MALDI mass spectrometry‐based sequence analysis of arginine‐containing glycopeptides: improved fragmentation of glycan and peptide chains by modifying arginine residue

This paper describes an improved method for the sequence analysis of Arg‐containing glycopeptide by MALDI mass spectrometry (MS). The method uses amino group derivatization (4‐aza‐6‐(2,6‐dimethyl‐1‐piperidinyl)‐5‐oxohexanoic acid N‐succinimidyl ester) and removal (carboxypeptidase B) or modification (peptidylarginine deiminase 4) of the arginine residue of the peptide. The derivatization attaches a basic tertiary amine moiety onto the peptides, and the enzymatic treatment removes or modifies the arginine residue. Fragmentation of the resulting glycopeptide under low‐energy collision‐induced dissociation yielded a simplified ion series of both the glycan and the peptide that can facilitate their sequencing. The feasibility of the method was studied using α₁‐acid glycoprotein‐derived N‐linked glycopeptides, and glycan and peptide in each glycopeptide were successfully sequenced by MALDI tandem MS (MS/MS). Copyright © 2013 John Wiley & Sons, Ltd.     

Quantitative mapping of glycoprotein micro‐heterogeneity and macro‐heterogeneity: an evaluation of mass spectrometry signal strengths using synthetic peptides and glycopeptides

Mass spectrometry (MS) is used to quantify the relative distribution of glycans attached to particular protein glycosylation sites (micro‐heterogeneity) and evaluate the molar site occupancy (macro‐heterogeneity) in glycoproteomics. However, the accuracy of MS for such quantitative measurements remains to be clarified. As a key step towards this goal, a panel of related tryptic peptides with and without complex, biantennary, disialylated N‐glycans was chemically synthesised by solid‐phase peptide synthesis. Peptides mimicking those resulting from enzymatic deglycosylation using PNGase F/A and endo D/F/H were synthetically produced, carrying aspartic acid and N‐acetylglucosamine‐linked asparagine residues, respectively, at the glycosylation site. The MS ionisation/detection strengths of these pure, well‐defined and quantified compounds were investigated using various MS ionisation techniques and mass analysers (ESI‐IT, ESI‐Q‐TOF, MALDI‐TOF, ESI/MALDI‐FT‐ICR‐MS). Depending on the ion source/mass analyser, glycopeptides carrying complex‐type N‐glycans exhibited clearly lower signal strengths (10–50% of an unglycosylated peptide) when equimolar amounts were analysed. Less ionisation/detection bias was observed when the glycopeptides were analysed by nano‐ESI and medium‐pressure MALDI. The position of the glycosylation site within the tryptic peptides also influenced the signal response, in particular if detected as singly or doubly charged signals. This is the first study to systematically and quantitatively address and determine MS glycopeptide ionisation/detection strengths to evaluate glycoprotein micro‐heterogeneity and macro‐heterogeneity by label‐free approaches. These data form a much needed knowledge base for accurate quantitative glycoproteomics. Copyright © 2013 John Wiley & Sons, Ltd.     

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.     

MALDI‐MS profiling of serum O‐glycosylation and N‐glycosylation in COG5‐CDG

Congenital disorders of glycosylation (CDG) are due to defective glycosylation of glycoconjugates. Conserved oligomeric Golgi (COG)‐CDG are genetic diseases due to defects of the COG complex subunits 1–8 causing N‐glycan and O‐glycan processing abnormalities. In COG‐CDG, isoelectric focusing separation of undersialylated glycoforms of serum transferrin and apolipoprotein C‐III (apoC‐III) allows to detect N‐glycosylation and O‐glycosylation defects, respectively. COG5‐CDG (COG5 subunit deficiency) is a multisystem disease with dysmorphic features, intellectual disability of variable degree, seizures, acquired microcephaly, sensory defects and autistic behavior. We applied matrix‐assisted laser desorption/ionization‐MS for a high‐throughput screening of differential serum O‐glycoform and N‐ glycoform in five patients with COG5‐CDG. When compared with age‐matched controls, COG5‐CDG showed a significant increase of apoC‐III_0a (aglycosylated glycoform), whereas apoC‐III₁ (mono‐sialylated glycoform) decreased significantly. Serum N‐glycome of COG5‐CDG patients was characterized by the relative abundance of undersialylated and undergalactosylated biantennary and triantennary glycans as well as slight increase of high‐mannose structures and hybrid glycans. Using advanced and well‐established MS‐based approaches, the present findings reveal novel aspects on O‐glycan and N‐glycan profiling in COG5‐CDG patients, thus providing an increase of current knowledge on glycosylation defects caused by impairment of COG subunits, in support of clinical diagnosis. Copyright © 2017 John Wiley & Sons, Ltd.     

