Direct in-gel fluorescence detection and cellular imaging of O-GlcNAc-modified proteins

We report an advanced chemoenzymatic strategy for the direct fluorescence detection, proteomic analysis, and cellular imaging of O-GlcNAc-modified proteins. O-GlcNAc residues are selectively labeled with fluorescent or biotin tags using an engineered galactosyltransferase enzyme and [3 + 2] azide-alkyne cycloaddition chemistry. We demonstrate that this approach can be used for direct in-gel detection and mass spectrometric identification of O-GlcNAc proteins, identifying 146 novel glycoproteins from the mammalian brain. Furthermore, we show that the method can be exploited to quantify dynamic changes in cellular O-GlcNAc levels and to image O-GlcNAc-glycosylated proteins within cells. As such, this strategy enables studies of O-GlcNAc glycosylation that were previously inaccessible and provides a new tool for uncovering the physiological functions of O-GlcNAc.     

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).     

Direct determination of glycosylation sites in O-fucosylated glycopeptides using nano-electrospray quadrupole time-of-flight mass spectrometry

O-Fucosylation is an unusual posttranslational modification present in several proteins that play important roles in physiological processes such as coagulation, cell signaling and metastasis. Although the exact function of the modification is still unclear, the number of proteins found to be modified is increasing, and there is a need for further structural and functional analyses. Here we report on a rapid and straightforward approach in the analysis of glycosylation status and determination of glycosylation sites in O-fucosylated glycopeptides using nano-electrospray quadrupole time-of-flight (nano-ESI Q-TOF) mass spectrometry. In a single measurement of previously chemically untreated O-fucosylated peptides originating from the thrombospondin-1 repeats, we were able to determine the glycosylation status of the analyzed peptide, the glycosylation site, and the glycan structure. The abundance of glycosylated peptide fragment ions in MS(2) spectra suggests that nano-ESI Q-TOF mass spectrometry can be used as a general approach in structural studies of O-fucosylation in proteins.     

Profiling surface glycans on live cells and tissues using quantum dot-lectin nanoconjugates

The surface of mammalian cells is densely coated with complex glycans, which are directly involved in cell-cell or cell-protein interactions that trigger various biological responses. Here, we present a novel glycomics approach that uses quantum dot (Qdot)-lectin nanoconjugates to interrogate the surface glycans of tissues and patterned cells. Our approach allows highly sensitive in situ monitoring of specific lectin-glycan interactions and quantitative information on surface glycans for each examined cell line and tissue. The results clearly show significant changes in glycosylation for each cell line and tissue sample. We expect that these results will be applicable in cancer diagnostics and promote the development of new analytical tools for glycomics.     

Specific labeling of cell surface proteins with chemically diverse compounds

The specific and covalent labeling of fusion proteins with synthetic molecules opens up new ways to study protein function in the living cell. Here we present a novel method that allows for the specific and exclusive extracellular labeling of proteins on the surfaces of live cells with a large variety of synthetic molecules including fluorophores, protein ligands, or quantum dots. The approach is based on the specific labeling of fusion proteins of acyl carrier protein with synthetic molecules through post-translational modification catalyzed by phosphopantetheine transferase. The specificity and versatility of the labeling should allow it to become an important tool for studying and manipulating cell surface proteins and for complementing existing approaches in cell surface engineering.     

Approach to the comprehensive analysis of glycoproteins isolated from human serum using a multi-lectin affinity column

Glycosylation is one the most common post-translational modifications (PTM) and glycoproteins play fundamental roles in a diversity of biological processes. The development of an analytical approach to the study of variation of glycosylation patterns in serum samples has been hindered by the structural heterogeneity of this post-translational modification and the complexity of serum proteome. We have used the ability of different lectins to recognize specific glycosylation motifs to develop a specific affinity system that can achieve a comprehensive capture of serum glycoproteins. In a preliminary investigation, we evaluated the ability of five commonly used immobilized lectins to capture glycoproteins from human serum. SDS-PAGE analysis showed each lectin was able to enrich a subset of the serum glycoproteome and overlaps in lectin specificity were indeed observed. Based on these results and with the goal of studying the extent of the human serum glycoproteome, we then developed a multi-lectin affinity column containing Concanavalin A (Con A), Wheat germ and Jacalin lectin. The selection of lectins was also based on the known N-linked and O-linked glycan structures that are considered representative of the serum proteome. We then demonstrated that the capture of glycoproteins was specific, efficient and reproducible with this multi-lectin column. The results obtained with this affinity step indicated that about 10% of human serum proteins are glycosylated (weight/weight) and, after removal of six high abundance proteins, including albumin, at least 50% of the remaining proteins were glycosylated. We then evaluated the use of this affinity column to monitor changes in the pattern of glycosylation in serum samples by a controlled, stepwise release of the captured proteins from the multi-lectin affinity column with specific displacers.     

