Recent advances in methods for the analysis of protein o‐glycosylation at proteome level

O‐Glycosylation, which refers to the glycosylation of the hydroxyl group of side chains of Serine/Threonine/Tyrosine residues, is one of the most common post‐translational modifications. Compared with N‐linked glycosylation, O‐glycosylation is less explored because of its complex structure and relatively low abundance. Recently, O‐glycosylation has drawn more and more attention for its various functions in many sophisticated biological processes. To obtain a deep understanding of O‐glycosylation, many efforts have been devoted to develop effective strategies to analyze the two most abundant types of O‐glycosylation, i.e. O‐N‐acetylgalactosamine and O‐N‐acetylglucosamine glycosylation. In this review, we summarize the proteomics workflows to analyze these two types of O‐glycosylation. For the large‐scale analysis of mucin‐type glycosylation, the glycan simplification strategies including the ‘‘SimpleCell’’ technology were introduced. A variety of enrichment methods including lectin affinity chromatography, hydrophilic interaction chromatography, hydrazide chemistry, and chemoenzymatic method were introduced for the proteomics analysis of O‐N‐acetylgalactosamine and O‐N‐acetylglucosamine glycosylation.     

Glycosylation characterization of recombinant human erythropoietin produced in glycoengineered Pichia pastoris by mass spectrometry

Glycosylation plays a critical role in the in vivo efficacy of both endogenous and recombinant erythropoietin (EPO). Using mass spectrometry, we characterized the N‐/O‐linked glycosylation of recombinant human EPO (rhEPO) produced in glycoengineered Pichia pastoris and compared with the glycosylation of Chinese hamster ovary (CHO) cell‐derived rhEPO. While the three predicted N‐linked glycosylation sites (Asn24, Asn38 and Asn83) showed complete site occupancy, Pichia‐ and CHO‐derived rhEPO showed distinct differences in the glycan structures with the former containing sialylated bi‐antennary glycoforms and the latter containing a mixture of sialylated bi‐, tri‐ and tetra‐antennary structures. Additionally, the N‐linked glycans from Pichia‐produced rhEPO were similar across all three sites. A low level of O‐linked mannosylation was detected on Pichia‐produced rhEPO at position Ser126, which is also the O‐linked glycosylation site for endogenous human EPO and CHO‐derived rhEPO. In summary, the mass spectrometric analyses revealed that rhEPO derived from glycoengineered Pichia has a highly uniform bi‐antennary N‐linked glycan composition and preserves the orthogonal O‐linked glycosylation site present on endogenous human EPO and CHO‐derived rhEPO. Copyright © 2013 John Wiley & Sons, Ltd.     

Effect of the elapsed time between sampling and formalin fixation on the N‐glycosylation profile of mouse tissue specimens

Formalin‐fixed, paraffin‐embedded (FFPE) samples are generally used for histology‐study, however, they also possess important molecular diagnostics information. While it has been reported that the N‐glycan moieties of glycoproteins is not affected by the FFPE process, no information is available about the effect of the elapsed time between sampling and fixation on the resulting N‐glycosylation profile. In this study, lung, brain, heart, spleen, liver, kidney, and intestine mouse tissue specimens were used for N‐glycan profiling analysis and the elapsed sampling time effect was investigated with the lung tissue. N‐glycan extraction from the tissue samples was performed by glycoprotein retrieval from the FFPE specimens using radioimmunoprecipitation assay (RIPA) buffer followed PNGase F digestion. The released oligosaccharides were fluorophore labeled and analyzed by capillary electrophoresis‐laser induced fluorescent detection (CE‐LIF). N‐glycosylation profiles of freshly collected lung‐tissue samples (zero time point), as well as 1 and 2 h after sampling were compared by carbohydrate profiling and exoglycosidase treatment based deep glycomic analysis. It was found that up to two hours of room temperature storage of tissue specimens apparently did not cause changes in the N‐glycosylation profiles of complex carbohydrates, but resulted in considerable decrease in the amount of linear glucose oligomers and high mannose type glycans present in the samples.     