Distinctive MS/MS Fragmentation Pathways of Glycopeptide‐Generated Oxonium Ions Provide Evidence of the Glycan Structure

Post‐translational glycosylation of proteins play key roles in cellular processes and the site‐specific characterisation of glycan structures is critical to understanding these events. Given the challenges regarding identification of glycan isomers, glycoproteomic studies generally rely on the assumption of conserved biosynthetic pathways. However, in a recent study, we found characteristically different HexNAc oxonium ion fragmentation patterns that depend on glycan structure. Such patterns could be used to distinguish between glycopeptide structural isomers. To acquire a mechanistic insight, deuterium‐labelled glycopeptides were prepared and analysed. We found that the HexNAc‐derived m/z 126 and 144 oxonium ions, differing in mass by H₂O, had completely different structures and that high‐mannose N‐glycopeptides generated abundant Hex‐derived oxonium ions. We describe the oxonium ion decomposition mechanisms and the relative abundance of oxonium ions as a function of collision energy for a number of well‐defined glycan structures, which provide important information for future glycoproteomic studies.     

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.     

Fast Epitope Mapping for the Anti‐MUC1 Monoclonal Antibody by Combining a One‐Bead‐One‐Glycopeptide Library and a Microarray Platform

Anti‐MUC1 monoclonal antibodies (mAbs) are powerful tools that can be used to recognize cancer‐related MUC1 molecules, the O‐glycosylation status of which is believed to affect binding affinity. We demonstrate the feasibility of using a rapid screening methodology to elucidate those effects. The approach involves i) “one‐bead‐one‐compound”‐based preparation of bilayer resins carrying glycopeptides on the shell and mass‐tag tripeptides coding O‐glycan patterns in the core, ii) on‐resin screening with an anti‐MUC1 mAb, iii) separating positive resins by utilizing secondary antibody conjugation with magnetic beads, and (iv) decoding the mass‐tag that is detached from the positive resins pool by using mass spectrometric analysis. We tested a small library consisting of 27 MUC1 glycopeptides with different O‐glycosylations against anti‐MUC1 mAb clone VU‐3C6. Qualitative mass‐tag analysis showed that increasing the number of glycans leads to an increase in the binding affinity. Six glycopeptides selected from the library were validated by using a microarray‐based assay. Our screening provides valuable information on O‐glycosylations of epitopes leading to high affinity with mAb.     

Cross‐metathesis of C‐Glycosides and Peptides

Peptides bearing an acryloyl residue at their N‐terminus were coupled with various C‐glycosides in an equimolar ratio via cross‐metathesis. The newly formed olefin was obtained with high E/Z selectivity in satisfying to high yields with low homodimerization of the starting materials. The posttranslational cross‐metathesis approach was shown to be suitable for the combinatorial synthesis of a small library of C‐glycopeptides.     

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.     

Convergent chemo-enzymatic synthesis of mannosylated glycopeptides; targeting of putative vaccine candidates to antigen presenting cells

