Two new dianthramide glucosides with cardiomyocytes protective activity from Aconitum carmichaelii

Two new dianthramide glucosides, N-(2′-β-d-glucopyranosyl-5′-hydroxysalicyl)-4-hydroxy-3-methoxyanthranilic acid methyl ester (1) and N-(2′-β-d-glucopyranosyl-5′-hydroxysalicyl)-4-hydroxyanthranilic acid methyl ester (2), together with five known glycosides, were isolated from the lateral roots of Aconitum carmichaelii. Their structures were elucidated by spectroscopic analyses. In the in vitro assays, compounds 1 and 2 showed activity against pentobarbital sodium-induced cardiomyocytes damage by recovering beating rhythm and increasing the cell viability.     

Sites of metabolism of N-nitrosodiethylamine in mice

Low-temperature whole-body autoradiography and autoradiography with heated sections in C57B1 mice injected with N-[¹⁴C]nitrosodiethylamine showed a homogenously distributed volatile radioactivity in most tissues — indicating an ability of the non-metabolized substance to freely pass the biological membranes and distribute evenly in the intra- and extracellular tissue-water. A high level of non-volatile metabolites was found in several tissues: the nasal and tracheal mucosa, the mucosa of the bronchial tree, the salivary glands, the liver, the mucosa of the oesophagus and the tongue, and the lacrimal glands. Studies in vitro indicated that these tissues had a capacity to degrade N-[¹⁴C]nitrosodiethylamine (¹⁴CO₂-production and incorporation of radioactivity in the acid-insoluble material of the tissues were used as indices of the metabolism), whereas several other tissues, which did not accumulate metabolites at short survival intervals in vivo, were devoid of significant metabolic capacity. The relationship between metabolic ability and carcinogenic response of the tissues for N-nitrosodiethylamine is discussed on basis of the obtained results.     

Glycoproteines sulfates des membranes de l'oeuf de poule et de l'oviducte Isolement et caracterisation de glycopeptides sulfatesSulfated glycoproteins form egg shell membranes and hen oviduct. Isolation and characterization of sulfated glycopeptides

Sulfated glycopeptides were isolated from pronaisc and tryptic digests of egg shell membranes and hen oviduct. They were precipitated by cationic detergents and separated by preparative electrophoresis, after removal of small quantities of glucuronoglycosaminoglycans detected only in the oviduct (isthmus and magnum). The principal isolated sulfated glycopeptides were divided according to increasing electrophoretic mobilities into two groups A and B. The homogeneity of the purified glycopeptides was confirmed by gel filtration and polyacrylamide gel electrophoresis.Glycopeptides from pool preparation of tissue are analysed and carbohydrate and amino acids average values are estimated. Hexosamines (mainly N-acetylglucosamine), hexoses (galactose, glucose, mannose) and fucose were found in Glycopeptides A. The molar ratio of hexose/hexosamine was 0.4. N-Acetylneuraminic acid and sulfate were also present in Glycopeptides A. The molar ratio of sulfate/hexosamine ranged from 0.1 to 0.25. The Glycopeptides A composition indicated the presence of chains with many glycosyl groups and a few of amino acids residues. The carbohydrate components of Glycopeptides B from egg shell membranes and magnum were found to be hexosamines (N-acetylgalactosamine and N-acetylglucosamine in equimolar proportions), hexoses (galactose mainly and glucose), N-acetylneuraminic acid, and fucose. The molar ratio of hexose/hexosamine was 1. Sulfate was also present and the molar ratio of N-acetylneuraminic acid and sulfate to hexosamine was ranged from 0.8 to 1. The main amino acid residues in these glycopeptides were serine and threonine with destruction of these hydroxyamino acids during alkali treatment. Glycopeptides B probably consist of short carbohydrate chains, linked to the polypeptide through O-glycosidic bonds involving N-acetylgalactosamine and serine and threonine. Approximately 40% of the amino acid residues were linked to carbohydrate chains.Glycopeptides B from egg shell membranes magnum and egg white were very similar in their carbohydrate and amino acid composition and in their properties.Gylcopeptides A from egg shell membranes, isthmus and magnum showed similarities and divergences especially in the amino acid composition. These results suggest that magnum and isthmus in oviduct are both concerned with the synthesis of egg shell membrane glycoproteins.     