Protein glycosylation: new challenges and opportunities

Protein glycosylation is the most complex post-translational modification process. More than 50% of proteins in humans are glycosylated, while bacteria such as E. coli does not have this modification machinery. Many small-molecule natural products also require glycosylation in order to express their function. Development of effective synthetic tools for use in understanding the effect of glycosylation on the structure and function of biomolecules will lead to the development of new strategies to tackle major problems associated with carbohydrate-mediated biological recognitions.     

Preparing to read the ubiquitin code: a middle‐out strategy for characterization of all lysine‐linked diubiquitins

Multiple studies demonstrate that ubiquitination of proteins codes for regulation of cell differentiation, apoptosis, endocytosis and many other cellular functions. There is great interest in and considerable effort being given to defining the relationships between the structures of polyubiquitin modifications and the fates of the modified proteins. Does each ubiquitin modification achieve a specific effect, much like phosphorylation, or is ubiquitin like glycosylation, where there is heterogeneity and redundancy in the signal? The sensitive analytical tools needed to address such questions readily are not yet mature. To lay the foundation for mass spectrometry (MS)‐based studies of the ubiquitin code, we have assembled seven isomeric diubiquitins with all‐native sequences and isopeptide linkages. Using these compounds as standards enables the development and testing of a new MS‐based strategy tailored specifically to characterize the number and sites of isopeptide linkages in polyubiquitin chains. Here, we report the use of Asp‐selective acid cleavage, separation by reverse phase high‐performance liquid chromatography and characterization by tandem MS to distinguish and characterize all seven isomeric lysine‐linked ubiquitin dimers. Copyright © 2014 John Wiley & Sons, Ltd.     

Glycosylation of the Arg-gingipains of Porphyromonas gingivalis and comparison with glycoconjugate structure and synthesis in other bacteria

Post-translational modification of proteins by covalent attachment of sugars to the protein backbone (protein glycosylation) is the most common post-translational modification in the eucaryotic cell. However, the addition of carbohydrates to proteins of Eubacteria and Archaea has been demonstrated and accepted only recently. There is now a rapidly expanding list of bacterial glycoproteins that have been characterised from a variety of different organisms including many important pathogens. The Arg-gingipains of Porphyromonas gingivalis are recent additions to this list. In this review we present a summary of our investigations on the structure of the glycan additions to these proteolytic enzymes, the genetics of the glycosylation process and some of the effects on enzyme function and recognition. These findings are placed in the context of the current status of understanding of glycoconjugate structure and synthesis in other bacteria. Given the importance of glycosylation of eucaryotic proteins to their stability, structure, resistance to proteolysis and recognition, the modifications to the proteases described in the present report are likely to have a functional role in the properties of these enzymes in periodontal disease.     

Glycosyl phenylthiosulfonates (glyco-PTS): novel reagents for glycoprotein synthesis

Controlled site-selective glycosylation can be achieved by combining site-directed cysteine mutagenesis with chemical modification of the introduced thiol; a new class of more efficient chemoselective reagents, glycosyl phenylthiosulfonates, allow rapid glycosylations of representative simple thiols, peptides and proteins.     

Decellularization of intact tissue enables MALDI imaging mass spectrometry analysis of the extracellular matrix

Matrix‐assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) is a powerful molecular mapping technology that offers unbiased visualization of the spatial arrangement of biomolecules in tissue. Although there has been a significant increase in the number of applications employing this technology, the extracellular matrix (ECM) has received little attention, likely because ECM proteins are mostly large, insoluble and heavily cross‐linked. We have developed a new sample preparation approach to enable MALDI IMS analysis of ECM proteins in tissue. Prior to freezing and sectioning, intact tissues are decellularized by incubation in sodium dodecyl sulfate. Decellularization removes the highly abundant, soluble species that dominate a MALDI IMS spectrum while preserving the structural integrity of the ECM. In situ tryptic hydrolysis and imaging of tryptic peptides are then carried out to accommodate the large sizes of ECM proteins. This new approach allows the use of MALDI IMS for identification of spatially specific changes in ECM protein expression and modification in tissue. Copyright © 2015 John Wiley & Sons, Ltd.     

