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Isotope labeling of both 15 N and 13 C in selected amino acids in a protein, known as sparse labeling, is an alternative to uniform labeling and is particularly useful for proteins that must be expressed using mammalian cells, including glycoproteins. High levels of enrichment in the selected amino acids enable multidimensional heteronuclear NMR measurements of glycoprotein three-dimensional structure. Mass spectrometry provides a means to quantify the degree of enrichment. Mass spectrometric measurements of tryptic peptides of a selectively labeled glycoprotein expressed in HEK293 cells revealed complicated isotope patterns which consisted of many overlapping isotope patterns from intermediately labeled peptides, which complicates the determination of the label incorporation. Two challenges are uncovered by these measurements. Metabolic scrambling of amino groups can reduce the 15 N content of enriched amino acids or increase the 15 N in nontarget amino acids. Also, undefined, unlabeled medium components may dilute the enrichment level of labeled amino acids. The impact of this unexpected metabolic scrambling was overcome by simulating isotope patterns for all isotope-labeled peptide states and generating linear combinations to fit to the data. This method has been used to determine the percent incorporation of 15 N and 13 C labels and has identified several metabolic scrambling effects that were previously undetected in NMR experiments. Ultrahigh mass resolution is also utilized to obtain isotopic fine structure, from which enrichment levels of 15 N and 13 C can be assigned unequivocally. Finally, tandem mass spectrometry can be used to confirm the location of heavy isotope labels in the peptides.

Uniform enrichment of 15 N and 13 C in proteins is commonly employed for 2D heteronuclear NMR measurements of the three-dimensional protein structure. Achieving a high degree of enrichment of both elements is important for obtaining high quality data. Uniform labeling of proteins and glycoproteins expressed in higher organisms (yeast or mammalian cell lines) is more challenging than expression in Escherichia coli , a prokaryote that grows on simple, chemically defined media but does not provide appropriate eukaryotic modifications. It is difficult to achieve complete incorporation of both heavy isotopes, and quality control measures are important for quantitating the level of their enrichment. Mass spectrometry measurements of the isotopic distribution of the intact protein or its proteolytic fragments provide the means to assess the enrichment level. A mass accuracy of 1 ppm or better is shown to be required to distinguish the correct combination of 13 C and 15 N enrichment due to subtle shifts in peak centroids with differences in the underlying, but unresolved, isotopic fine structure. A simple computer program was developed to optimize the fitting of experimental isotope patterns to statistically derived distributions. This method can determine the isotopic abundance from isotope patterns and isotopologue masses in conventional MS data for peptides, intact proteins, and glycans. For this purpose, MATLAB's isotope simulator, isotopicdist, has been modified to permit the variation of 15 N and 13 C enrichment levels and to perform a two-dimensional grid search of enrichment levels of both isotopes. We also incorporated an alternate isotope simulator, js-emass, into MATLAB for use in the same fitting program. Herein we benchmark this technique on natural abundance ubiquitin and uniformly [ 15 N, 13 C]-labeled ubiquitin at both the intact and peptide level, outline considerations for data quality and mass accuracy, and report several improvements we have made to the previously reported analysis of the [ 15 N, 13 C]-enriched human IgG Fc domain, a glycoprotein that has been expressed in Saccharomyces cerevisiae .

We investigated the light-absorption properties of brown carbon (BrC) as part of the Georgia Wildland-Fire Simulation Experiment. We constructed fuel beds representative of three ecoregions in the Southeastern U.S. and varied the fuel-bed moisture content to simulate either prescribed fires or drought-induced wildfires. Based on decreasing fire radiative energy normalized by fuel-bed mass loading (FRE norm ), the combustion conditions were grouped into wildfire (Wild), prescribed fire (Rx), and wildfire involving duff ignition (WildDuff). The emitted BrC ranged from weakly absorbing (WildDuff) to moderately absorbing (Rx and Wild) with the imaginary part of the refractive index ( k ) values that were well-correlated with FRE norm . We apportioned the BrC into water-soluble (WSBrC) and water-insoluble (WIBrC). Approximately half of the WSBrC molecules detected using electrospray-ionization mass spectrometry were potential chromophores. Nevertheless, k of WSBrC was an order of magnitude smaller than k of WIBrC. Furthermore, k of WIBrC was well-correlated with FRE norm while k of WSBrC was not, suggesting different formation pathways between WIBrC and WSBrC. Overall, the results signify the importance of combustion conditions in determining BrC light-absorption properties and indicate that variables in wildland fires, such as moisture content and fuel-bed composition, impact BrC light-absorption properties to the extent that they influence combustion conditions., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

A large proportion of human proteins contain post-translational modifications that cannot be synthesized by prokaryotes. Thus, mammalian expression systems are often employed to characterize structure/function relationships using NMR spectroscopy. Here we define the selective isotope labeling of secreted, post-translationally modified proteins using human embryonic kidney (HEK)293 cells. We determined that alpha-[ 15 N]- atoms from 10 amino acids experience minimal metabolic scrambling (C, F, H, K, M, N, R, T, W, Y). Two more interconvert to each other (G, S). Six others experience significant scrambling (A, D, E, I, L, V). We also demonstrate that tuning culture conditions suppressed V and I scrambling. These results define expectations for 15 N-labeling in HEK293 cells., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Despite the prevalence and importance of glycoproteins in human biology, methods for isotope labeling suffer significant limitations. Common prokaryotic platforms do not produce mammalian post-translation modifications that are essential to the function of many human glycoproteins, including immunoglobulin G1 (IgG1). Mammalian expression systems require complex media and thus introduce significant costs to achieve uniform labeling. Expression with Pichia is available, though expertise and equipment requirements surpass E. coli culture. We developed a system utilizing Saccharomyces cerevisiae, [ 13 C]-glucose, and [ 15 N]-ammonium chloride with complexity comparable to E. coli. Here we report two vectors for expressing the crystallizable fragment (Fc) of IgG1 for secretion into the culture medium, utilizing the ADH2 or DDI2 promoters. We also report a strategy to optimize the expression yield using orthogonal Taguchi arrays. Lastly, we developed two different media formulations, a standard medium which provides 86-92% 15 N and 30% 13 C incorporation into the polypeptide, or a rich medium which provides 98% 15 N and 95% 13 C incorporation as determined by mass spectrometry. This advance represents an expression and optimization strategy accessible to experimenters with the capability to grow and produce proteins for NMR-based experiments using E. coli., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