The combination of solid phase peptide synthesis and endo-β-N-acetylglucosaminidase (ENGase) catalysed glycosylation is a powerful convergent synthetic method allowing access to glycopeptides bearing full-length N-glycan structures. Mannose-terminated N-glycan oligosaccharides, produced by either total or semi-synthesis, were converted into oxazoline donor substrates. A peptide from the human cytomegalovirus (CMV) tegument protein pp65 that incorporates a well-characterised T cell epitope, containing N-acetylglucosamine at specific Asn residues, was accessed by solid phase peptide synthesis, and used as an acceptor substrate. High-yielding enzymatic glycosylation afforded glycopeptides bearing defined homogeneous high-mannose N-glycan structures. These high-mannose containing glycopeptides were tested for enhanced targeting to human antigen presenting cells (APCs), putatively mediated via the mannose receptor, and for processing by the APCs for presentation to human CD8+ T cells specific for a 9-mer epitope within the peptide. Binding assays showed increased binding of glycopeptides to APCs compared to the non-glycosylated control. Glycopeptides bearing high-mannose N-glycan structures at a single site outside the T cell epitope were processed and presented by the APCs to allow activation of a T cell clone. However, the addition of a second glycan within the T cell epitope resulted in ablation of T cell activation. We conclude that chemo-enzymatic synthesis of mannosylated glycopeptides enhances uptake by human APCs while preserving the immunogenicity of peptide epitopes within the glycopeptides, provided those epitopes are not themselves glycosylated.     

Identification and structural characterization of novel O‐ and N‐glycoforms in the urine of a Schindler disease patient by Orbitrap mass spectrometry

Schindler disease is an inherited metabolic disorder caused by the deficient activity of α‐N‐acetylgalactosaminidase enzyme. An accurate diagnosis requires, besides clinical examination, complex and costly biochemical and molecular genetic tests. In the last years, mass spectrometry (MS) based on nanofluidics and high‐resolution instruments has become a successful alternative for disease diagnosis based on the investigation of O‐glycopeptides in patient urine. A complex mixture of glycoforms extracted from the urine of a 3‐year‐old patient was investigated by Orbitrap MS equipped with Nanospray Flex Ion Source in the negative ion mode. For structural characterization of several molecular species, collision‐induced dissociation MS²–MS³ was carried out using collision energy values within 20–60 eV range. By our approach, 39 novel species associated to this condition were identified, among which O‐glycopeptides, free O‐glycans and one structure corresponding to an N‐glycan never characterized in the context of Schindler disease. The experiments conducted at a resolution of 60 000 allowed the discrimination and identification of a total number of 69 different species with an average mass accuracy of 9.87 ppm, an in‐run reproducibility of almost 100%, an experiment‐to‐experiment and day‐to‐day reproducibility of about 95%. This study brings contributions in the diagnosis of Schindler disease through the elucidation of potential biomarker species in urine. Our multistage MS results completed with 39 new glycoforms the inventory of potential biomarker structures associated to Schindler disease. For the first time, an N‐glycan was identified and structurally characterized in Schindler patient urine, which opens new research directions in the field. Copyright © 2015 John Wiley & Sons, Ltd.     

A Practical One‐Step Synthesis of 1,2‐Oxazoline Derivatives from Unprotected Sugars and Its Application to Chemoenzymatic β‐N‐Acetylglucosaminidation of Disialo‐oligosaccharide

A facile and practical method for synthesis of sugar oxazolines (=dihydrooxazoles) from the corresponding N‐acetyl‐2‐amino sugars has been developed by using 2‐chloro‐1,3‐dimethyl‐1H‐benzimidazol‐3‐ium chloride (CDMBI) as a dehydrative condensing agent. The intramolecular dehydrative reaction between the 2‐acetamido group and the anomeric OH group of unprotected N‐acetyl‐2‐amino sugars took place smoothly in H₂O, leading to the formation of a 1,2‐oxazoline (=4,5‐dihydrooxazole) moiety in good yield. Since the reaction proceeds in H₂O without using any protecting groups, the resulting oxazolines can be utilized as effective glycosyl donors for the subsequent enzymatic glycosylation. We have successfully demonstrated a highly efficient chemoenzymatic transglycosylation of a disialo‐oligosaccharide moiety to p‐nitrophenyl N‐acetylglucosaminide catalyzed by a mutant endo‐N‐acetylglucosaminidase without isolating disialo‐oligosaccharide oxazoline as synthetic intermediate.     

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.     