A bioinformatics approach to the development of immunoassays for specified risk material in canned meat products

A bioinformatics approach to developing antibodies to specific proteins has been evaluated for the production of antibodies to heat-processed specified risk tissues from ruminants (brain and eye tissue). The approach involved the identification of proteins specific to ruminant tissues by interrogation of the annotation fields within the Swissprot database. These protein sequences were then interrogated for peptide sequences that were unique to the protein. Peptides were selected that met these criteria as close as possible and that were also theoretically resistant to either pepsin or trypsin. The selected peptides were synthesised and used as immunogens to raise monoclonal antibodies. Antibodies specific for the synthetic peptides were raised to half of the selected peptides. These antibodies have each been incorporated into a competitive enzyme-linked immunosorbent assay (ELISA) and shown to be able to detect the heat-processed parent protein after digestion with either pepsin or trypsin. One antibody, specific for alpha crystallin peptide (from bovine eye tissue), was able to detect the peptide in canned meat products spiked with 10% eye tissue. These results, although preliminary in nature, show that bioinformatics in conjunction with enzyme digestion can be used to develop ELISA for proteins in high-temperature processed foods and demonstrate that the approach is worth further study.     

Carbohydrate analysis of glycoproteins A review

Many of the products prepared by biotechnological approaches, including recombinant genetic engineering, cell tissue culture, and monoclonal technologies, are glycoproteins. As little as five years ago, glycosylation was believed to play no significant role in the function of glycoproteins. Recent large scale testing of glycoprotein-based pharmaceuticals has indicated that both the extent and type of glycosylation can play a central role in glycoprotein activity. Although methods for compositional and sequence analysis of proteins and nucleic acids are generally available, similar methods have yet to be developed for carbohydrate oligomers and polymers. This review focuses on new, developing methods for the analysis and sequencing of the carbohydrate portion of glycoproteins. Included are: (1) the release of oligosaccharides and hydrolysis of carbohydrate chains using enzymatic and chemical methods; (2) fractionation by LPLC, electrophoresis, HPLC, and lectin affinity chromatography; (3) detection through the preparation of derivatives or by new electrochemical methods; (4) analysis by spectroscopic methods, including MS and high-field NMR; and (5) their sequencing through the use of multiple, well-integrated techniques. The ultimate goal of the analytical approaches discussed is to firmly establish structure and, thus, permit the study of structure-function relationships and eventually to allow the intelligent application of carbohydrate remodeling techniques in the preparation of new glycoproteins.     

Selective in vitro glycosylation of recombinant proteins: semi-synthesis of novel homogeneous glycoforms of human erythropoietin

BACKGROUND: A natural glycoprotein usually exists as a spectrum of glycosylated forms, where each protein molecule may be associated with an array of oligosaccharide structures. The overall range of glycoforms can have a variety of different biophysical and biochemical properties, although details of structure-function relationships are poorly understood, because of the microheterogeneity of biological samples. Hence, there is clearly a need for synthetic methods that give access to natural and unnatural homogeneously glycosylated proteins. The synthesis of novel glycoproteins through the selective reaction of glycosyl iodoacetamides with the thiol groups of cysteine residues, placed by site-directed mutagenesis at desired glycosylation sites has been developed. This provides a general method for the synthesis of homogeneously glycosylated proteins that carry saccharide side chains at natural or unnatural glycosylation sites. Here, we have shown that the approach can be applied to the glycoprotein hormone erythropoietin, an important therapeutic glycoprotein with three sites of N-glycosylation that are essential for in vivo biological activity. RESULTS: Wild-type recombinant erythropoietin and three mutants in which glycosylation site asparagine residues had been changed to cysteines (His(10)-WThEPO, His(10)-Asn24Cys, His(10)-Asn38Cys, His(10)-Asn83CyshEPO) were overexpressed and purified in yields of 13 mg l(-1) from Escherichia coli. Chemical glycosylation with glycosyl-beta-N-iodoacetamides could be monitored by electrospray MS. Both in the wild-type and in the mutant proteins, the potential side reaction of the other four cysteine residues (all involved in disulfide bonds) were not observed. Yield of glycosylation was generally about 50% and purification of glycosylated protein from non-glycosylated protein was readily carried out using lectin affinity chromatography. Dynamic light scattering analysis of the purified glycoproteins suggested that the glycoforms produced were monomeric and folded identically to the wild-type protein. CONCLUSIONS: Erythropoietin expressed in E. coli bearing specific Asn-->Cys mutations at natural glycosylation sites can be glycosylated using beta-N-glycosyl iodoacetamides even in the presence of two disulfide bonds. The findings provide the basis for further elaboration of the glycan structures and development of this general methodology for the synthesis of semi-synthetic glycoproteins.     