Proliferative forms of glomerulonephritis are characterized by the influx of leukocytes, albuminuria, and loss of kidney function. The glomerular endothelial glycocalyx is a thick carbohydrate layer that covers the endothelium and is comprised of heparan sulfate (HS), which plays a pivotal role in glomerular inflammation by facilitating endothelial-leukocyte trafficking. We hypothesize that the exogenous glomerular glycocalyx may reduce the glomerular influx of inflammatory cells during glomerulonephritis. Indeed, administration of mouse glomerular endothelial cell (mGEnC)-derived glycocalyx constituents, or the low-molecular-weight heparin enoxaparin, reduced proteinuria in mice with experimental glomerulonephritis. Glomerular influx of granulocytes and macrophages, as well as glomerular fibrin deposition, was reduced by the administration of mGEnC-derived glycocalyx constituents, thereby explaining the improved clinical outcome. HS glx also inhibited granulocyte adhesion to human glomerular endothelial cells in vitro . Notably, a specific HS glx fraction inhibited both CD11b and L-selectin binding to activated mGEnCs. Mass spectrometry analysis of this specific fraction revealed six HS oligosaccharides, ranging from tetra- to hexasaccharides with 2-7 sulfates. In summary, we demonstrate that exogenous HS glx reduces albuminuria during glomerulonephritis, which is possibly mediated via multiple mechanisms. Our results justify the further development of structurally defined HS-based therapeutics for patients with (acute) inflammatory glomerular diseases, which may be applicable to non-renal inflammatory diseases as well., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Maciej-Hulme, Van Gemst, Sanderson, Rops, Berden, Smeets, Amster, Rabelink and Van Der Vlag.)

Untargeted metabolomics studies are unbiased but identifying the same feature across studies is complicated by environmental variation, batch effects, and instrument variability. Ideally, several studies that assay the same set of metabolic features would be used to select recurring features to pursue for identification. Here, we developed an anchored experimental design. This generalizable approach enabled us to integrate three genetic studies consisting of 14 test strains of Caenorhabditis elegans prior to the compound identification process . An anchor strain, PD1074, was included in every sample collection, resulting in a large set of biological replicates of a genetically identical strain that anchored each study. This enables us to estimate treatment effects within each batch and apply straightforward meta-analytic approaches to combine treatment effects across batches without the need for estimation of batch effects and complex normalization strategies. We collected 104 test samples for three genetic studies across six batches to produce five analytical datasets from two complementary technologies commonly used in untargeted metabolomics. Here, we use the model system C. elegans to demonstrate that an augmented design combined with experimental blocks and other metabolomic QC approaches can be used to anchor studies and enable comparisons of stable spectral features across time without the need for compound identification. This approach is generalizable to systems where the same genotype can be assayed in multiple environments and provides biologically relevant features for downstream compound identification efforts. All methods are included in the newest release of the publicly available SECIMTools based on the open-source Galaxy platform., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Shaver, Garcia, Gouveia, Morse, Liu, Asef, Borges, Leach, Andersen, Amster, Fernández, Edison and McIntyre.)

N -glycosylation is a common posttranslational modification of secreted proteins in eukaryotes. This modification targets asparagine residues within the consensus sequence, N-X-S/T. While this sequence is required for glycosylation, the initial transfer of a high-mannose glycan by oligosaccharyl transferases A or B (OST-A or OST-B) can lead to incomplete occupancy at a given site. Factors that determine the extent of transfer are not well understood, and understanding them may provide insight into the function of these important enzymes. Here, we use mass spectrometry (MS) to simultaneously measure relative occupancies for three N -glycosylation sites on the N-terminal IgV domain of the recombinant glycoprotein, hCEACAM1. We demonstrate that addition is primarily by the OST-B enzyme and propose a kinetic model of OST-B N -glycosylation. Fitting the kinetic model to the MS data yields distinct rates for glycan addition at most sites and suggests a largely stochastic initial order of glycan addition. The model also suggests that glycosylation at one site influences the efficiency of subsequent modifications at the other sites, and glycosylation at the central or N-terminal site leads to dead-end products that seldom lead to full glycosylation of all three sites. Only one path of progressive glycosylation, one initiated by glycosylation at the C-terminal site, can efficiently lead to full occupancy for all three sites. Thus, the hCEACAM1 domain provides an effective model system to study site-specific recognition of glycosylation sequons by OST-B and suggests that the order and efficiency of posttranslational glycosylation is influenced by steric cross-talk between adjoining acceptor sites.

Methanogens and anaerobic methane-oxidizing archaea (ANME) are important players in the global carbon cycle. Methyl-coenzyme M reductase (MCR) is a key enzyme in methane metabolism, catalyzing the last step in methanogenesis and the first step in anaerobic methane oxidation. Divergent mcr and mcr-like genes have recently been identified in uncultured archaeal lineages. However, the assembly and biochemistry of MCRs from uncultured archaea remain largely unknown. Here we present an approach to study MCRs from uncultured archaea by heterologous expression in a methanogen, Methanococcus maripaludis. Promoter, operon structure, and temperature were important determinants for MCR production. Both recombinant methanococcal and ANME-2 MCR assembled with the host MCR forming hybrid complexes, whereas tested ANME-1 MCR and ethyl-coenzyme M reductase only formed homogenous complexes. Together with structural modeling, this suggests that ANME-2 and methanogen MCRs are structurally similar and their reaction directions are likely regulated by thermodynamics rather than intrinsic structural differences., (© 2022. The Author(s).)

Human roundabout 1 (hRobo1) is an extracellular receptor glycoprotein that plays important roles in angiogenesis, organ development, and tumor progression. Interaction between hRobo1 and heparan sulfate (HS) has been shown to be essential for its biological activity. To better understand the effect of HS binding we engineered a lanthanide-binding peptide sequence (Loop) into the Ig2 domain of hRobo1. Native mass spectrometry was used to verify that loop introduction did not inhibit HS binding or conformational changes previously suggested by gas phase ion mobility measurements. NMR experiments measuring long-range pseudocontact shifts were then performed on 13 C-methyl labeled hRobo1-Ig1-2-Loop in HS-bound and unbound forms. The magnitude of most PCSs for methyl groups in the Ig1 domain increase in the bound state confirming a change in the distribution of interdomain geometries. A grid search over Ig1 orientations to optimize the fit of data to a single conformer for both forms produced two similar structures, both of which differ from existing X-ray crystal structures and structures inferred from gas-phase ion mobility measurements. The structures and degree of fit suggest that the hRobo1-Ig1-2 structure changes slightly and becomes more rigid on HS binding. This may have implications for Robo-Slit signaling., (© 2022. The Author(s).)

