Coupling an ion chromatography to high resolution mass spectrometry (IC-MS) for the discovery of potentially prebiotic oligosaccharides in Chardonnay grape marc.

Oligosaccharides are carbohydrates made of three to twenty monosaccharide units linked through glycosidic bonds. Emerging research into the potential prebiotic activity of oligosaccharides is creating opportunities to use industrial byproducts as value-added products. Grape marc is a residue left after winemaking and has been shown to provide health benefits to humans. In this study, we analyzed the oligosaccharides in Chardonnay grape marc by utilizing a hyphenated platform in which an ion chromatography (IC) system is coupled to an Orbitrap mass spectrometer (MS). With this platform, we obtained a structural library including 32 oligosaccharides with unique compositions of monosaccharides and 61 oligosaccharide structures. Notably, the ion chromatographic separation provided resolution of charged isomers while maintaining separation capacity for small, neutral oligosaccharides. High-quality tandem MS also facilitated the identification of oligosaccharides with structural modifications including methylation and the presence of sugar alditols and hexuronic acids. The data acquired by the IC-MS system were also compared with previously published LC-MS data. We found that these two platforms are largely complementary and, in combination, provide a more comprehensive characterization of oligosaccharides than either platform achieves alone.

Towards a more complete glycome: Advances in ion chromatography-mass spectrometry (IC-MS) for improved separation and analysis of carbohydrates.

To date, few tools are available for the analysis of the glycome without derivatization, a process which is known to introduce issues such as differential loss of sialic acid and incomplete labeling. We have previously reported the use of ion chromatography-mass spectrometry (IC-MS) to analyze native sialylated and sulfated glycans. Here, we introduce improvements to IC column technology, enabling the separation of neutral glycans while maintaining charge separation capabilities. When implemented in an IC-MS workflow, this enables the structural characterization of a broad array of chemically distinct glycans. With the newly developed IC column and modified IC-MS instrumentation configuration, we qualitatively investigated O-glycome profiles in bovine fetuin and porcine gastric mucins. The improved chromatographic resolution in combination with high-resolution MS data present a powerful tool for glycan structural identification.

VectorMOD: Method for Bottom-Up Proteomic Characterization of rAAV Capsid Post-Translational Modifications and Vector Impurities.

Progress in recombinant AAV gene therapy product and process development has advanced our understanding of the basic biology of this critical delivery vector. The discovery of rAAV capsid post-translational modifications (PTMs) has spurred interest in the field for detailed rAAV-specific methods for vector lot characterization by mass spectrometry given the unique challenges presented by this viral macromolecular complex. Recent concerns regarding immunogenic responses to systemically administered rAAV at high doses has highlighted the need for investigators to catalog and track potentially immunogenic vector lot components including capsid PTMs and PTMs on host cell protein impurities. Here we present a simple step-by-step guide for academic rAAV laboratories and Chemistry, Manufacturing and Control (CMC) groups in industry to perform an in-house or outsourced bottom-up mass spectrometry workflow to characterize capsid PTMs and process impurities.

Methods Matter: Standard Production Platforms for Recombinant AAV Produce Chemically and Functionally Distinct Vectors.

Different approaches are used in the production of recombinant adeno-associated virus (rAAV). The two leading approaches are transiently transfected human HEK293 cells and live baculovirus infection of Spodoptera frugiperda (Sf9) insect cells. Unexplained differences in vector performance have been seen clinically and preclinically. Thus, we performed a controlled comparative production analysis varying only the host cell species but maintaining all other parameters. We characterized differences with multiple analytical approaches: proteomic profiling by mass spectrometry, isoelectric focusing, cryo-EM (transmission electron cryomicroscopy), denaturation assays, genomic and epigenomic sequencing of packaged genomes, human cytokine profiling, and functional transduction assessments in vitro and in vivo, including in humanized liver mice. Using these approaches, we have made two major discoveries: (1) rAAV capsids have post-translational modifications (PTMs), including glycosylation, acetylation, phosphorylation, and methylation, and these differ between platforms; and (2) rAAV genomes are methylated during production, and these are also differentially deposited between platforms. Our data show that host cell protein impurities differ between platforms and can have their own PTMs, including potentially immunogenic N-linked glycans. Human-produced rAAVs are more potent than baculovirus-Sf9 vectors in various cell types in vitro (p < 0.05-0.0001), in various mouse tissues in vivo (p < 0.03-0.0001), and in human liver in vivo (p < 0.005). These differences may have clinical implications for rAAV receptor binding, trafficking, expression kinetics, expression durability, vector immunogenicity, as well as cost considerations.

An RNA-centric dissection of host complexes controlling flavivirus infection.

