Improved sample preparation method for glycan analysis of glycoproteins by CE‐LIF and CE‐MS

CE is a high‐resolution separation technique broadly used in the biotechnology industry for carbohydrate analysis. The standard sample preparation protocol for CE analysis of glycans released from glycoproteins generally requires derivatization times of overnight at 37°C, using ≥100 fold excess of fluorophore reagent, 8‐aminopyrene‐1,3,6‐trisulfonic‐acid, if the sample is unknown, or it is a regulated biotherapeutic product, possibly containing terminal sialic acid(s). In this paper, we report on significant improvements for the standard CE sample preparation method of glycan analysis. By replacing the conventionally used acetic acid catalyst with citric acid, as low as 1:10 glycan to fluorophore molar ratio (versus the typical 1:≥100 ratio) maintained the >95% derivatization yield at 55°C with only 50 min reaction time. Terminal sialic acid loss was negligible at 55°C during the derivatization process, and indicating that the kinetics of labeling at 55°C was faster than the loss of sialic acid from the glycan. The reduced relative level of 8‐aminopyrene‐1,3,6‐trisulfonic‐acid simplified the removal of excess reagent, important in both CE‐LIF (electrokinetic injection bias) and CE‐MS (ion suppression). Coupling CE‐ ESI‐MS confirmed that the individual peaks separated by CE corresponded to single glycans and increased the confidence of structural assignment based on glucose unit values.     

Metabolic Labeling and Imaging of N‐Linked Glycans in Arabidopsis Thaliana

Molecular imaging of glycans has been actively pursued in animal systems for the past decades. However, visualization of plant glycans remains underdeveloped, despite that glycosylation is essential for the life cycle of plants. Metabolic glycan labeling in Arabidopsis thaliana by using N‐azidoacetylglucosamine (GlcNAz) as the chemical reporter is reported. GlcNAz is metabolized through the salvage pathway of N‐acetylglucosamine (GlcNAc) and incorporated into N‐linked glycans, and possibly intracellular O‐GlcNAc. Click‐labeling with fluorescent probes enables visualization of newly synthesized N‐linked glycans. N‐glycosylation in the root tissue was discovered to possess distinct distribution patterns in different developmental zones, suggesting that N‐glycosylation is regulated in a developmental stage‐dependent manner. This work shows the utility of metabolic glycan labeling in elucidating the function of N‐linked glycosylation in plants.     

Tracking Surface Glycans on Live Cancer Cells with Single‐Molecule Sensitivity

Using a combination of metabolically labeled glycans, a bioorthogonal copper(I)‐catalyzed azide–alkyne cycloaddition, and the controlled bleaching of fluorescent probes conjugated to azide‐ or alkyne‐tagged glycans, a sufficiently low spatial density of dye‐labeled glycans was achieved, enabling dynamic single‐molecule tracking and super‐resolution imaging of N‐linked sialic acids and O‐linked N‐acetyl galactosamine (GalNAc) on the membrane of live cells. Analysis of the trajectories of these dye‐labeled glycans in mammary cancer cells revealed constrained diffusion of both N‐ and O‐linked glycans, which was interpreted as reflecting the mobility of the glycan rather than to be caused by transient immobilization owing to spatial inhomogeneities on the plasma membrane. Stochastic optical reconstruction microscopy (STORM) imaging revealed the structure of dynamic membrane nanotubes.     

Live‐Cell Stimulated Raman Scattering Imaging of Alkyne‐Tagged Biomolecules

Alkynes can be metabolically incorporated into biomolecules including nucleic acids, proteins, lipids, and glycans. In addition to the clickable chemical reactivity, alkynes possess a unique Raman scattering within the Raman‐silent region of a cell. Coupling this spectroscopic signature with Raman microscopy yields a new imaging modality beyond fluorescence and label‐free microscopies. The bioorthogonal Raman imaging of various biomolecules tagged with an alkyne by a state‐of‐the‐art Raman imaging technique, stimulated Raman scattering (SRS) microscopy, is reported. This imaging method affords non‐invasiveness, high sensitivity, and molecular specificity and therefore should find broad applications in live‐cell imaging.     