The predominant protein expression host for NMR spectroscopy is Escherichia coli, however, it does not synthesize appropriate post-translation modifications required for mammalian protein function and is not ideal for expressing naturally secreted proteins that occupy an oxidative environment. Mammalian expression platforms can address these limitations; however, these are not amenable to cost-effective uniform 15  N labeling resulting from highly complex growth media requirements. Yeast expression platforms combine the simplicity of bacterial expression with the capabilities of mammalian platforms, however yeasts require optimization prior to isotope labeling. Yeast expression will benefit from methods to boost protein expression levels and developing labeling conditions to facilitate growth and high isotope incorporation within the target protein. In this work, we describe a novel platform based on the yeast Saccharomyces cerevisiae that simultaneously expresses the Kar2p chaperone and protein disulfide isomerase in the ER to facilitate the expression of secreted proteins. Furthermore, we developed a growth medium for uniform 15  N labeling. We recovered 2.2 mg/L of uniformly 15  N-labeled human immunoglobulin (Ig)G1 Fc domain with 90.6% 15  N labeling. NMR spectroscopy revealed a high degree of similarity between the yeast and mammalian-expressed IgG1 Fc domains. Furthermore, we were able to map the binding interaction between IgG1 Fc and the Z domain through chemical shift perturbations. This platform represents a novel cost-effective strategy for 15  N-labeled immunoglobulin fragments., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

The antibody-binding Fc γ receptors (FcγRs) trigger life-saving immune responses and many therapeutic monoclonal antibodies require FcγR engagement for full effect. One proven strategy to improve the efficacy of antibody therapies is to increase receptor binding affinity, in particular binding to FcγRIIIa/CD16a. Currently, affinities are measured using recombinantly-expressed soluble extracellular FcγR domains and CD16a-mediated antibody-dependent immune responses are characterized using cultured cells. It is notable that CD16a is highly processed with multiple N-glycosylation sites, and preventing individual N-glycan modifications affects affinity. Furthermore, multiple groups have demonstrated that CD16a N-glycan composition is variable and composition impacts antibody binding affinity. The level of N-glycosylation at each site is not known though computational prediction indicates low to moderate potential at each site based on primary sequence (40-70%). Here we quantify occupancy of the extracellular domains using complementary mass spectrometry-based methods. All five sites of the tighter-binding CD16a V158 allotype showed 65-100% N-glycan occupancy in proteomics-based experiments. These observations were confirmed using intact protein mass spectrometry that demonstrated the predominant species corresponded to CD16a V158 with five N-glycans, with a smaller contribution from CD16a with four N-glycans. Occupancy was likewise high for the membrane-bound receptor at all detected N-glycosylation sites using CD16a purified from cultured human natural killer cells. Occupancy of the N162 site, critical for antibody binding, appeared independent of N169 occupancy based on analysis of the T171A mutant protein. The weaker-binding CD16a F158 allotype showed higher occupancy of >93% at each site., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Authors.)

Traveling wave ion-mobility mass spectrometry (TWIMS) combined with native mass spectrometry (MS) has emerged as a powerful tool for analyzing biomolecules, including complexes of protein and heparan sulfate (HS). This technique allows determination of the stoichiometry of the protein-HS interaction and information on the overall 3D molecular envelope., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

Placental malaria infection is mediated by the binding of the malarial VAR2CSA protein to the placental glycosaminoglycan, chondroitin sulfate. Recombinant subfragments of VAR2CSA (rVAR2) have also been shown to bind specifically and with high affinity to cancer cells and tissues, suggesting the presence of a shared type of oncofetal chondroitin sulfate (ofCS) in the placenta and in tumors. However, the exact structure of ofCS and what determines the selective tropism of VAR2CSA remains poorly understood. In this study, ofCS was purified by affinity chromatography using rVAR2 and subjected to detailed structural analysis. We found high levels of N-acetylgalactosamine 4-O-sulfation (∼80-85%) in placenta- and tumor-derived ofCS. This level of 4-O-sulfation was also found in other tissues that do not support parasite sequestration, suggesting that VAR2CSA tropism is not exclusively determined by placenta- and tumor-specific sulfation. Here, we show that both placenta and tumors contain significantly more chondroitin sulfate moieties of higher molecular weight than other tissues. In line with this, CHPF and CHPF2, which encode proteins required for chondroitin polymerization, are significantly upregulated in most cancer types. CRISPR/Cas9 targeting of CHPF and CHPF2 in tumor cells reduced the average molecular weight of cell-surface chondroitin sulfate and resulted in a marked reduction of rVAR2 binding. Finally, utilizing a cell-based glycocalyx model, we showed that rVAR2 binding correlates with the length of the chondroitin sulfate chains in the cellular glycocalyx. These data demonstrate that the total amount and cellular accessibility of chondroitin sulfate chains impact rVAR2 binding and thus malaria infection., Competing Interests: Conflict of interest The University of California San Diego and J. D. E. have a financial interest in TEGA Therapeutics, Inc. The terms of this arrangement have been reviewed and approved by the University of California, San Diego, in accordance with its conflict-of-interest policies. A. S., T. M. C., and T. G. T. have financial interest in VAR2Pharmaceuticals that holds the rights to use VAR2CSA for cancer treatment and diagnostic purposes., (Published by Elsevier Inc.)