Identification of new<Emphasis Type="Italic"> O</Emphasis>-GlcNAc modified proteins using a click-chemistry-based tagging

The O-linked β-N-acetylglucosamine (O-GlcNAc) modification is an abundant post-translational modification in eukaryotic cells. This dynamic glycosylation plays a fundamental role in the activity of many nuclear and cytoplasmic proteins and is associated with pathologies like type II diabetes, Alzheimer’s disease or some cancers. However the exact link between O-GlcNAc-modified proteins and their function in cells is largely undefined for most cases. Here we report a strategy based on the 1,3-dipolar cycloaddition, called click chemistry, between unnatural N-acetylglucosamine (GlcNAc) analogues (substituted with an azido or alkyne group) and the corresponding biotinylated probe to specifically detect, enrich and identify O-GlcNAc-modified proteins. This bio-orthogonal conjugation confirms that only azido analogue of GlcNAc is metabolized by the cell. Thanks to the biotin probe, affinity purification on streptavidin beads allowed us to identify 32 O-GlcNAc-azido-tagged proteins by LC-MS/MS analysis in an MCF-7 cellular model, 14 of which were previously unreported. This work illustrates the use of the click-chemistry-based strategy combined with a proteomic approach to get further insight into the pattern of O-GlcNAc-modified proteins and the biological significance of this post-translational modification. Figure Detection of biotinylated O-GlcNAz proteins in MCF-7 cells Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Caroline Gurcel and Anne-Sophie Vercoutter-Edouart contributed equally to this work.     

“Clickable” and Antifouling Block Copolymer Brushes as a Versatile Platform for Peptide‐Specific Cell Attachment

To tailor cell–surface interactions, precise and controlled attachment of cell‐adhesive motifs is required, while any background non‐specific cell and protein adhesion has to be blocked effectively. Herein, a versatile and highly reproducible antifouling surface modification based on “clickable” groups and hierarchically structured diblock copolymer brushes for the controlled attachment of cells is reported. The polymer brush architecture combines an antifouling bottom block of poly(2‐hydroxyethyl methacrylate) poly(HEMA) and an ultrathin azide‐bearing top block, which can participate in well‐established “click” reactions including the highly selective copper‐catalyzed alkyne‐azide cycloaddition (CuAAC) reaction under mild conditions. This straightforward approach allows the rapid conjugation of a cell‐adhesive, alkyne‐bearing cyclic RGD peptide motif, enabling subsequent specific attachment of NIH 3T3 fibroblasts, their extensive proliferation and confluent cell sheet formation after 48 h of incubation. The generally applicable strategy presented in this report can be employed for surface functionalization with diverse alkyne‐bearing biological moieties via CuAAC or copper‐free alkyne‐azide cycloaddition protocols, making it a versatile functionalization approach and a promising tool for tissue engineering, biomaterial implant design, and other applications that require surfaces supporting highly specific cell attachment.     