The Implications of (2S,4S)‐Hydroxyproline 4‐O‐Glycosylation for Prolyl Amide Isomerization

The conformations of peptides and proteins are often influenced by glycans O‐linked to serine (Ser) or threonine (Thr). (2S,4R)‐4‐Hydroxyproline (Hyp), together with L ‐proline (Pro), are interesting targets for O‐glycosylation because they have a unique influence on peptide and protein conformation. In previous work we found that glycosylation of Hyp does not affect the N‐terminal amide trans/cis ratios (K_trans/cis) or the rates of amide isomerization in model amides. The stereoisomer of Hyp—(2S,4S)‐4‐hydroxyproline (hyp)—is rarely found in nature, and has a different influence both on the conformation of the pyrrolidine ring and on K_trans/cis. Glycans attached to hyp would be expected to be projected from the opposite face of the prolyl side chain relative to Hyp; the impact this would have on K_trans/cis was unknown. Measurements of ³J coupling constants indicate that the glycan has little impact on the C^γ‐endo conformation produced by hyp. As a result, it was found that the D ‐galactose residue extending from a C^γ‐endo pucker affects both K_trans/cis and the rate of isomerization, which is not found to occur when it is projected from a C^γ‐exo pucker; this reflects the different environments delineated by the proline side chain. The enthalpic contributions to the stabilization of the trans amide isomer may be due to disruption of intramolecular interactions present in hyp; the change in enthalpy is balanced by a decrease in entropy incurred upon glycosylation. Because the different stereoisomers—Hyp and hyp—project the O‐linked carbohydrates in opposite spatial orientations, these glycosylated amino acids may be useful for understanding of how the projection of a glycan from the peptide or protein backbone exerts its influence.     

Total Synthesis of a Hyperbranched N‐Linked Hexasaccharide Attached to ATCV‐1 Major Capsid Protein without Precedent

The N‐glycans attached to some chloroviruses comprise a hyperbranched core structure without precedent. We are interested in the chemical synthesis of the hexasaccharide attached to ATCV‐1 (Acanthocystis turfacea Chlorella virus 1) for its distinct structure. After exploring four routes, the target hexasaccharide 2 was successfully synthesized for the first time in overall 10% yield over 8 steps from thioglycoside building blocks. This synthetic protocol is characterized by the three‐component one‐pot glycosylation and the regioselective glycosylation reactions. The disclosed synthetic approach to this new type of N‐glycans will facilitate the in‐depth understanding of their biological functions.     

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.     

Chemical Synthesis of Asparagine‐Linked Archaeal N‐Glycan from Methanothermus fervidus

Several N‐linked glycoproteins have been identified in archaea and there is growing evidence that the N‐glycan is involved in survival and functioning of archaea in extreme conditions. Chemical synthesis of the archaeal N‐glycans represents a crucial step towards understanding the putative function of protein glycosylation in archaea. Herein the first total synthesis of the archaeal L ‐asparagine linked hexasaccharide from Methanothermus fervidus is reported using a highly convergent [3+3] glycosylation approach in high overall yields. The synthesis relies on efficient preparation of regioselectively protected thioglycoside building blocks for orthogonal glycosylations and late stage N‐aspartylation.     

Nucleophile‐Directed Stereocontrol Over Glycosylations Using Geminal‐Difluorinated Nucleophiles

The glycosylation reaction is the key transformation in oligosaccharide synthesis, but it is still difficult to control in many cases. Stereocontrol during cis‐glycosidic linkage formation relies almost exclusively on tuning the glycosylating agent or the reaction conditions. Herein, we use nucleophile‐directed stereocontrol to manipulate the stereoselectivity of glycosylation reactions. Placing two fluorine atoms in close proximity to the hydroxy group of an aliphatic amino alcohol lowers the oxygen nucleophilicity and reverses the stereoselectivity of glycosylations to preferentially form the desired cis‐glycosides with a broad set of substrates. This concept was applied to the design of a cis‐selective linker for automated glycan assembly. Fluorination of an amino alcohol linker does not impair glycan immobilization and lectin binding as illustrated by glycan microarray experiments. These fluorinated linkers enable the facile generation of α‐terminating synthetic glycans for the formation of glycoconjugates.     