Flaviviruses, including dengue virus (DENV) and Zika virus (ZIKV), cause severe human disease. Co-opting cellular factors for viral translation and viral genome replication at the endoplasmic reticulum is a shared replication strategy, despite different clinical outcomes. Although the protein products of these viruses have been studied in depth, how the RNA genomes operate inside human cells is poorly understood. Using comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS), we took an RNA-centric viewpoint of flaviviral infection and identified several hundred proteins associated with both DENV and ZIKV genomic RNA in human cells. Genome-scale knockout screens assigned putative functional relevance to the RNA-protein interactions observed by ChIRP-MS. The endoplasmic-reticulum-localized RNA-binding proteins vigilin and ribosome-binding protein 1 directly bound viral RNA and each acted at distinct stages in the life cycle of flaviviruses. Thus, this versatile strategy can elucidate features of human biology that control the pathogenesis of clinically relevant viruses.

Chemical Modulation of Protein O-GlcNAcylation via OGT Inhibition Promotes Human Neural Cell Differentiation.

The enzymes that determine protein O-GlcNAcylation, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), act on key transcriptional and epigenetic regulators, and both are abundantly expressed in the brain. However, little is known about how alterations in O-GlcNAc cycling affect human embryonic stem cell (hESC) neural differentiation. Here, we studied the effects of perturbing O-GlcNAcylation during neural induction of hESCs using the metabolic inhibitor of OGT, peracetylated 5-thio-N-acetylglucosamine (Ac4-5SGlcNAc). Treatment of hESCs with Ac4-5SGlcNAc during induction limited protein O-GlcNAcylation and also caused a dramatic decrease in global levels of UDP-GlcNAc. Concomitantly, a subpopulation of neural progenitor cells (NPCs) acquired an immature neuronal morphology and expressed early neuronal markers such as ß-III tubulin (TUJ1) and microtubule associated protein 2 (MAP2), phenotypes that took longer to manifest in the absence of OGT inhibition. These data suggest that chemical inhibition of OGT and perturbation of protein O-GlcNAcylation accelerate the differentiation of hESCs along the neuronal lineage, thus providing further insight into the dynamic molecular mechanisms involved in neuronal development.

Characterizing peptide neutral losses induced by negative electron-transfer dissociation (NETD).

We implemented negative electron-transfer dissociation (NETD) on a hybrid ion trap/Orbitrap mass spectrometer to conduct ion/ion reactions using peptide anions and radical reagent cations. In addition to sequence-informative ladders of a•- and x-type fragment ions, NETD generated intense neutral loss peaks corresponding to the entire or partial side-chain cleavage from amino acids constituting a given peptide. Thus, a critical step towards the characterization of this recently introduced fragmentation technique is a systematic study of synthetic peptides to identify common neutral losses and preferential fragmentation pathways. Examining 46 synthetic peptides with high mass accuracy and high resolution analysis permitted facile determination of the chemical composition of each neutral loss. We identified 19 unique neutral losses from 14 amino acids and three modified amino acids, and assessed the specificity and sensitivity of each neutral loss using a database of 1542 confidently identified peptides generated from NETD shotgun experiments employing high-pH separations and negative electrospray ionization. As residue-specific neutral losses indicate the presence of certain amino acids, we determined that many neutral losses have potential diagnostic utility. We envision this catalogue of neutral losses being incorporated into database search algorithms to improve peptide identification specificity and to further advance characterization of the acidic proteome.

Analysis of the acidic proteome with negative electron-transfer dissociation mass spectrometry.

We describe the first implementation of negative electron-transfer dissociation (NETD) on a hybrid ion trap-orbitrap mass spectrometer and its application to high-throughput sequencing of peptide anions. NETD, coupled with high pH separations, negative electrospray ionization (ESI), and an NETD compatible version of OMSSA, is part of a complete workflow that includes the formation, interrogation, and sequencing of peptide anions. Together these interlocking pieces facilitated the identification of more than 2000 unique peptides from Saccharomyces cerevisiae representing the most comprehensive analysis of peptide anions by tandem mass spectrometry to date. The same S. cerevisiae samples were interrogated using traditional, positive modes of peptide LC-MS/MS analysis (e.g., acidic LC separations, positive ESI, and collision activated dissociation), and the resulting peptide identifications of the different workflows were compared. Due to a decreased flux of peptide anions and a tendency to produce lowly charged precursors, the NETD-based LC-MS/MS workflow was not as sensitive as the positive mode methods. However, the use of NETD readily permits access to underrepresented acidic portions of the proteome by identifying peptides that tend to have lower pI values. As such, NETD improves sequence coverage, filling out the acidic portions of proteins that are often overlooked by the other methods.