Simultaneous determination of NG‐monomethyl‐l‐arginine,NG,NG‐dimethyl‐l‐arginine,NG,NG′‐dimethyl‐l‐arginine,and l‐arginine using monolithic silica disk‐packed spin columns and a monolithic silica column

We have developed and validated a high‐performance liquid chromatography method that uses monolithic silica disk‐packed spin columns and a monolithic silica column for the simultaneous determination of N^G‐monomethyl‐l ‐arginine, N^G,N^G‐dimethyl‐l ‐arginine, and N^G,N^G′‐dimethyl‐l ‐arginine in human plasma. For solid‐phase extraction, our method employs a centrifugal spin column packed with monolithic silica bonded to propyl benzenesulfonic acid as a cation exchanger. After pretreatment, the methylated arginines are converted to fluorescent derivatives with 4‐fluoro‐7‐nitro‐2,1,3‐benzoxadiazole, and then the derivatives are separated on a monolithic silica column. l ‐Arginine concentration was also determined in diluted samples. Standard calibration curves revealed that the assay was linear in the concentration range 0.2–1.0 μM for methylated arginines and 40–200 μM for l ‐arginine. Linear regression of the calibration curve yielded equations with correlation coefficients of 0.999 (r²). The sensitivity was satisfactory, with a limit of detection ranging from 3.75 to 9.0 fmol for all four compounds. The RSDs were 4.3–4.8% (intraday) and 3.0–6.8% (interday). When this method was applied to samples from six healthy donors, the detected concentrations of N^G‐monomethyl‐l ‐arginine, N^G,N^G‐dimethyl‐l ‐arginine, N^G,N^G′‐dimethyl‐l ‐arginine and l ‐arginine were 0.05 ± 0.01, 0.41 ± 0.07, 0.59 ± 0.11, and 83.8 ± 30.43 μM (n = 6), respectively.     

Site-specific qualitative and quantitative analysis of the N- and O-glycoforms in recombinant human erythropoietin

Recombinant human erythropoietin (rhEPO) has been extensively used as a pharmaceutical product for treating anemia. Glycosylation of rhEPO affects the biological activity, immunogenicity, pharmacokinetics, and in-vivo clearance rate of rhEPO. Characterization of the glycosylation status of rhEPO is of great importance for quality control. In this study, we established a fast and comprehensive approach for reliable characterization and relative quantitation of rhEPO glycosylation, which combines multiple-enzyme digestion, hydrophilic-interaction chromatography (HILIC) enrichment of glycopeptides, and tandem mass spectrometry (MS) analysis. The N-linked and O-linked intact glycopeptides were analyzed with high-resolution and high-accuracy (HR–AM) mass spectrometry using an Orbitrap. In total, 74 intact glycopeptides from four glycosylation sites at N₂₄, N₃₈, N₈₃, and O₁₂₆ were identified, with the simultaneous determination of peptide sequences and glycoform compositions. The extracted ion chromatograms based on the HR–AM data enabled relative quantification of glycoforms. Our results could be extended to quality control of rhEPO or could help establish detection approaches for glycosylation of other proteins. Graphical Abstract

?     

Fe3O4@L‐arginine magnetic nanoparticles: A novel and magnetically retrievable catalyst for the synthesis of 1′3‐diaryl‐2N‐azaphenalene

The catalytic activity of l ‐arginine‐coated nano‐Fe₃O₄ particles (Fe₃O₄@l ‐arginine) proves they are a novel magnetic catalyst without the use of heat and reflux for the synthesis of 1,3‐diaryl‐2‐N‐azaphenalene derivatives and n‐acyl‐1,3‐diaryl‐2‐N‐azaphenylene derivatives in a one‐pot pseudo‐five‐component condensation reaction of compounds of 2,7‐naphthalene diol, aldehydes, and ammonia derivatives (ammonium acetate or ammonium hydrogen phosphate) and solvent (water and alcohol) with high yield and short reaction times, economical, and simple workup. The structure and magnetic properties of the obtained nanoparticles were characterized via Fourier transform infrared spectroscopy (IR) and field emission scanning electron microscopy (FE‐SEM). The results demonstrated that the average size of the synthesized magnetite nanoparticles is about 21 nm. In addition, the heterogeneous catalyst can be easily recovered magnetically and can be reused for further runs without significant loss of its catalytic activity.     