Fourier transform ion cyclotron resonance (FT-ICR) and Orbitrap mass spectrometry (MS) are among the highest-performing analytical platforms used in metabolomics. Non-targeted metabolomics experiments, however, yield extremely complex datasets that make metabolite annotation very challenging and sometimes impossible. The high-resolution accurate mass measurements of the leading MS platforms greatly facilitate this process by reducing mass errors and spectral overlaps. When high resolution is combined with relative isotopic abundance (RIA) measurements, heuristic rules, and constraints during searches, the number of candidate elemental formula(s) can be significantly reduced. Here, we evaluate the performance of Orbitrap ID-X and 12T solariX FT-ICR mass spectrometers in terms of mass accuracy and RIA measurements and how these factors affect the assignment of the correct elemental formulas in the metabolite annotation pipeline. Quality of the mass measurements was evaluated under various experimental conditions (resolution: 120, 240, 500 K; automatic gain control: 5 × 10 4 , 1 × 10 5 , 5 × 10 5 ) for the Orbitrap MS platform. High average mass accuracy (<1 ppm for UPLC-Orbitrap MS and <0.2 ppm for direct infusion FT-ICR MS) was achieved and allowed the assignment of correct elemental formulas for over 90% ( m / z 75-466) of the 104 investigated metabolites. 13 C 1 and 18 O 1 RIA measurements further improved annotation certainty by reducing the number of candidates. Overall, our study provides a systematic evaluation for two leading Fourier transform (FT)-based MS platforms utilized in metabolite annotation and provides the basis for applying these, individually or in combination, to metabolomics studies of biological systems.

Glycosaminoglycans (GAGs) are linear polysaccharides that participate in a broad range of biological functions. Their incomplete biosynthesis pathway leads to nonuniform chains and complex mixtures. For this reason, the characterization of GAGs has been a difficult hurdle for the analytical community. Recently, ultraviolet photodissociation (UVPD) has emerged as a useful tool for determining sites of modification within a GAG chain. Here, we investigate the ability for UVPD to distinguish chondroitin sulfate epimers and the effects of UVPD experimental parameters on fragmentation efficiency. Chondroitin sulfate A (CS-A) and chondroitin sulfate B (CS-B), commonly referred to as dermatan sulfate (DS), differ only in C-5 uronic acid stereochemistry. This uronic acid difference can influence GAG-protein binding and therefore can alter the specific biological function of a GAG chain. Prior tandem mass spectrometry methods investigated for the elucidation of GAG structures also have difficulty differentiating 4- O from 6- O sulfation in chondroitin sulfate GAGs. Preliminary data using UVPD to characterize GAGs showed a promising ability to characterize 4- O sulfation in CS-A GAGs. Here, we look in depth at the capability of UVPD to distinguish chondroitin sulfate C-5 diastereomers and the role of key experimental parameters in making this distinction. Results using a 193 nm excimer laser and a 213 nm solid-state laser are compared for this study. The effect of precursor ionization state, the number of laser pulses (193 or 213 nm UVPD), and the use of the low-pressure versus high-pressure trap are investigated.

Preparation of samples for nuclear magnetic resonance (NMR) characterization of larger proteins requires enrichment with less abundant, NMR-active, isotopes such as 13C and 15N. This is routine for proteins that can be expressed in bacterial culture where low-cost isotopically enriched metabolic substrates can be used. However, it can be expensive for glycosylated proteins expressed in mammalian culture where more costly isotopically enriched amino acids are usually used. We describe a simple, relatively inexpensive procedure in which standard commercial media is supplemented with 13C-enriched glucose to achieve labeling of all glycans plus all alanines of the N-terminal domain of the highly glycosylated protein, CEACAM1. We demonstrate an ability to detect partially occupied N-glycan sites, sites less susceptible to processing by an endoglycosidase, and some unexpected truncation of the amino acid sequence. The labeling of both the protein (through alanines) and the glycans in a single culture requiring no additional technical expertise past standard mammalian expression requirements is anticipated to have several applications, including structural and functional screening of the many glycosylated proteins important to human health., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Glycosaminoglycans (GAGs) are linear polysaccharides found in a variety of organisms. GAGs contribute to biochemical pathway regulation, cell signaling, and disease progression. GAG sequence information is imperative for determining structure-function relationships. Recent advances in electron-activation techniques paired with high-resolution mass spectrometry allow for full sequencing of GAG structures. Electron detachment dissociation (EDD) and negative electron transfer dissociation (NETD) are two electron-activation methods that have been utilized for GAG characterization. Both methods produce an abundance of informative glycosidic and cross-ring fragment ions without producing a high degree of sulfate decomposition. Here, we provide detailed protocols for using EDD and NETD to sequence GAG chains. In addition to protocols directly involving performing these MS/MS methods, protocols include sample preparation, method development, internal calibration, and data analysis. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Preparation of glycosaminoglycan samples Basic Protocol 2: FTICR method development Basic Protocol 3: Internal calibration with NaTFA Basic Protocol 4: Electron Detachment Dissociation (EDD) of GAG samples Basic Protocol 5: Negative electron transfer dissociation (NETD) of GAG samples Basic Protocol 6: Analysis of MS/MS data., (© 2021 Wiley Periodicals LLC.)

This review covers recent developments in glycosaminoglycan (GAG) analysis via mass spectrometry (MS). GAGs participate in a variety of biological functions, including cellular communication, wound healing, and anticoagulation, and are important targets for structural characterization. GAGs exhibit a diverse range of structural features due to the variety of O- and N-sulfation modifications and uronic acid C-5 epimerization that can occur, making their analysis a challenging target. Mass spectrometry approaches to the structure assignment of GAGs have been widely investigated, and new methodologies remain the subject of development. Advances in sample preparation, tandem MS techniques (MS/MS), online separations, and automated analysis software have advanced the field of GAG analysis. These recent developments have led to remarkable improvements in the precision and time efficiency for the structural characterization of GAGs., Competing Interests: Conflicts of interest The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Glycosaminoglycans (GAGs) participate in a broad range of physiological processes, and their structures are of interest to researchers in structural biology and medicine. Although they are abundant in tissues and extracellular matrices, their structural heterogeneity makes them challenging analytes. Mass spectrometry, and more specifically, tandem mass spectrometry, is particularly well suited for their analysis. Many tandem mass spectrometry techniques have been examined for their suitability toward the structural characterization of GAGs. Threshold activation methods such as collision-induced dissociation (CID) produce mainly glycosidic cleavages and do not yield a broad range of structurally informative cross-ring fragments. Considerable research efforts have been directed at finding other means of dissociating gas-phase GAG ions to produce more comprehensive structural information. Here, we compare the structural information on GAGs obtained by charge-transfer dissociation (CTD) and electron detachment dissociation (EDD). EDD has previously been applied to GAGs and is known to produce both glycosidic and cross-ring cleavages in similar abundance. CTD has not previously been used to analyze GAGs but has been shown to produce abundant cross-ring cleavages and no sulfate loss when applied to another class of sulfated carbohydrates like algal polysaccharides. In contrast to EDD, which is restricted to FTICR mass spectrometers, CTD can be implemented on other platforms, such as ion trap mass spectrometers (ITMS). Here, we show the capability of CTD-ITMS to produce structurally significant details of the sites of modification in both heparan sulfate (HS) and chondroitin sulfate (CS) standards ranging in length from degree of polymerization (dp) 4 to dp6. EDD and CTD both yield more structural information than CID and yield similar fractional abundances to one another for glycosidic fragments, cross-ring fragments, and neutral losses.

