Development of ultrahigh resolution FTICR mass spectrometry methods for clinical proteomics

Fourier transform ion cyclotron resonance (FTICR) MS provides (a) ultrahigh mass resolving power that allows the analysis of large proteins and complex mixtures; (b) ultrahigh mass measurement accuracy and precision that allow a more reliable identification of the detected species; (c) a wide dynamic range that is favourable for the detection of low abundant components. Despite these excellent characteristics, MALDI-FTICR MS has been rarely used for peptide and protein profiling of large cohorts of samples. In this thesis, the development and application of novel MALDI-FTICR-MS methods for high-throughput analysis of human serum peptides and proteins is described. Due to the large biological variability of human serum, the analysis of a large cohort is needed to obtain reliable statistical results.UBL - phd migration 201

Glycoform analysis of intact erythropoietin by MALDI FT-ICR mass spectrometry

Recombinant human erythropoietin (EPO) is a complex therapeutic glycoprotein with three N- and one O-glycosylation sites. Glycosylation of EPO influences its safety and efficacy and is defined as a critical quality attribute. Thus, analytical methods for profiling EPO glycosylation are highly demanded. Owing to the complexity of the intact protein, information about EPO glycosylation is commonly derived from released glycan and glycopeptide analysis using mass spectrometry (MS). Alternatively, comprehensive insights into the glycoform heterogeneity of intact EPO are obtained using ESI MS-based methods with or without upfront separation of EPO glycoforms. MALDI MS, typically performed with TOF mass analyzers, has been also used for the analysis of intact EPO but, due to the poor glycoform resolution, has only provided limited glycoform information. Here, we present a MALDI FT-ICR MS method for the glycosylation profiling of intact EPO with improved glycoform resolution and without loss of sialic acid residues commonly observed in MALDI analysis. Three EPO variants were characterized in-depth and up to 199 glycoform compositions were assigned from the evaluation of doubly-charged ions, without any deconvolution of the mass spectra. Key glycosylation features such as sialylation, acetylation, and N- acetyllactosamine repeats were determined and found to agree with previously reported data obtained from orthogonal analyses. The developed method allowed for a fast and straightforward data acquisition and evaluation and can be potentially used for the high-throughput comparison of EPO samples throughout its manufacturing process. (c) 2021 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).Proteomic

Intact and subunit-specific analysis of bispecific antibodies by sheathless CE-MS

Bispecific antibodies (BsAb) are next-generation, antibody-based pharmaceuticals which come with a great functional versatility and often a vast structural heterogeneity. Although engineering of the primary sequence of BsAbs guides the proper pairing of the different chains, several side products can often be observed contributing to the macroheterogeneity of these products. Furthermore, changes in the amino acid sequence can result in different protein modifications which can affect the properties of the antibody and further increase the structural complexity. A multi-methods approach can be used for the characterization of their heterogeneity but new analytical strategies are needed for a more accurate and in-depth analysis.Here, we present a combination of intact antibody and subunit-specific mass measurements using sheathless capillary electrophoresis-mass spectrometry for assessing the macro- and microheterogeneity of BsAbs. Two homologous BsAbs with the same bispecificity but slightly different amino acid sequences were analyzed. Intact measurements were performed using a positively coated capillary and a background electrolyte (BGE) consisting of 3% acetic acid. For intact BsAbs, the separation permitted the characterization of free light chains, homo- and heterodimers as well as incomplete assemblies. For subunit-specific measurements, BsAbs were hinge region cleaved using two different enzymes (SpeB and IdeS) followed by disulfide-bond reduction. The six different subunits (Lc1, Lc2, Fd'1, Fd'2, (Fc/2)1 and (Fc/2)2) were separated using the same positively-coated capillary and a BGE consisting of 20% acetic acid and 10% methanol. Mass measurements of hinge region cleaved antibodies were performed at isotopic resolution (resolving power 140000 at m/z 1100) for a more confident analysis of low abundance proteoforms. For both BsAbs several proteoforms with e.g. pyroglutamic acid (Pyro-Glu) or glycation which could not be properly assigned at the intact level, were accurately determined in the subunits showing the complementarity of both approaches. (C) 2020 Elsevier B.V. All rights reserved.Proteomic

Analysis of Synthetic Monodisperse Polysaccharides by Wide Mass Range Ultrahigh-Resolution MALDI Mass Spectrometry

Carbohydrates, such as oligo- and polysaccharides, are highly abundant biopolymers that are involved in numerous processes. The study of their structure and functions is commonly based on a material that is isolated from complex natural sources. However, a more precise analysis requires pure compounds with well-defined structures that can be obtained from chemical or enzymatic syntheses. Novel synthetic strategies have increased the accessibility of larger monodisperse polysaccharides, posing a challenge to the analytical methods used for their molecular characterization. Here, we present wide mass range ultrahigh-resolution matrix-assisted laser desorption/ionization (MALDI) Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS) as a powerful platform for the analysis of synthetic oligo- and polysaccharides. Synthetic carbohydrates 16-, 64-, 100-, and 151-mers were mass analyzed and characterized by MALDI in-source decay FT-ICR MS. Detection of fragment ions generated from glycosidic bond cleavage (or cross-ring cleavage) provided information of the monosaccharide content and the linkage type, allowing for the corroboration of the carbohydrate compositions and structures

IgA N- and O-glycosylation profiling reveals no association with the pregnancy-related improvement in rheumatoid arthritis