Divergent Chemoenzymatic Synthesis of Asymmetrical‐Core‐Fucosylated and Core‐Unmodified N‐Glycans

A divergent chemoenzymaytic approach for the preparation of core‐fucosylated and core‐unmodified asymmetrical N‐glycans from a common advances precursor is described. An undecasaccharide was synthesized by sequential chemical glycosylations of an orthogonally protected core fucosylated hexasaccharide that is common to all mammalian core fucosylated N‐glycans. Antennae‐selective enzymatic extension of the undecasaccharide using a panel of glycosyl transferases afforded core fucosylated asymmetrical triantennary N‐glycan isomers, which are potential biomarkers for breast cancer. A unique aspect of our approach is that a fucosidase (FucA1) has been identified that selectively can cleave a core‐fucoside without affecting the fucoside of a sialyl Lewis^X epitope to give easy access to core‐unmodified compounds.     

Synthesis of a Sialic Acid Containing Complex‐Type N‐Glycan on a Solid Support

On solid ground : A new solid‐phase synthesis of N‐linked glycans featuring 1) highly stereoselective β‐mannosylation and microfluidic α‐sialylation and 2) efficient glycosylation of the N‐phenyltrifluoroacetimidate units on JandaJel resin is reported. Reagent concentration effects by a fluorous solvent are effectively applied, and the use of these methods results in the first synthesis of a sialic acid containing complex‐type N‐glycan on a solid support.

     

Dinuclear Complexes of Copper(I) with Crown Ether‐containing N‐Thiophosphorylated Bis‐thioureas and 2,2′‐Bipyridine or 1,10‐Phenanthroline: Synthesis,Characterization, and Picrate Extraction Properties

Reaction of O,O′‐diisopropylthiophosphoric acid isothiocyanate (iPrO)₂P(S)NCS with 1,10‐diaza‐18‐crown‐6, 1,7‐diaza‐18‐crown‐6, or 1,7‐diaza‐15‐crown‐5 leads to the N‐thiophosphorylated bis‐thioureas N,N′‐bis[C(S)NHP(S)(OiPr)₂]‐1,10‐diaza‐18‐crown‐6 ( H₂L^I ), N,N′‐bis[C(S)NHP(S)(OiPr)₂]‐1,7‐diaza‐18‐crown‐6 ( H₂L^II ) and N,N′‐bis[C(S)NHP(S)(OiPr)₂]‐1,7‐diaza‐15‐crown‐5 ( H₂L^III ). Reaction of the potassium salts of H₂L^I–III with a mixture of CuI and 2,2′‐bipyridine ( bpy ) or 1,10‐phenanthroline ( phen ) in aqueous EtOH/CH₂Cl₂ leads to the dinuclear complexes [Cu₂(bpy)₂L^I–III] and [Cu₂(phen)₂L^I–III] . The structures of these compounds were investigated by ¹H, ³¹P{¹H} NMR spectroscopy, and elemental analysis. The crystal structures of H₂L^I and [Cu₂(phen)₂L^I] were determined by single‐crystal X‐ray diffraction. Extraction capacities of the obtained compounds in comparison to the related compounds 1,10‐diaza‐18‐crown‐6, N,N′‐bis[C(=CMe₂)CH₂P(O)(OiPr)₂]‐1,10‐diaza‐18‐crown‐6, N,N′‐bis[C(S)NHP(O)(OiPr)₂]‐1,10‐diaza‐18‐crown‐6 towards the picrate salts LiPic, NaPic, KPic. and NH₄Pic were also studied.     

A Unified Strategy for the Synthesis of Mucin Cores 1–4 Saccharides and the Assembled Multivalent Glycopeptides

By displaying different O‐glycans in a multivalent mode, mucin and mucin‐like glycoproteins are involved in a plethora of protein binding events. The understanding of the roles of the glycans and the identification of potential glycan binding proteins are major challenges. To enable future binding studies of mucin glycan and glycopeptide probes, a method that gives flexible and efficient access to all common mucin core‐glycosylated amino acids was developed. Based on a convergent synthesis strategy starting from a shared early stage intermediate by differentiation in the glycoside acceptor reactivity, a common disaccharide building block allows for the creation of extended glycosylated amino acids carrying the mucin type‐2 cores 1–4 saccharides. Formation of a phenyl‐sulfenyl‐N‐Troc (Troc=trichloroethoxycarbonyl) byproduct during N‐iodosuccinimide‐promoted thioglycoside couplings was further characterized and a new methodology for the removal of the Troc group is described. The obtained glycosylated 9‐fluorenylmethoxycarbonyl (Fmoc)‐protected amino acid building blocks are incorporated into peptides for multivalent glycan display.     