Visualization of Protein‐Specific Glycosylation inside Living Cells

Protein glycosylation is a ubiquitous post‐translational modification that is involved in the regulation of many aspects of protein function. In order to uncover the biological roles of this modification, imaging the glycosylation state of specific proteins within living cells would be of fundamental importance. To date, however, this has not been achieved. Herein, we demonstrate protein‐specific detection of the glycosylation of the intracellular proteins OGT, Foxo1, p53, and Akt1 in living cells. Our generally applicable approach relies on Diels–Alder chemistry to fluorescently label intracellular carbohydrates through metabolic engineering. The target proteins are tagged with enhanced green fluorescent protein (EGFP). Förster resonance energy transfer (FRET) between the EGFP and the glycan‐anchored fluorophore is detected with high contrast even in presence of a large excess of acceptor fluorophores by fluorescence lifetime imaging microscopy (FLIM).     

Glycan labeling strategies and their use in identification and quantification

Most methods for the analysis of oligosaccharides from biological sources require a glycan derivatization step: glycans may be derivatized to introduce a chromophore or fluorophore, facilitating detection after chromatographic or electrophoretic separation. Derivatization can also be applied to link charged or hydrophobic groups at the reducing end to enhance glycan separation and mass-spectrometric detection. Moreover, derivatization steps such as permethylation aim at stabilizing sialic acid residues, enhancing mass-spectrometric sensitivity, and supporting detailed structural characterization by (tandem) mass spectrometry. Finally, many glycan labels serve as a linker for oligosaccharide attachment to surfaces or carrier proteins, thereby allowing interaction studies with carbohydrate-binding proteins. In this review, various aspects of glycan labeling, separation, and detection strategies are discussed.     

Multiplexing N‐glycan analysis by DNA analyzer

Analysis of N‐glycan structures has been gaining attentions over the years due to their critical importance to biopharma‐based applications and growing roles in biological research. Glycan profiling is also critical to the development of biosimilar drugs. The detailed characterization of N‐glycosylation is mandatory because it is a nontemplate driven process and that significantly influences critical properties such as bio‐safety and bio‐activity. The ability to comprehensively characterize highly complex mixtures of N‐glycans has been analytically challenging and stimulating because of the difficulties in both the structure complexity and time‐consuming sample pretreatment procedures. CE‐LIF is one of the typical techniques for N‐glycan analysis due to its high separation efficiency. In this paper, a 16‐capillary DNA analyzer was coupled with a magnetic bead glycan purification method to accelerate the sample preparation procedure and therefore increase N‐glycan assay throughput. Routinely, the labeling dye used for CE‐LIF is 8‐aminopyrene‐1,3,6‐trisulfonic acid, while the typical identification method involves matching migration times with database entries. Two new fluorescent dyes were used to either cross‐validate and increase the glycan identification precision or simplify sample preparation steps. Exoglycosidase studies were carried out using neuramididase, galactosidase, and fucosidase to confirm the results of three dye cross‐validation. The optimized method combines the parallel separation capacity of multiple‐capillary separation with three labeling dyes, magnetic bead assisted preparation, and exoglycosidase treatment to allow rapid and accurate analysis of N‐glycans. These new methods provided enough useful structural information to permit N‐glycan structure elucidation with only one sample injection.     