Bacillus licheniformis peptidoglycan characterization by CZE–MS: Assessment with the benchmark RP‐HPLC‐MS method

Peptidoglycan or murein is an essential polymer found in bacterial cell wall. It is a dynamic structure that is continuously remodeled or modified during bacterial cell growth or in presence of cell wall stresses. These modifications are still poorly understood mainly due to the peptidoglycan, which is rather non‐soluble, and the difficulties to separate the hydrophilic glycopeptides (muropeptides) by reversed phase liquid chromatography, generated by the enzymatic digestion using mutanolysin, an N‐acetyl‐muramidase, cleaving the β_1→4 bound between N‐acetylglucosamine and N‐acetylmuramic acid. Here, we report the use of CZE–MS for an easy and fast screening of muropeptides generated by the action of muramidase on the Bacillus licheniformis cell wall. Electron transfer and CID–MS were also used to unambiguously identify and localize the presence or the absence of amidation and acetylation moieties on muropeptide variants. The reference method to analyse muropeptides by reversed phase chromatography was also tested and the advantages and disadvantages of both methods were evaluated.     

Spectrofluorimetric Study on the Weak Interaction between ATP and Na-4-Tosyl-L-arginine Methyl Ester Hydrochloride

In this paper, some new results on the selective weak interaction between Na-4-tosyl-L-arginine methyl ester hydrochloride （TAME） and adenosine-5＇-triphosphate （ATP） have been reported. Fluorescence spectrophotometry and Fourier transform infrared （FT-IR） spectroscopy were used to investigate this kind of weak interaction. In fluorescence experiments, obvious fluorescence quenching phenomena were observed when TAME was added, which indicated the weak interactions between TAME and ATP. It has been identified by fluorescence titration experiments that TAME exhibited high selectivity to ATP over ADP and AMP. FT-IR spectral results showed that an ATP-TAME adduct was formed. The experimental results indicated that the interaction sites were the guanidinium group of TAME main-chain and the γ-phosphate group of ATP, and the interaction took place through hydrogen bonding and electrostatic force. In addition, the effects of metal ions on the weak interaction between ATP and TAME, or between ATP and analogues of L-arginine were studied.     

One‐Pot Protection‐Glycosylation Reactions for Synthesis of Lipid II Analogues

(2,6‐Dichloro‐4‐methoxyphenyl)(2,4‐dichlorophenyl)methyl trichloroacetimidate ( 3 ) and its polymer‐supported reagent 4 can be successfully applied to a one‐pot protection‐glycosylation reaction to form the disaccharide derivative 7 d for the synthesis of lipid II analogues. The temporary protecting group or linker at the C‐6 position and N‐Troc protecting group of 7 d can be cleaved simultaneously through a reductive condition. Overall yields of syntheses of lipid II ( 1 ) and neryl‐lipid II N^ε‐dansylthiourea are significantly improved by using the described methods.     

Analysis of recombinant human erythropoietin glycopeptides by capillary electrophoresis electrospray–time of flight-mass spectrometry

Capillary electrophoresis electrospray–mass spectrometry was used to detect and characterize the great variety of O- and N-glycopeptide glycoforms of recombinant human erythropoietin (rhEPO) using an orthogonal accelerating time-of-flight mass spectrometer to obtain their exact molecular masses (CE–TOF-MS). rhEPO was digested with trypsin and Glu-C and analyzed by CE–TOF-MS to detect O₁₂₆, N₈₃, N₂₄–N₃₈ and N₂₄ and N₃₈ glycopeptide glycoforms, respectively. Neuraminidase was first used to enhance the detection of the glycopeptides and detect all possible glycoforms contained in each glycosylation site. O₁₂₆ and N₈₃ glycopeptides were extensively characterized. Twelve sialoforms corresponding to 5 different glycoforms were detected in N₈₃, and for the first time, a sulfated sialoform of this glycopeptide was also detected. In the case of O₁₂₆, different sialoforms with different types of sialic acids (Neu5Gc and Neu5Ac) were detected and an estimation of the relative percentage of Neu5Gc versus Neu5Ac was also carried out for this glycopeptide. N₂₄ and N₃₈ glycosylation sites were also characterized by CE–TOF-MS after Glu-C digestion and these results permitted to rule out some glycan combinations for N₂₄–N₃₈ glycopeptide glycoforms. This study provided a reliable glycopeptide map of rhEPO and may be regarded as an excellent starting point to analyze rhEPO glycopeptides in biological fluids and detect the use of this hormone in sports.