Selective Inhibition of Aggregation and Toxicity of a Tau‐Derived Peptide using Its Glycosylated Analogues

Protein glycosylation is a ubiquitous post‐translational modification that regulates the folding and function of many proteins. Misfolding of protein monomers and their toxic aggregation are the hallmark of many prevalent diseases. Thus, understanding the role of glycans in protein aggregation is highly important and could contribute both to unraveling the pathology of protein misfolding diseases as well as providing a means for modifying their course for therapeutic purposes. Using β‐O‐linked glycosylated variants of the highly studied Tau‐derived hexapeptide motif VQIVYK, which served as a simplified amyloid model, we demonstrate that amyloid formation and toxicity can be strongly attenuated by a glycan unit, depending on the nature of the glycan itself. Importantly, we show for the first time that not only do glycans hinder self‐aggregation, but the glycosylated peptides are capable of inhibiting aggregation of the non‐modified corresponding amyloid scaffold.     

N‐ and O‐linked glycosylation site profiling of the human basic salivary proline‐rich protein 3M

In the present study, we show that the heterogeneous mixture of glycoforms of the basic salivary proline‐rich protein 3M, encoded by PRB3‐M locus, is a major component of the acidic soluble fraction of human whole saliva in the first years of life. Reversed‐phase high‐performance liquid chromatography with high‐resolution electrospray ionization mass spectrometry analysis of the intact proteoforms before and after N‐deglycosylation with Peptide‐N‐Glycosidase F and tandem mass spectrometry sequencing of peptides obtained after Endoproteinase GluC digestion allowed the structural characterization of the peptide backbone and identification of N‐ and O‐glycosylation sites. The heterogeneous mixture of the proteoforms derives from the combination of 8 different neutral and sialylated glycans O‐linked to Threonine 50, and 33 different glycans N‐linked to Asparagine residues at positions 66, 87, 108, 129, 150, 171, 192, and 213.     

β‐Selective Glycosylation by Using O‐Aryl‐Protected Glycosyl Donors

New glycosyl donors have been developed that contained several para‐substituted O‐aryl protecting groups and their stereoselectivity for the glycosylation reaction was evaluated. A highly β‐selective glycosylation reaction was achieved by using thioglycosides that were protected by 4‐nitrophenyl (NP) groups, which were introduced by using the corresponding diaryliodonium triflate. Analysis of the stereoselectivities of several glycosyl donors indicated that the β‐glycosides were obtained through an S_N2‐type displacement from the corresponding α‐glycosyl triflate. The NP group could be removed by reduction of the nitro group and acylation, followed by oxidation with ceric ammonium nitrate (CAN).     

Highly Stereoselective β‐Mannopyranosylation via the 1‐α‐Glycosyloxy‐isochromenylium‐4‐gold(I) Intermediates

While the gold(I)‐catalyzed glycosylation reaction with 4,6‐O‐benzylidene tethered mannosyl ortho‐alkynylbenzoates as donors falls squarely into the category of the Crich‐type β‐selective mannosylation when Ph₃PAuOTf is used as the catalyst, in that the mannosyl α‐triflates are invoked, replacement of the ^?OTf in the gold(I) complex with less nucleophilic counter anions (i.e., ^?NTf₂, ^?SbF₆, ^?BF₄, and ^?BAr₄^F) leads to complete loss of β‐selectivity with the mannosyl ortho‐alkynylbenzoate β‐donors. Nevertheless, with the α‐donors, the mannosylation reactions under the catalysis of Ph₃PAuBAr₄^F (BAr₄^F=tetrakis[3,5‐bis(trifluoromethyl)phenyl]borate) are especially highly β‐selective and accommodate a broad scope of substrates; these include glycosylation with mannosyl donors installed with a bulky TBS group at O3, donors bearing 4,6‐di‐O‐benzoyl groups, and acceptors known as sterically unmatched or hindered. For the ortho‐alkynylbenzoate β‐donors, an anomerization and glycosylation sequence can also ensure the highly β‐selective mannosylation. The 1‐α‐mannosyloxy‐isochromenylium‐4‐gold(I) complex ( Cα ), readily generated upon activation of the α‐mannosyl ortho‐alkynylbenzoate ( 1 α ) with Ph₃PAuBAr₄^F at ?35 °C, was well characterized by NMR spectroscopy; the occurrence of this species accounts for the high β‐selectivity in the present mannosylation.     