Targeted Imaging and Proteomic Analysis of Tumor‐Associated Glycans in Living Animals

Although it has been well known that dynamic changes in glycosylation are associated with tumor progression, it remains challenging to selectively visualize the cancer glycome in vivo. Herein, a strategy for the targeted imaging of tumor‐associated glycans by using ligand‐targeted liposomes encapsulating azidosugars is described. The intravenously injected liposomal nanoparticles selectively bound to the cancer‐cell‐specific receptors and installed azides into the melanoma glycans in a xenograft mouse model in a tissue‐specific manner. Subsequently, a copper‐free click reaction was performed in vivo to chemoselectively conjugate the azides with a near‐infrared fluorescent dye. The glycosylation dynamics during tumor growth were monitored by in vivo fluorescence imaging. Furthermore, the newly synthesized sialylated glycoproteins were enriched during tumor growth and identified by glycoproteomics. Compared with the labeling methods using free azidosugars, this method offers improved labeling efficiency and high specificity and should facilitate the elucidation of the functional role of glycans in cancer biology.     

Universal proteolysis and MSn for N‐ and O‐ glycan branching analysis

The continually growing list of critical glycosylation‐related processes has made analytical methodology for detailed glycan characterization an area of increasing interest. Glycosylation is a post translational modification of unsurpassed complexity due to the variety of compositions and linkages formed by these biopolymers. Structural characterization of glycan isomers has been achieved using ion trap mass spectrometry and MSⁿ of released, permethylated glycans. However, N‐ and O‐glycans require different sample preparation strategies; and release of the glycans may be hindered, result in degradation of the glycan, and/or produce limited yields of permethylated product. In the current report, we demonstrate universal proteolysis of both N‐ and O‐linked glycoproteins to individual glycoamino acids. These samples were shown to be directly amenable to permethylation and MSⁿ analysis for isomeric structural determination. Universal proteolysis and permethylation provides an identical sample preparation strategy for both classes of glycans that avoids potential pitfalls of commonly used release methods. This methodology should be applicable to all glycoproteins and serve as an alternative to glycan release for MSⁿ branching analysis. Published 2013. This article is a U.S. Government work and is in the public domain in the USA.     

Direct meso‐Alkynylation of Metalloporphyrins Through Gold Catalysis for Hemoprotein Engineering

A method was developed for the direct functionalization of metalloporphyrins at the methine protons (meso positions) to yield asymmetric alkynylated derivatives by using gold catalysis and hypervalent iodine reagents. This single‐step procedure was applied to b‐type heme and the product was incorporated into a gas‐sensor heme protein. The terminal alkyne allows fluorophore labeling through copper(I)‐catalyzed azide–alkyne cycloaddition (CuAAC). Hemoproteins with this type of engineered cofactor have several potential applications in labeling and imaging technologies. Additionally, the alkyne provides a handle for modulating porphyrin electron density, which affects cofactor redox potential and ligand affinity. This method will be helpful for investigating the chemistry of natural heme proteins and for designing artificial variants with altered properties and reactivities.     

A Single Excitation‐Duplexed Imaging Strategy for Profiling Cell Surface Protein‐Specific Glycoforms

This work develops a site‐specific duplexed luminescence resonance energy transfer system on cell surface for simultaneous imaging of two kinds of monosaccharides on a specific protein by single near‐infrared excitation. The single excitation‐duplexed imaging system utilizes aptamer modified upconversion luminescent nanoparticles as an energy donor to target the protein, and two fluorescent dye acceptors to tag two kinds of cell surface monosaccharides by a dual metabolic labeling technique. Upon excitation at 980 nm, only the dyes linked to protein‐specific glycans can be lit up by the donor by two parallel energy transfer processes, for in situ duplexed imaging of glycoforms on specific protein. Using MUC1 as the model, this strategy can visualize distinct glycoforms of MUC1 on various cell types and quantitatively track terminal monosaccharide pattern. This approach provides a versatile platform for profiling protein‐specific glycoforms, thus contributing to the study of the regulation mechanisms of protein functions by glycosylation.     