Heparins are the most pharmaceutically important polysaccharides. These heparin-based anticoagulant/antithrombotic agents include unfractionated heparins, low molecular weight heparins (LMWHs) and ultralow molecular weight heparins (ULMWHs). Heparins exhibit their pharmacological and biological activities through interaction with heparin-binding proteins. The prototypical heparin-binding protein is antithrombin III (AT), responsible for heparin's anticoagulant/antithrombotic activity. This study describes a filter-trapping method to isolate the chains in enoxaparin, a LMWH, which bind to AT. We demonstrate this method using the ULMWH, fondaparinux, which consists of a single well defined AT binding site. The interacting chains of enoxaparin are then characterized by activity assays, top-down liquid chromatography-mass spectrometry, and capillary zone electrophoresis mass spectrometry. This filter-trapping assay is an improvement over affinity chromatography for isolating heparin chains interacting with heparin binding proteins., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Urinary glycosaminoglycans (GAGs) can reflect the health condition of a human being, and the GAGs composition can be directly related to various diseases. In order to effectively utilize such information, a detailed understanding of urinary GAGs in healthy individuals can provide insight into the levels and structures of human urinary GAGs. In this study, urinary GAGs were collected and purified from healthy males and females of adults and young adults. The total creatinine-normalized urinary GAG content, molecular weight distribution and disaccharide compositions were determined. Using capillary zone electrophoresis (CZE)-mass spectrometry (MS) and CZE-MS/MS relying on negative electron transfer dissociation, the major components of healthy human urinary GAGs were determined. The structures of 10 GAG oligosaccharides representing the majority of human urinary GAGs were determined., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The role of glycosaminoglycans (GAGs) in major biological functions is numerous and diverse, yet structural characterization of them by mass spectrometric techniques proves to be challenging. Characterization of GAG structure from tandem mass spectrometry is a tedious and time-consuming process but one that can be automated in a database-independent, high-throughput fashion through the assistance of software implementing a genetic algorithm (J. Am. Soc. Mass Spectrom. 29, 1802-1911, 2018). This work presents the manner in which this data is interpreted by the software, specifically addressing the development of a scoring algorithm. The significance of glycosidic and cross-ring fragment ions and the implications that specific fragments provide for assigning the positions of modifications are discussed. The scoring algorithm is tested for statistical merit using the widely accepted expectation value as the criterion for quality. Using MS/MS data for well-characterized standards, this scoring approach is shown to assign the correct structure, with a low likelihood (1 in 10 12 chances) that the assigned structure matches the data due to random chance. The integrated software that automates the structure assignment is called Glycosaminoglycan-Unambiguous Identification Technology (G-UNIT).

Glycosaminoglycans (GAGs) are important biological molecules that are highly anionic and occur in nature as complex mixtures. A platform that combines capillary zone electrophoresis (CZE) separations with mass spectrometry (MS) and gas-phase sequencing by using negative electron transfer dissociation (NETD) is shown to be efficacious for the structural analysis of GAG mixtures. CZE is a separation method well suited to the highly negatively charged nature of GAGs. NETD is an electron-based ion activation method that enables the generation of informative fragments with retention of the labile sulfate half-ester modification that determine specific GAG function. Here we combine for the first time NETD and CZE for assigning the structures of GAG oligomers present in mixtures. The speed of ion activation by NETD is found to couple well with the narrow peaks resulting from CZE migration. The platform was optimized with mixtures of GAG tetrasaccharide standards. The potential of the platform is demonstrated by the analysis of enoxaparin, a complex mixture of low molecular weight heparins, which was separated by CZE within 30 minutes and characterized by NETD MS/MS in one online experiment. 37 unique molecular compositions have been identified in enoxaparin using CZE-MS and 9 structures have been assigned with CZE-NETD-MS/MS.

Structural characterization of sulfated glycosaminoglycans (GAGs) by mass spectrometry has long been a formidable analytical challenge owing to their high structural variability and the propensity for sulfate decomposition upon activation with low-energy ion activation methods. While derivatization and complexation workflows have aimed to generate informative spectra using low-energy ion activation methods, alternative ion activation methods present the opportunity to obtain informative spectra from native GAG structures. Both electron- and photon-based activation methods, including electron detachment dissociation (EDD), negative electron transfer dissociation (NETD), and extreme ultraviolet photon activation, have been explored previously to overcome the limitations associated with low-energy activation methods for GAGs and other sulfated oligosaccharides. Further, implementation of such methods on high-resolution mass spectrometers has aided the interpretation of the complex spectra generated. Here, we explore ultraviolet photodissociation (UVPD) implemented on an Orbitrap mass spectrometer as another option for structural characterization of GAGs. UVPD spectra for both dermatan and heparan sulfate structures display extensive fragmentation including both glycosidic and cross-ring cleavages with the extent of sulfate retention comparable to that observed by EDD and NETD. In addition, the relatively short activation time of UVPD makes it promising for higher throughput analysis of GAGs in complex mixtures.

Residual dipolar couplings (RDCs) provide both structural and dynamical information useful in the characterization of biological macromolecules. While most data come from the interaction of simple pairs of directly bonded spin-1/2 nuclei ( 1 H- 15 N, 1 H- 13 C, 1 H- 1 H), it is possible to acquire data from interactions among the multiple spins of 13 C-labeled methyl groups ( 1 H 3 - 13 C). This is especially important because of the advantages that observation of 13 C-labeled methyl groups offers in working with very large molecules. Here we consider some of the options for measurement of methyl RDCs in large and often fully protonated proteins and arrive at a pulse sequence that exploits both J-modulation and direct detection of 13 C. Its utility is illustrated by application to a fully protonated two domain fragment from the mammalian glycoprotein, Robo1, 13 C-methyl-labeled in all valines.