基于三甲基苯磺酰羟胺消除反应的氧连接氮乙酰葡萄糖胺修饰肽段的精准鉴定

氧连接氮乙酰葡萄糖胺(O-GlcNAc)是一种重要的蛋白质翻译后修饰,它在维持机体正常的生命活动中发挥着重要作用。许多研究证实,O-GlcNAc糖基化修饰稳态的破坏与人类多种疾病的发生相关,大规模富集鉴定O-GlcNAc糖基化修饰蛋白有助于发现新的临床疾病诊断标志物。由于O-GlcNAc糖基化修饰丰度较低,形成的糖苷键不稳定,O-GlcNAc糖基化修饰蛋白/肽段的富集鉴定面临一定挑战。近年来,全乙酰化的非天然糖代谢标记技术被广泛应用于O-GlcNAc糖基化修饰蛋白/肽段的富集鉴定。然而,最新的研究发现,在细胞代谢标记过程中,全乙酰化的非天然单糖会同时标记半胱氨酸的巯基而引入半胱氨酸巯基-叠氮糖人为修饰物。该副反应在一定程度上干扰了O-GlcNAc糖基化修饰蛋白/肽段的富集鉴定。鉴于此,研究发展了一种通过三甲基苯磺酰羟胺(MSH)特异性氧化消除半胱氨酸巯基-叠氮糖人为修饰物的方法,进而显著提高O-GlcNAc糖基化修饰肽段的精准鉴定。该方法建立于温和的磷酸钠缓冲液(50 mmol/L, pH=8)体系,利用过量的MSH,于95 ℃避光振荡反应30 min,可完全消除半胱氨酸巯基-叠氮糖人为修饰物。该方法应用于Hela细胞中,可有效消除叠氮全乙酰化半乳糖胺(Ac₄GalNAz)代谢产生的半胱氨酸巯基-叠氮糖人为修饰物,从而成功富集鉴定到157条O-GlcNAc糖基化修饰肽段,归属于130个蛋白质。该方法有效去除了半胱氨酸巯基-叠氮糖人为修饰物对代谢标记结果的干扰,为非天然糖代谢标记技术在糖蛋白组学分析中的应用提供了新的研究策略。     

Site-selective glycosylation of hemoglobin with variable molecular weight oligosaccharides: potential alternative to PEGylation

Poly(ethylene glycol) (PEG) conjugation (i.e., PEGylation) is a commonly used strategy to increase the circulatory half-life of therapeutic proteins and colloids; however, few viable alternatives exist to replicate its functions. Herein, we report a method for the rapid site-selective glycosylation of proteins with variously sized carbohydrates, up to a molecular weight (MW) of 10,000, thus serving as a potential alternative for PEGylation. More importantly, the method developed has two unique features. First, traditional protecting group strategies that typically accompany the modification of the carbohydrate fragments are circumvented, allowing for the facile site-selective glycosylation of a desired protein with variously sized glycans. Second, the methodology employed is not limited by oligosaccharide size; consequently, glycans of MW similar to that of PEG, used in the PEGylation of therapeutic proteins, can be employed. To demonstrate the usefulness of this technology, hemoglobin (Hb) was site-selectively glycosylated with a series of carbohydrates of increasing MW (from 504 to ～10,000). Hb was selected on the basis of the vast wealth of biochemical and biophysical knowledge present in the literature and because of its use as a precursor in the synthesis/formulation of artificial red blood cell substitutes. Following the successful site-selective glycosylation of Hb, the impact of increasing the glycan MW on Hb's biophysical properties was investigated in vitro.     

Differential Glycosite Profiling—A Versatile Method to Compare Membrane Glycoproteomes

Glycosylation is the most prevalent and varied form of post-translational protein modifications. Protein glycosylation regulates multiple cellular functions, including protein folding, cell adhesion, molecular trafficking and clearance, receptor activation, signal transduction, and endocytosis. In particular, membrane proteins are frequently highly glycosylated, which is both linked to physiological processes and of high relevance in various disease mechanisms. The cellular glycome is increasingly considered to be a therapeutic target. Here we describe a new strategy to compare membrane glycoproteomes, thereby identifying proteins with altered glycan structures and the respective glycosites. The workflow started with an optimized procedure for the digestion of membrane proteins followed by the lectin-based isolation of glycopeptides. Since alterations in the glycan part of a glycopeptide cause mass alterations, analytical size exclusion chromatography was applied to detect these mass shifts. N-glycosidase treatment combined with nanoUPLC-coupled mass spectrometry identified the altered glycoproteins and respective glycosites. The methodology was established using the colon cancer cell line CX1, which was treated with 2-deoxy-glucose—a modulator of N-glycosylation. The described methodology is not restricted to cell culture, as it can also be adapted to tissue samples or body fluids. Altogether, it is a useful module in various experimental settings that target glycan functions.