Coumarin‐Based Fluorogenic Probes for No‐Wash Protein Labeling

A fluorescent protein‐labeling strategy was developed in which a protein of interest (POI) is genetically tagged with a short peptide sequence presenting two Cys residues that can selectively react with synthetic fluorogenic reagents. These fluorogens comprise a fluorophore and two maleimide groups that quench fluorescence until they both undergo thiol addition during the labeling reaction. Novel fluorogens were prepared and kinetically characterized to demonstrate the importance of a methoxy substituent on the maleimide in suppressing reactivity with glutathione, an intracellular thiol, while maintaining reactivity with the dithiol tag. This system allows the rapid and specific labeling of intracellular POIs.     

Live‐cell MRI with Xenon Hyper‐CEST Biosensors Targeted to Metabolically Labeled Cell‐Surface Glycans

The targeting of metabolically labeled glycans with conventional MRI contrast agents has proved elusive. In this work, which further expands the utility of xenon Hyper‐CEST biosensors in cell experiments, we present the first successful molecular imaging of such glycans using MRI. Xenon Hyper‐CEST biosensors are a novel class of MRI contrast agents with very high sensitivity. We designed a multimodal biosensor for both fluorescent and xenon MRI detection that is targeted to metabolically labeled sialic acid through bioorthogonal chemistry. Through the use of a state of the art live‐cell bioreactor, it was demonstrated that xenon MRI biosensors can be used to image cell‐surface glycans at nanomolar concentrations.     

Recognition of Complex Core‐Fucosylated N‐Glycans by a Mini Lectin

The mini fungal lectin PhoSL was recombinantly produced and characterized. Despite a length of only 40 amino acids, PhoSL exclusively recognizes N‐glycans with α1,6‐linked fucose. Core fucosylation influences the intrinsic properties and bioactivities of mammalian N‐glycoproteins and its level is linked to various cancers. Thus, PhoSL serves as a promising tool for glycoprofiling. Without structural precedence, the crystal structure was solved using the zinc anomalous signal, and revealed an interlaced trimer creating a novel protein fold termed β‐prism III. Three biantennary core‐fucosylated N‐glycan azides of 8 to 12 sugars were cocrystallized with PhoSL. The resulting highly resolved structures gave a detailed view on how the exclusive recognition of α1,6‐fucosylated N‐glycans by such a small protein occurs. This work also provided a protein consensus motif for the observed specificity as well as a glimpse into N‐glycan flexibility upon binding.     

Poly(amidoamine) dendrimer‐coated magnetic nanoparticles for the fast purification and selective enrichment of glycopeptides and glycans

Glycosylated proteins modulate various important functions of organisms. To reveal the functions of glycoproteins, in‐depth characterization studies are necessary. Although mass spectrometry is a very efficient tool for glycoproteomic and glycomic studies, efficient sample preparation methods are required prior to analyses. In the study, poly(amidoamine) dendrimer‐coated magnetic nanoparticles were presented for the specific enrichment and fast purification of glycopeptides and glycans. The enrichment and purification performance of the developed method was evaluated both at the glycopeptide, and the glycan level using several standard glycoprotein digests and released glycan samples. The poly(amidoamine) dendrimer‐coated magnetic nanoparticles not only showed selective affinity (Immunoglobulin G/Bovine Serum Albumin, 1/10 by weight) to glycopeptides and released glycans but also good sensitivity (0.4 ng/µL for Immunoglobulin G) for glycoproteomic and glycomic applications. Thirty‐five glycopeptides of Immunoglobulin G were detected after enrichment with poly(amidoamine) dendrimer‐coated magnetic nanoparticles. In addition, 55 ¹⁸O tagged deamidated glycopeptides belonging to human plasma glycoproteome were confirmed. Finally, fifty 2‐aminobenzoic acid, and 30 procainamide‐labelled human plasma N‐glycans released from human plasma glycoproteins were determined after purifications. The results indicate that the proposed enrichment and purification method using poly(amidoamine) dendrimer‐coated magnetic nanoparticles could be simply adjusted to sample preparation methods.     

