaption

In der Automobilindustrie werden Kundenwünsche zunehmend mittels Elektrik/Elektronik-Komponenten und zugehöriger Software realisiert. Dies führt in der fortlaufenden Entwicklung zu einer steigenden Komplexität und Variabilität der Software. Software-Produktlinien (SPL) beschreiben eine Methodik, um solch variantenreiche Softwaresysteme zu beherrschen. Ein durchgängiges Variantenmanagement betrifft sämtliche Entwicklungsphasen und deren Abstraktionsebenen. So ermöglicht es ein verbessertes Tracing, zielgenaue Change-Impact-Analysen und durchgängige Fehlerbeseitigungen. Um die Anforderungen an ein durchgängiges Variantenmanagement besser zu verstehen, untersuchten wir eine existierende SPL und fanden voneinander isoliert erstellte Merkmalmodelle vor. Als Ursachen hierfür lassen sich u. a. differenzierte Sichtweisen auf Softwareproduktvarianten in der Entwicklung sowie die fehlende Prozessverankerung der Merkmalmodellierung ausmachen. Eine Harmonisierung der isoliert erstellten Merkmalmodelle gestaltet sich aufwändig und erschwert ein durchgängiges Variantenmanagement.In diesem Beitrag werden industrielle Herausforderungen zum Erreichen eines durchgängigen Variantenmanagement in der Praxis erläutert. Ziel ist es, bestehende Merkmalmodelle zu harmonisieren und Assoziationen zwischen Merkmalen über die gesamten Entwicklungsphasen und den vorhandenen Abstraktionsebenen herzustellen. Für eine standardisierte Merkmalmodellierung wird zudem eine Adaption des Entwicklungsprozesses beschrieben

Shotgun ion mobility mass spectrometry sequencing of heparan sulfate saccharides

Despite evident regulatory roles of heparan sulfate (HS) saccharides in numerous biological processes, definitive information on the bioactive sequences of these polymers is lacking, with only a handful of natural structures sequenced to date. Here, we develop a “Shotgun” Ion Mobility Mass Spectrometry Sequencing (SIMMS2) method in which intact HS saccharides are dissociated in an ion mobility mass spectrometer and collision cross section values of fragments measured. Matching of data for intact and fragment ions against known values for 36 fully defined HS saccharide structures (from di- to decasaccharides) permits unambiguous sequence determination of validated standards and unknown natural saccharides, notably including variants with 3O-sulfate groups. SIMMS2 analysis of two fibroblast growth factor-inhibiting hexasaccharides identified from a HS oligosaccharide library screen demonstrates that the approach allows elucidation of structure-activity relationships. SIMMS2 thus overcomes the bottleneck for decoding the informational content of functional HS motifs which is crucial for their future biomedical exploitation

Artery tertiary lymphoid organs control multi-layered territorialized atherosclerosis B cell responses in aged ApoE-/- mice

Objective: Explore aorta B cell immunity in aged ApoE-/- mice. Approach and Results: Transcript maps, FACS, immunofluorescence analyses, cell transfers, and Ig-ELISPOT assays showed multi-layered atherosclerosis B cell responses in artery tertiary lymphoid organs (ATLOs). Aging-associated aorta B cell-related transcriptomes were identified and transcript atlases revealed highly territorialized B cell responses in ATLOs versus atherosclerotic lesions: ATLOs showed upregulation of bona fide B cell genes including Cd19, Ms4a1 (Cd20), Cd79a/b, and Ighm though intima plaques preferentially expressed molecules involved in non-B effector responses towards B cell-derived mediators, i.e. Fcgr3 (Cd16), Fcer1g (Cd23), and the C1q family. ATLOs promoted B cell recruitment. ATLO B-2 B cells included naïve, transitional, follicular, germinal center, switched IgG1+, IgA+, and IgE+ memory cells, plasmablasts, and long-lived plasma cells (PCs). ATLOs recruited large numbers of B-1 cells whose subtypes were skewed towards IL-10+ B-1b cells versus IL-10- B-1a cells. ATLO B-1 cells and PCs constitutively produced IgM and IgG and a fraction of PCs expressed IL-10. Moreover, ApoE-/- mice showed increased germinal center B cells in renal lymph nodes, IgM-producing PCs in the bone marrow, and higher IgM and anti-MDA-LDL IgG serum titers. Conclusions: ATLOs orchestrate dichotomic, territorialized, and multi-layered B cell responses in the diseased aorta; germinal center reactions indicate generation of autoimmune B cells within the diseased arterial wall during aging

Fulminant Cardiotoxicity in a Patient With Cardiac Lymphoma Treated With CAR-T Cells

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Value of fecal calprotectin in the evaluation of patients with abdominal discomfort: an observational study

<p>Abstract</p> <p>Background</p> <p>The evaluation of patients with abdominal discomfort is challenging and patient selection for endoscopy based on symptoms is not reliable. We evaluated the diagnostic value of fecal calprotectin in patients with abdominal discomfort.</p> <p>Methods</p> <p>In an observational study, 575 consecutive patients with abdominal discomfort referred for endoscopy to the Department of Gastroenterology & Hepatology at the University Hospital Basel in Switzerland, were enrolled in the study. Calprotectin was measured in stool samples collected within 24 hours before the investigation using an enzyme-linked immunosorbent assay. The presence of a clinically significant finding in the gastrointestinal tract was the primary endpoint of the study. Final diagnoses were adjudicated blinded to calprotectin values.</p> <p>Results</p> <p>Median calprotectin levels were higher in patients with significant findings (N = 212, median 97 μg/g, IQR 43-185) than in patients without (N = 326, 10 μg/g, IQR 10-23, P < 0.001). The area under the receiver operating characteristics curve (AUC) to identify a significant finding was 0.877 (95% CI, 0.85-0.90). Using 50 μg/g as cut off yielded a sensitivity of 73% and a specificity of 93% with good positive and negative likelihood ratios (10.8 and 0.29, respectively). Fecal calprotectin was useful as a diagnostic parameter both for findings in the upper intestinal tract (AUC 0.730, 0.66-0.79) and for the colon (AUC 0.912, 0.88-0.94) with higher diagnostic precision for the latter (P < 0.001). In patients > 50 years, the diagnostic precision remained unchanged (AUC 0.889 vs. 0.832, P = 0.165).</p> <p>Conclusion</p> <p>In patients with abdominal discomfort, fecal calprotectin is a useful non-invasive marker to identify clinically significant findings of the gastrointestinal tract, irrespective of age.</p