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In-Depth Mass Spectrometry-Based Glycoproteomics: Advances in Sample Preparation, Measurement and Data-Analysis of Glycoproteins

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In-Depth Mass Spectrometry-Based Glycoproteomics: Advances in Sample Preparation, Measurement and Data-Analysis of Glycoproteins In-Depth Mass Spectrometry-Based Glycoproteomics: Advances in Sample Preparation, Measurement and Data-Analysis of Glycoproteins Dissertation zur Erlangung des akademischen Grades Doktoringenieur (Dr.-Ing.) von: Dipl.-Ing. Marcus Hoffmann geboren am: 21. Oktober 1984 in Aschersleben genehmigt durch die Fakultät für Verfahrens- und Systemtechnik der Otto-von-Guericke Universität Magdeburg Promotionskommission: Prof. Dr. rer. nat. habil. Helmut Weiß (Vorsitz) Prof. Dr.-Ing. Udo Reichl (Gutachter) Dr. rer. nat. Erdmann Rapp (Gutachter) Prof. Dr. rer. nat. Manfred Wuhrer (Gutachter) eingereicht am: 26. November 2019 vorgelegte Dissertation Promotionskolloquium am: 29. Juni 2020 Abstract Protein glycosylation is a universal and essential feature that is prominent in all domains of life (eukarya, bacteria, archaea, viruses). The co- and posttranslational attachment of glycans to specific amino acids may thereby profoundly alter the biophysical characteristics, and with this also the functioning of the proteins they are bound to. This may include structural and modulatory effects, such as proper protein folding or protection from proteolytic digestion, along with extrinsic and intrinsic recognition effects, such as recognition of antigens, protein trafficking and turnover, intra- and extracellular signaling and adhesion, as well as molecular mimicry. Protein glycosylation therefore plays an important role in many physiological but also pathophysiological processes. To understand the functional implications of the protein glyco- sylation and to take advantage of these insights for clinical diagnosis and prognosis, as well as for biopharmaceutical and vaccine development and quality control, the continuous development and improvement of glycoanalytical methods and workflows is required. This thesis describes the development of two mass spectrometry-based glycoproteomic workflows that enable the in-depth and site-specific analysis of N- and O-glycoproteins. Further, the results of applying of those workflows for the glycoproteomic analysis of clinically and bio- pharmaceutically relevant samples are presented. In the first part of this thesis a glycoproteomic workflow, developed for the site-specific O- glycosylation analysis of human blood plasma glycoproteins, is introduced and the results of this analysis are presented and discussed. To this end pooled human blood plasma of healthy donors was proteolytically digested using an enzyme with a broad cleavage specificity (proteinase K), followed by a precipitation step, as well as a glycopeptide enrichment and fractionation step via hydrophilic interaction liquid chromatography (HILIC). The latter was optimized for intact O-glycopeptides carrying short mucin-type core-1 and 2 O-glycans, which represent the vast majority of O-glycans on human blood plasma proteins. Enriched O- glycopeptide fractions were subjected to mass spectrometric analysis using reversed-phase liquid chromatography coupled online to an ion trap (IT) mass spectrometer operated in positive-ion mode. Peptide identity and glycan composition were derived from low-energy collision-induced dissociation fragment ion spectra acquired in multistage mode. To pinpoint the O-glycosylation sites glycopeptides were fragmented using electron transfer dissociation. Acquired fragment ion spectra were annotated by database searches and manual assignment. Overall, 31 O-glycosylation sites and regions belonging to 22 proteins were identified, with the majority being acute-phase proteins. Strikingly, also 11 novel O-glycosylation sites and regions were identified. In total 23 of the 31 O-glycosylation sites/regions could be pinpointed. Interestingly, the use of proteinase K, which cleaves primarily after aliphatic, aromatic and hydrophobic amino acids, proved to be particularly beneficial in this context. The identified O- glycan compositions most probably correspond to mono- and disialylated core-1 mucin-type O-glycans (T-antigen). In summary, the developed workflow allows the identification and characterization of the major population of the human blood plasma O-glycoproteome. I Thereby, these results provide novel insights, which may help to unravel glycosylation-related structure-function relationships, e.g. during glycoproteomic biomarker discovery studies. In the second part of the thesis a glycoproteomic workflow, combining lower and stepped collisional energy fragmentation on an Orbitrap mass spectrometer, is introduced for the in- depth and site-specific analysis of intact N- and O-glycopeptides. Further, using a set of four representative and biopharmaceutically-relevant