Capillary zone electrophoresis (CZE) paired with mass spectrometry (MS) is a powerful analytical technique for examining mixtures of ionic analytes such as glycosaminoglycans. This study examines the mechanics of the electrospray process for a sheath flow CZE-MS interface under reverse polarity negative ionization conditions. Liquid flow in a sheath flow nano-electrospray CZE-MS interface is driven by two mechanisms, electroosmotic flow and electrospray nebulization. The contribution of these two processes to the overall flow of solution to the electrospray tip is influenced by the surface coatings of the sheath flow emitter tip and by the solvent composition. We have investigated the application of this interface to the reverse polarity separation of glycosaminoglycans and find that the role of electroosmotic flow is far less than has been reported previously, and the electrospray process itself is the largest contributor to the flow of the sheath liquid.

An enzyme- and click chemistry-mediated methodology for the site-specific nitroxide spin labeling of glycoproteins has been developed and applied. The procedure relies on the presence of single N-glycosylation sites that are present natively in proteins or that can be engineered into glycoproteins by mutational elimination of all but one glycosylation site. Recombinantly expressing glycoproteins in HEK293S (GnT1-) cells results in N-glycans with high-mannose structures that can be processed to leave a single GlcNAc residue. This can in turn be modified by enzymatic addition of a GalNAz residue that is subject to reaction with an alkyne-carrying TEMPO moiety using copper(I)-catalyzed click chemistry. To illustrate the procedure, we have made an application to a two-domain construct of Robo1, a protein that carries a single N-glycosylation site in its N-terminal domains. The construct has also been labeled with 15 N at amide nitrogens of lysine residues to provide a set of sites that are used to derive an effective location of the paramagnetic nitroxide moiety of the TEMPO group. This, in turn, allowed measurements of paramagnetic perturbations to the spectra of a new high affinity heparan sulfate ligand. Calculation of distance constraints from these data facilitated determination of an atomic level model for the docked complex.

Roundabout 1 (Robo1) interacts with its receptor Slit to regulate axon guidance, axon branching, and dendritic development in the nervous system and to regulate morphogenesis and many cell functions in the nonneuronal tissues. This interaction is known to be critically regulated by heparan sulfate (HS). Previous studies suggest that HS is required to promote the binding of Robo1 to Slit to form the minimal signaling complex, but the molecular details and the structural requirements of HS for this interaction are still unclear. Here, we describe the application of traveling wave ion mobility spectrometry (TWIMS) to study the conformational details of the Robo1-HS interaction. The results suggest that Robo1 exists in two conformations that differ by their compactness and capability to interact with HS. The results also suggest that the highly flexible interdomain hinge region connecting the Ig1 and Ig2 domains of Robo1 plays an important functional role in promoting the Robo1-Slit interaction. Moreover, variations in the sulfation pattern and size of HS were found to affect its binding affinity and selectivity to interact with different conformations of Robo1. Both MS measurements and CIU experiments show that the Robo1-HS interaction requires the presence of a specific size and pattern of modification of HS. Furthermore, the effect of N-glycosylation on the conformation of Robo1 and its binding modes with HS is reported. Graphical Abstract ᅟ.

A chemoenzymatic approach has been developed for the preparation of diverse libraries of heparan sulfate (HS) oligosaccharides. It employs chemically synthesized oligosaccharides having a chemical entity at a GlcN residue, which in unanticipated manners influences the site of modification by NST, C5-Epi/2-OST and 6-OST 1 /6-OST 3 , thus resulting in oligosaccharides differing in N/O-sulfation and epimerization pattern. The enzymatic transformations defined fine substrate requirements of NST, C5-Epi, 2-OST, and 6-OST., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Reverse polarity capillary zone electrophoresis coupled to negative ion mode mass spectrometry (CZE-MS) is shown to be an effective and sensitive tool for the analysis of glycosaminoglycan mixtures. Covalent modification of the inner wall of the separation capillary with neutral or cationic reagents produces a stable and durable surface that provides reproducible separations. By combining CZE-MS with a cation-coated capillary and a sheath flow interface, a rapid and reliable method has been developed for the analysis of sulfated oligosaccharides from dp4 to dp12. Several different mixtures have been separated and detected by mass spectrometry. The mixtures were selected to test the capability of this approach to resolve subtle differences in structure, such as sulfation position and epimeric variation of the uronic acid. The system was applied to a complex mixture of heparin/heparan sulfate oligosaccharides varying in chain length from dp3 to dp12 and more than 80 molecular compositions were identified by accurate mass measurement., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Despite decades of accumulated knowledge about proteins and their post-translational modifications (PTMs), numerous questions remain regarding their molecular composition and biological function. One of the most fundamental queries is the extent to which the combinations of DNA-, RNA- and PTM-level variations explode the complexity of the human proteome. Here, we outline what we know from current databases and measurement strategies including mass spectrometry-based proteomics. In doing so, we examine prevailing notions about the number of modifications displayed on human proteins and how they combine to generate the protein diversity underlying health and disease. We frame central issues regarding determination of protein-level variation and PTMs, including some paradoxes present in the field today. We use this framework to assess existing data and to ask the question, "How many distinct primary structures of proteins (proteoforms) are created from the 20,300 human genes?" We also explore prospects for improving measurements to better regularize protein-level biology and efficiently associate PTMs to function and phenotype.