Visualizing Cholesterol Uptake by Self‐Assembling Rhodamine B‐Labeled Polymer Inside Living Cells via FLIM‐FRET Microscopy

Atherosclerosis is a widespread and hazardous disease characterized by the formation of arterial plaques mostly composed of fat, cholesterol, and calcium ions. The direct solubilization of cholesterol represents a promising, atheroprotective strategy to subside lipid blood levels and reverse atherosclerosis. This study deals with the in‐depth analysis of polymer‐mediated cholesterol dissolution inside living human cells. To this end, a recently described multifunctional block‐polymer is labeled with Rhodamine B (RhoB) to investigate its interaction with cells via fluorescence microscopy. This gives insight into the cellular internalization process of the polymer, which appears to be clathrin‐ and caveolae/raft‐dependent endocytosis. In cell single particle tracking reveals an active transport of RhoB polymer including structures. Förster resonance energy transfer (FRET) measurements of cells treated with a fluorophore‐tagged cholesterol derivative and the RhoB polymer indicates the uptake of cholesterol by the polymeric particles. Hence, these results present a first step toward possible applications of cholesterol‐absorbing polymers for treating atherosclerosis.     

Super‐Resolution Imaging of Plasma Membrane Glycans

Much of the physiology of cells is controlled by the spatial organization of the plasma membrane and the glycosylation patterns of its components, however, studying the distribution, size, and composition of these components remains challenging. A bioorthogonal chemical reporter strategy was used for the efficient and specific labeling of membrane‐associated glycoconjugates with modified monosaccharide precursors and organic fluorophores. Super‐resolution fluorescence imaging was used to visualize plasma membrane glycans with single‐molecule sensitivity. Our results demonstrate a homogeneous distribution of N‐acetylmannosamine (ManNAc)‐, N‐acetylgalactosamine (GalNAc)‐, and O‐linked N‐acetylglucosamine (O‐GlcNAc)‐modified plasma membrane proteins in different cell lines with densities of several million glycans on each cell surface.     

Super‐Chelators for Advanced Protein Labeling in Living Cells

Live‐cell labeling, super‐resolution microscopy, single‐molecule applications, protein localization, or chemically induced assembly are emerging approaches, which require specific and very small interaction pairs. The minimal disturbance of protein function is essential to derive unbiased insights into cellular processes. Herein, we define a new class of hexavalent N‐nitrilotriacetic acid (hexaNTA) chelators, displaying the highest affinity and stability of all NTA‐based small interaction pairs described so far. Coupled to bright organic fluorophores with fine‐tuned photophysical properties, the super‐chelator probes were delivered into human cells by chemically gated nanopores. These super‐chelators permit kinetic profiling, multiplexed labeling of His₆‐ and His₁₂‐tagged proteins as well as single‐molecule‐based super‐resolution imaging.     

Hydrophobic Derivatization of <Emphasis Type="Italic">N</Emphasis>-linked Glycans for Increased Ion Abundance in Electrospray Ionization Mass Spectrometry

A library of neutral, hydrophobic reagents was synthesized for use as derivatizing agents in order to increase the ion abundance of N-linked glycans in electrospray ionization mass spectrometry (ESI MS). The glycans are derivatized via hydrazone formation and are shown to increase the ion abundance of a glycan standard more than 4-fold. Additionally, the data show that the systematic addition of hydrophobic surface area to the reagent increases the glycan ion abundance, a property that can be further exploited in the analysis of glycans. The results of this study will direct the future synthesis of hydrophobic reagents for glycan analysis using the correlation between hydrophobicity and theoretical non-polar surface area calculation to facilitate the development of an optimum tag for glycan derivatization. The compatibility and advantages of this method are demonstrated by cleaving and derivatizing N-linked glycans from human plasma proteins. The ESI-MS signal for the tagged glycans are shown to be significantly more abundant, and the detection of negatively charged sialylated glycans is enhanced.