glycoproteins (immunoglobulin gamma, fibrinogen, lactotransferrin, and ribonuclease B), the benefits and limitations of this workflow are highlighted. For these representative glycoproteins several new glycosylation sites and regions with their respective glycoforms were discovered and characterized – in addition to confirming already known glycosylation sites and glycoforms (elucidation of glycan micro- and macroheterogeneity). Moreover, for the enrichment of glycopeptides a modified and improved version of a cotton HILIC-based solid phase extraction protocol is described. For the unambiguous identification of N-glycopeptides the use of a conserved fragmentation signature 0,2 + [Mpeptide+H+ X GlcNAc] , that has rarely been employed in Orbitrap-based glycoproteomic analyses up to now, is proposed. This signature represents a valuable indicator for the determination of the correct peptide mass and is particularly important when analyzing glycopeptides obtained by proteases with broad or no cleavage specificity (e.g. glycopeptides generated by proteinase K digest). In this thesis, it is shown for the first time that this fragmentation signature can consistently be found across all N-glycopeptides analyzed, but not for O-glycopeptides. Moreover, we have systematically and comprehensively evaluated the use of the relative abundance of oxonium ions to retrieve glycan structure information, e.g. differentiation of hybrid- and high-mannose-type N-glycans, differentiation between antenna GlcNAc and bisecting GlcNAc, or differentiation between N-glycopeptides and mucin-type O- glycopeptides. These new findings may increase confidence and comprehensiveness in manual and software-assisted glycoproteomics. Overall, the developed analytical workflow along with the analyzed representative glycoproteins and the given insights into diagnostic glycopeptide fragment ions serve as a guide to in-depth glycoproteomic analysis for a broad range of glycoproteins. The workflow may therefore be beneficial to basic and clinical glycoproteomic research and diagnostics, but also to biopharmaceutical research and process development as well as quality control. In summary, in this thesis two mass spectrometry-based glycoproteomic workflows were developed and applied which enable the in-depth and site-specific analysis of N- and O- glycoproteins. Thereby, several advances in sample preparation, measurement and data- analysis were achieved that increase the confidence, depth and comprehensiveness of glycoproteomic analyses. II Kurzfassung Die Protein-Glykosylierung ist ein universelles und essentielles Merkmal, das in allen Organismen (Eukarya, Bakteria, Archaea), einschließlich Viren, zu finden ist. Die ko- und posttranslationale Bindung von Glykanen an bestimmte Aminosäuren kann dabei die biophysikalischen Eigenschaften und damit auch die Funktion der Proteine, an die sie gebunden sind, grundlegend verändern. Dies schließt strukturelle und modulatorische Effekte, wie z.B. die richtige Proteinfaltung oder den Schutz vor Proteasen, aber auch extrinsische und intrinsische Erkennungseffekte wie z.B. die Erkennung von Antigenen, sowie intra- und extrazelluläre Signalgebung, Adhäsion oder molekulare Mimikry mit ein. Die Glykosylierung von Proteinen spielt daher eine wesentliche Rolle in einer Vielzahl physiologischer sowie pathophysiologischer Prozesse. Um die funktionellen Auswirkungen der Proteinglyko- sylierung zu verstehen und diese Erkenntnisse für die klinische Diagnose und Prognose sowie für die Entwicklung von Biopharmazeutika und Impfstoffen und deren Qualitätskontrolle nutzen können, ist die kontinuierliche Entwicklung und Verbesserung glykoanalytischer Methoden und Arbeitsabläufe erforderlich. Diese Dissertation beschreibt die Entwicklung zweier Arbeitsabläufe für die detaillierte und ortsspezifische Glykoproteom-Analyse von N- und O-Glykoproteinen mittels Massenspektrometrie (MS). Ferner wird die Anwendung beider Arbeitsabläufe für die Glykoproteom-Analyse von klinisch- und biopharmazeutisch-relevanten Proben gezeigt. Im ersten Teil der Arbeit wird die ortsspezifische O-Glykoproteom-Analyse von Glykoproteinen aus menschlichem Blutplasma beschrieben. Zu diesem Zweck wurde menschliches Blutplasma gesunder Spender unter Verwendung eines proteolytischen Enzyms mit breiter Spezifität (Proteinase K) proteolytisch verdaut, gefolgt von einem Präzipitationsschritt sowie einem Glykopeptid-Anreicherungs- und Fraktionierungsschritt mittels hydrophiler Interaktionsflüssigkeitschromatographie (HILIC). Letzterer wurde für intakte O-Glycopeptide mit O-Glykanen vom Mucin-Typ (Core 1 und 2) optimiert, welche die große Mehrheit der O-Glykane auf menschlichen Blutplasmaproteinen darstellen. Die O- Glykopeptid-angereicherten Fraktionen wurden einer massenspektrometrischen Analyse unterzogen. Die O-Glykopeptide wurden dabei unter Verwendung von Umkehrphasen- Flüssigkeitschromatographie aufgetrennt
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