Fucosylated chondroitin sulfates are complex polysaccharides extracted from sea cucumber. They have been extensively studied for their anticoagulant properties and have been implicated in other biological activities. While nuclear magnetic resonance spectroscopy has been used to extensively characterize fucosylated chondroitin sulfate oligomers, we herein report the first detailed mass characterization of fucosylated chondroitin sulfate using high-resolution Fourier transform ion cyclotron resonance mass spectrometry. The two species of fucosylated chondroitin sulfates considered for this work include Pearsonothuria graeffei (FCS-Pg) and Isostichopus badionotus (FCS-Ib). Fucosylated chondroitin sulfate oligosaccharides were prepared by N-deacetylation-deaminative cleavage of the two fucosylated chondroitin sulfates and purified by repeated gel filtration. Accurate mass measurements obtained from electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry measurements confirmed the oligomeric nature of these two fucosylated chondroitin sulfate oligosaccharides with each trisaccharide repeating unit averaging four sulfates per trisaccharide. Collision-induced dissociation of efficiently deprotonated molecular ions through Na/H + exchange proved useful in providing structurally relevant glycosidic and cross-ring product ions, capable of assigning the sulfate modifications on the fucosylated chondroitin sulfate oligomers. Careful examination of the tandem mass spectrometry of both species deferring in the positions of sulfate groups on the fucose residue (FCS-Pg-3,4- OS) and (FCS-Ib-2,4- OS) revealed cross-ring products 0,2 A αf and 2,4 X 2αf which were diagnostic for (FCS-Pg-3,4- OS) and 0,2 X 2αf diagnostic for (FCS-Ib-2,4- OS). Mass spectrometry and tandem mass spectrometry data acquired for both species varying in oligomer length (dp3-dp15) are presented.

The current paradigm in FT-ICR cell design is to approximate the ideal three-dimensional quadratic trapping potential as closely as possible to maintain ion cloud spatial coherence and achieve long transients, either with hyperbolically shaped electrodes, shimming electrodes, or by dynamic harmonization. In sharp contrast, the FT-ICR analyzer cell with four narrow aperture detection electrodes (NADEL) introduces significant anharmonic terms to the trapping potential. This analyzer cell is capable of quadrupolar detection by which one can measure a signal that is close to the unperturbed cyclotron frequency. This is far less sensitive to trapping potential and space charge shifts than the reduced cyclotron frequency measured in conventional ICR cells. The quadrupolar mode of ion detection in NADEL cells has been examined previously by SIMION simulations of ion clouds with up to 500 ions per simulation. Here, the behavior of the NADEL analyzer cell is examined through particle-in-cell (PIC) simulations, which allows us to examine the behavior of large populations (tens of thousands) of ions with space charge considerations, and to calculate the induced charge on the NADEL detection electrodes, and thus the transient signal. PIC simulations confirm a unique spatial distribution of the ions, with a coherent motion that results in long transient signals. Dependence of the ion cloud and image current signal on cell design, ion energy, and magnetron radius are examined. Coalescence effects are compared with those found in a dynamically harmonized cell. The insensitivity of the measured cyclotron frequency to space-charge is demonstrated both with simulations and experimentally. Graphical Abstract ᅟ.

Glycomics represents one of the last frontiers and most challenging in omic analysis. Glycosylation occurs in the endoplasmic reticulum and the Golgi organelle and its control is neither well-understood nor predictable based on proteomic or genomic analysis. One of the most structurally complex classes of glycoconjugates is the proteoglycans (PGs) and their glycosaminoglycan (GAG) side chains. Previously, our laboratory solved the structure of the chondroitin sulfate chain of the bikunin PG. The current study examines the much more complex structure of the dermatan sulfate GAG chain of decorin PG. By utilizing sophisticated separation methods followed by compositional analysis, domain mapping, and tandem mass spectrometry coupled with analysis by a modified genetic algorithm approach, the structural motif for the decorin dermatan sulfate chain was determined. This represents the second example of a GAG with a prominent structural motif, suggesting that the structural variability of this class of glycoconjugates is somewhat simpler than had been expected.

The analysis of heparan sulfate (HS) glycosaminoglycans presents many challenges, due to the high degree of structural heterogeneity arising from their non-template biosynthesis. Complete structural elucidation of glycosaminoglycans necessitates the unambiguous assignments of sulfo modifications and the C-5 uronic acid stereochemistry. Efforts to develop tandem mass spectrometric-based methods for the structural analysis of glycosaminoglycans have focused on the assignment of sulfo positions. The present work focuses on the assignment of the C-5 stereochemistry of the uronic acid that lies closest to the reducing end. Prior work with electron-based tandem mass spectrometry methods, specifically electron detachment dissociation (EDD), have shown great promise in providing stereo-specific product ions, such as the B 3 ´ -CO 2 , which has been found to distinguish glucuronic acid (GlcA) from iduronic acid (IdoA) in some HS tetrasaccharides. The previously observed diagnostic ions are generally not observed with 2-O-sulfo uronic acids or for more highly sulfated heparan sulfate tetrasaccharides. A recent study using electron detachment dissociation and principal component analysis revealed a series of ions that correlate with GlcA versus IdoA for a set of 2-O-sulfo HS tetrasaccharide standards. The present work comprehensively investigates the efficacy of these ions for assigning the C-5 stereochemistry of the reducing end uronic acid in 33 HS tetrasaccharides. A diagnostic ratio can be computed from the sum of the ions that correlate to GlcA to those that correlate to IdoA. Graphical Abstract ᅟ.

The structural characterization of sulfated glycosaminoglycan (GAG) carbohydrates remains an important target for analytical chemists attributable to challenges introduced by the natural complexity of these mixtures and the defined need for molecular-level details to elucidate biological structure-function relationships. Tandem mass spectrometry has proven to be the most powerful technique for this purpose. Previously, electron detachment dissociation (EDD), in comparison to other methods of ion activation, has been shown to provide the largest number of useful cleavages for de novo sequencing of GAG oligosaccharides, but such experiments are restricted to Fourier transform ion cyclotron resonance mass spectrometers (FTICR-MS). Negative electron transfer dissociation (NETD) provides similar fragmentation results, and can be achieved on any mass spectrometry platform that is designed to accommodate ion-ion reactions. Here, we examine for the first time the effectiveness of NETD-Orbitrap mass spectrometry for the structural analysis of GAG oligosaccharides. Compounds ranging in size from tetrasaccharides to decasaccharides were dissociated by NETD, producing both glycosidic and cross-ring cleavages that enabled the location of sulfate modifications. The highly-sulfated, heparin-like synthetic GAG, Arixtra TM , was also successfully sequenced by NETD. In comparison to other efforts to sequence GAG chains without fully ionized sulfate constituents, the occurrence of sulfate loss peaks is minimized by judicious precursor ion selection. The results compare quite favorably to prior results with electron detachment dissociation (EDD). Significantly, the duty cycle of the NETD experiment is sufficiently short to make it an effective tool for on-line separations, presenting a straightforward path for selective, high-throughput analysis of GAG mixtures. Graphical Abstract ᅟ.