天然产物 6-脱氧-6氨基葡萄糖甘油糖脂的合成

天然氨基甘油糖脂sn-1,2-dipalmitoyl-3-(N-palmitoyl-6-dehydroxy-6-amino-α-glucosyl)glycerol 3 和 sn-1-palmitoyl-2-myristoyl-3-(N-stearoyl-6-dehydroxy-6-amino-α-glucosyl)glycerol 4 通过简便有效的合成策略首次被合成。其关键步骤为：三氯亚胺酯糖基供体 10 与 (S)-isopropyleneglycerol 在乙醚溶液中发生糖苷化反应,立体选择性的生成3-O-(2,3,4-tri-O-benzyl-6-dehydroxy-6-benzyloxycarbonylamino-α-D- glucopyranoyl)-1,2-O-isopropylene-sn- glycerol 7。中间体 7 经过脱除丙酮叉、与不同的脂肪酸缩合、脱除保护基和选择性的在氨基上酰化,最终得到目标化合物 3 和 4。     

Qualitative and quantitative comparison of N‐glycans between pig endothelial and islet cells by high‐performance liquid chromatography and mass spectrometry‐based strategy

N‐glycan structures released from miniature pig endothelial and islet cells were determined by matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF), negative ion electrospray ionization (ESI) MS/MS and normal‐phase high performance liquid chromatography (NP‐HPLC) combined with exoglycosidase digestion. Totally, the identified structures were 181 N‐glycans including 129 sialylated and 18 α‐galactosylated glycans from pig endothelial cells and 80 N‐glycans including 41 sialylated and one α‐galactosylated glycans from pig islet cells. The quantity of the α‐galactosylated glycans from pig islet cells was certainly neglectable compared to pig endothelial cells. A number of NeuGc‐terminated N‐glycans (80 from pig endothelial cells and 13 from pig islet cells) are newly detected by our mass spectrometric strategies. The detailed structural information will be a matter of great interest in organ or cell xenotransplantation using α 1,3‐galactosyltransferase gene‐knockout (GalT‐KO) pig. Copyright © 2009 John Wiley & Sons, Ltd.     

Construction of a High‐Mannose‐Type Glycan Library by a Renewed Top‐Down Chemo‐Enzymatic Approach

A comprehensive method for the construction of a high‐mannose‐type glycan library by systematic chemo‐enzymatic trimming of a single Man9‐based precursor was developed. It consists of the chemical synthesis of a non‐natural tridecasaccharide precursor, the orthogonal demasking of the non‐reducing ends, and trimming by glycosidases, which enabled a comprehensive synthesis of high‐mannose‐type glycans in their mono‐ or non‐glucosylated forms. It employed glucose, isopropylidene, and N‐acetylglucosamine groups for blocking the A‐, B‐, and C‐arms, respectively. After systematic trimming of the precursor, thirty‐seven high‐mannose‐type glycans were obtained. The power of the methodology was demonstrated by the enzymatic activity of human recombinant N‐acetylglucosaminyltransferase‐I toward M7–M3 glycans, clarifying the substrate specificity in the context of high‐mannose‐type glycans.     

{Bis­[2‐(2‐oxido‐1‐naphthylmethylidene­amino)­phenyl] disulfide}chloroiron(III)

The crystal structure of the title compound, {bis­[2‐(2‐oxido‐2‐naphthyl­idene­amino)­phenyl] di­sulfide‐κ⁵O,N,S,N′,O′}chloroiron(III), [FeCl(C₃₄H₂₂N₂O₂S₂)], has been determined. The structure consists of monomeric iron(III) complexes with distorted octahedral coordination. The di­sulfide functions as a pentadentate ligand and the Fe^III atom is coordinated through two N, two O and one S atom, and one chloride ion. The distance between the second S atom and the Fe^III atom is a non‐bonding 3.8473 (14) Å.