Background: The Fc glycosylation of immunoglobulin G (IgG) is well known to associate with rheumatoid arthritis (RA) disease activity. The same may be true for other classes of Igs. In the present study, we sought to determine whether the glycosylation of IgA was different between healthy subjects and patients with RA, as well as whether it was associated with RA disease activity, in particular with the pregnancy-associated improvement thereof or the flare after delivery. Methods: A recently developed high-throughput method for glycoprofiling of IgA1 was applied to affinity-captured IgA from sera of patients with RA (n = 252) and healthy control subjects (n = 32) collected before, during and after pregnancy. Results: IgA1 O-glycans bore more sialic acids in patients with RA than in control subjects. In addition, levels of bisecting N-acetylglucosamine of the N-glycans at asparagine 144 were higher in the patients with RA. The levels of several N-glycosylation traits were shown to change with pregnancy, similar to what has been shown before for IgG. However, the changes in IgA glycosylation were not associated with improvement or a flare of disease activity. Conclusions: The glycosylation of IgA differs between patients with RA and healthy control subjects. However, our data suggest only a minor, if any, association of IgA glycosylation with RA disease activity

Structural Characterization of Biofunctionalized Gold Nanoparticles by Ultrahigh-Resolution Mass Spectrometry

Bio-organic Synthesi

A semi-automated, high throughput approach for O-glycosylation profiling of in vitro established cancer cell lines by MALDI-FT-ICR MS

The study of protein O-glycosylation is important in biological research as O-glycans have been reported to regulate a multitude of molecular and cell biology processes occurring in cancer. It is known that alterations in O-glycosylation are involved in the development and progression of cancer. Their easy accessibility makes in vitro established cell lines suitable and useful models for studying biological mechanisms in disease. However, the O-glycosylation analysis of large numbers of samples, as required in systems biology and biomarker discovery studies, is often challenging. In the present study, O-glycans from three human colorectal cancer cell lines and two human pancreatic cancer cell lines were released by semi-automated, high throughput reductive beta-elimination and analysed using ultrahigh resolution MALDI-FT-ICR MS. Automated data integration and processing was performed using MassyTools, where the analyte was automatically included for relative quantitation based on a range of selection criteria including signal-to-noise ratio, mass error and isotopic pattern quality scores. A total of 126 O-glycan compositions, ranging from a single monosaccharide to large oligosaccharides exhibiting complex glycan motifs, were detected. The use of ultrahigh resolution MALDI-FTICR MS enabled glycan identification and quantitation in the matrix region of the spectrum. This approach has the potential to be used for O-glycosylation analysis of large numbers of samples, such as patient sample cohorts.Proteomic

Glycoform-resolved Fc gamma RIIIa affinity chromatography-mass spectrometry

Proteomic

Glycan and protein analysis of glycoengineered bacterial E. coli vaccines by MALDI-in-source decay FT-ICR mass spectrometry

Bacterial glycoconjugate vaccines have a major role in preventing microbial infections. Immunogenic bacterial glycans, suchas O-antigen polysaccharides, can be recombinantly expressed and combined with specific carrier proteins to produce effective vaccines. O-Antigen polysaccharides are typically polydisperse, and carrier proteins can have multiple glycosylation sites. Consequently, recombinant glycoconjugate vaccines have a high structural heterogeneity, making their characterization challenging. Sincedevelopment and quality control processes rely on such character-ization, novel strategies are needed for faster and informative analysis.Here, we present a novel approach employing minimal samplepreparation and ultrahigh-resolution mass spectrometry analysis forprotein terminal sequencing and characterization of the oligosaccharide repeat units of bacterial glycoconjugate vaccines. Threeglycoconjugate vaccine candidates, obtained from the bioconjugation of the O-antigen polysaccharides fromE. coliserotypes O2,O6A, and O25B with the genetically detoxified exotoxin A fromPseudomonas aeruginosa, were analyzed by MALDI-in-source decay(ISD) FT-ICR MS. Protein and glycan ISD fragment ions were selectively detected using 1,5-diaminonaphtalene and a 2,5-dihydroxybenzoic acid/2-hydroxy-5-methoxybenzoic acid mixture (super-DHB) as a MALDI matrix, respectively. The analysis of protein fragments required the absence of salts in the samples, while the presence of salt was key for the detection of sodiated glycanfragments. MS/MS analysis of O-antigen ISD fragments allowed for the detection of specific repeat unit signatures. The developed strategy requires minute sample amounts, avoids the use of chemical derivatizations, and comes with minimal hands-on time allowing for fast corroboration of key structural features of bacterial glycoconjugate vaccines during early- and late-stage developmentProteomic

High-throughput glycopeptide profiling of prostate-specific antigen from seminal plasma by MALDI-MS

An altered total seminal plasma glycosylation has been associated with male infertility, and the highly abundant seminal plasma glycoprotein prostate-specific antigen (PSA) plays an important role in fertilization. However, the exact role of PSA glycosylation in male fertility is not clear. To understand the involvement of PSA glycosylation in the fertilization process, analytical methods are required to study the glycosylation of PSA from seminal plasma with a high glycoform resolution and in a protein-specific manner. In this study, we developed a novel, high-throughput PSA glycopeptide workflow, based on matrix-assisted laser desorption/ionization-mass spectrometry, allowing the discrimination of sialic acid linkage isomers via the derivatization of glycopeptides. The method was successfully applied on a cohort consisting of seminal plasma from infertile and fertile men (N = 102). Forty-four glycopeptides were quantified in all samples, showing mainly complex-type glycans with high levels of fucosylation and sialylation. In addition, N,N-diacetyllactosamine (LacdiNAc) motives were found as well as hybrid-type and high mannose-type structures. Our method showed a high intra- and interday repeatability and revealed no difference in PSA glycosylation between fertile and infertile men. Next to seminal plasma, the method is also expected to be of use for studying PSA glycopeptides derived from other biofluids and/or in other disease contexts.Proteomic