Heparin is a polysaccharide that is widely used as an anticoagulant drug. The mechanism for heparin's anticoagulant activity is primarily through its interaction with a serine protease inhibitor, antithrombin III (AT), that enhances its ability to inactivate blood coagulation serine proteases, including thrombin (factor IIa) and factor Xa. The AT-binding site in the heparin is one of the most well-studied carbohydrate-protein binding sites and its structure is the basis for the synthesis of the heparin pentasaccharide drug, fondaparinux. Despite our understanding of the structural requirements for the heparin pentasaccharide AT-binding site, there is a lack of data on the natural variability of these binding sites in heparins extracted from animal tissues. The present work provides a detailed study on the structural variants of the tetrasaccharide fragments of this binding site afforded following treatment of a heparin with heparin lyase II. The 5 most commonly observed tetrasaccharide fragments of the AT-binding site are fully characterized, and a method for their quantification in heparin and low-molecular-weight heparin products is described., (Copyright © 2017 American Pharmacists Association®. All rights reserved.)

Most hyphenated analytical approaches that rely on liquid chromatography-MS require relatively long separation times, produce incomplete resolution of oligosaccharide mixtures, use eluents that are incompatible with electrospray ionization, or require oligosaccharide derivatization. Here we demonstrate the analysis of heparin oligosaccharides, including disaccharides, ultralow molecular weight heparin, and a low molecular weight heparin, using a novel electrokinetic pump-based CE-MS coupling eletrospray ion source. Reverse polarity CE separation and negative-mode electrospray ionization were optimized using a volatile methanolic ammonium acetate electrolyte and sheath fluid. The online CE hyphenated negative-ion electrospray ionization MS on an LTQ Orbitrap mass spectrometer was useful in disaccharide compositional analysis and bottom-up and top-down analysis of low molecular weight heparin. The application of this CE-MS method to ultralow molecular heparin suggests that a charge state distribution and the low level of sulfate group loss that is achieved make this method useful for online tandem MS analysis of heparins. Graphical abstract Most hyphenated analytical approaches that rely on liquid chromatography-MS require relatively long separation times, produce incomplete resolution of oligosaccharide mixtures, use eluents that are incompatible with electrospray ionization, or require oligosaccharide derivatization. Here we demonstrate the analysis of heparin oligosaccharides, including disaccharides, ultralow molecular weight heparin, and a low molecular weight heparin, using a novel electrokinetic pump-based CE-MS coupling eletrospray ion source. Reverse polarity CE separation and negative-mode electrospray ionization were optimized using a volatile methanolic ammonium acetate electrolyte and sheath fluid. The online CE hyphenated negative-ion electrospray ionization MS on an LTQ Orbitrap mass spectrometer was useful in disaccharide compositional analysis and bottom-up and top-down analysis of low molecular weight heparin. The application of this CE-MS method to ultralow molecular heparin suggests that a charge state distribution and the low level of sulfate group loss that is achieved make this method useful for online tandem MS analysis of heparins., Competing Interests: The authors declare that they have no conflict of interest.

Fibroblast growth factors (FGFs) regulate several cellular developmental processes by interacting with cell surface heparan proteoglycans and transmembrane cell surface receptors (FGFR). The interaction of FGF with heparan sulfate (HS) is known to induce protein oligomerization, increase the affinity of FGF towards its receptor FGFR, promoting the formation of the HS-FGF-FGFR signaling complex. Although the role of HS in the signaling pathways is well recognized, the details of FGF oligomerization and formation of the ternary signaling complex are still not clear, with several conflicting models proposed in literature. Here, we examine the effect of size and sulfation pattern of HS upon FGF1 oligomerization, binding stoichiometry and conformational stability, through a combination of ion mobility (IM) and theoretical modeling approaches. Ion mobility-mass spectrometry (IMMS) of FGF1 in the presence of several HS fragments ranging from tetrasaccharide (dp4) to dodecasaccharide (dp12) in length was performed. A comparison of the binding stoichiometry of variably sulfated dp4 HS to FGF1 confirmed the significance of the previously known high-affinity binding motif in FGF1 dimerization, and demonstrated that certain tetrasaccharide-length fragments are also capable of inducing dimerization of FGF1. The degree of oligomerization was found to increase in the presence of dp12 HS, and a general lack of specificity for longer HS was observed. Additionally, collision cross-sections (CCSs) of several FGF1-HS complexes were calculated, and were found to be in close agreement with experimental results. Based on the (CCSs) a number of plausible binding modes of 2:1 and 3:1 FGF1-HS are proposed. Graphical Abstract ᅟ.

An integrated methodology is described to establish ligand requirements for heparan sulfate (HS) binding proteins based on a workflow in which HS octasaccharides are produced by partial enzymatic degradation of natural HS followed by size exclusion purification, affinity enrichment using an immobilized HS-binding protein of interest, putative structure determination of isolated compounds by a hydrophilic interaction chromatography-high-resolution mass spectrometry platform, and chemical synthesis of well-defined HS oligosaccharides for structure-activity relationship studies. The methodology was used to establish the ligand requirements of human Roundabout receptor 1 (Robo1), which is involved in a number of developmental processes. Mass spectrometric analysis of the starting octasaccharide mixture and the Robo1-bound fraction indicated that Robo1 has a preference for a specific set of structures. Further analysis was performed by sequential permethylation, desulfation, and pertrideuteroacetylation followed by online separation and structural analysis by MS/MS. Sequences of tetrasaccharides could be deduced from the data, and by combining the compositional and sequence data, a putative octasaccharide ligand could be proposed (GlA-GlcNS6S-IdoA-GlcNS-IdoA2S-GlcNS6S-IdoA-GlcNAc6S). A modular synthetic approach was employed to prepare the target compound, and binding studies by surface plasmon resonance (SPR) confirmed it to be a high affinity ligand for Robo1. Further studies with a number of tetrasaccharides confirmed that sulfate esters at C-6 are critical for binding, whereas such functionalities at C-2 substantially reduce binding. High affinity ligands were able to reverse a reduction in endothelial cell migration induced by Slit2-Robo1 signaling.