On the structure of heparitin sulfates Analyses of the products formed from heparitin sulfates by two heparitinases and a heparinase from Flavobacterium heparinum

The analyses of the products formed from heparitin sulfates by the action of two heparitinases and a heparinase from Flavobacterium heparinum is reported. Heparitin sulfates A and B are degraded by heparitinase I yielding two disaccharides, one of them composed of N-acetylglucosamine and an unsaturated uronic, joined by α(1 → 4) linkage, and the other, with the same composition but with an O-sulfate at the hexosamine moiety. A third disaccharide is also formed from heparitin sulfate B, by the action of the same enzyme, composed of glucosamine N-sulfate and an unsaturated uronic acid joined probably by α(1 → 4) linkage. Besides these three disaccharides, heparitin sulfate B yields, by the action of heparitinase I, an oligosaccharide (with an average molecular weight of 6000) which is completely degraded by the heparitinase II yielding a disaccharide composed of glucosamine 2,6-disulfate and unsaturated uronic acid. All the disaccharides are further degraded by α-glycuronidase from Flavobacterium heparinum yielding the respective monosaccharides. Based on these and other analyses the possible structures of the heparitin sulfates are proposed.     

Studies on neuraminidase from influenza virus A(H3N2) obtained by two procedures

• 1.1. Neuraminidase was obtained by (A) bromelain solubilization or (B) by treatment with N-lauroylsarcosine.
• 2.2. 5-N-acetyl-2-O(3-methoxyphenyl)-α-d-neuraminic acid, employed as substrate, avoids the interference produced by the thiobarbituric acid method, and is not interfered by the ampholytes.
• 3.3. Only about 20% of original enzyme activity was lost after electrofocusing. The sample from procedure A showed two peaks, corresponding to pis 4.4 and 5.6. The sample from procedure B, having a higher activity, showed only one peak at pI 4.4.
• 4.4. Samples A and B showed different K_m and hydrolysis rate with N-acetylneuraminyl-lactose and glycophorin A. It was not found significantly different with other substrates: α₁-acid glycoprotein, brain gangliosides. 5-N-acetyl-2-O-(3-methoxyphenyl)-α-d-neuraminic acid and 2'-(4-methyl umbelliferyl)-α-d-N-acetylneuraminic acid.

     

Structural and synthetic studies on the retrofractamides—amide constituents of Piper retrofractum

Two new unsaturated amides, retrofractamides A and C, were isolated from the total above-ground parts of Piper retrofractum. Retrofractamide A was shown to be N-isobutyl-9(3′,4′-methylenedioxyphenyl)2E,4E,8E-nonatrienamide from spectroscopic and chemical investigations. The structure 6 for retrofractamide C was suggested from spectroscopic and chemical studies and was confirmed by a total stereoselective synthesis. The presence of sesamin and 3,4,5-trimethoxydihydrocinnamic acid as well as two higher homologues of retrofractamide A, viz. pipericide (retrofractamide B) and retrofractamide D was demonstrated. The synthesis of pipericide was also achieved.     

Affinity chromatography of sialoglycoproteins,utilising the interaction of serotonin with N-acetylneuraminic acid and its derivatives

Serotonin, immobilised on Sepharose 4B, has been used to study the affinity chromatography of neuraminic acid and its derivatives. Free N-acetylneuraminic acid and oligosaccharides, polysaccharides, and glycoproteins containing that sugar are specifically bound to the columns. Removal of neuraminic acid from sialoglyco-conjugates, or modification of the neuraminic acid residues by periodate oxidation, abolishes their ability to bind to the ligand. The presence of the N-acetyl group, but not the N-glycolyl group, and the integrity of the side chain (C-7–C-9) of the neuraminic acid are essential for binding to serotonin.     

ABH blood group antigens in N-glycan of human glycophorin A

We previously showed that a small proportion of the O-linked oligosaccharide chains of human glycophorin A (GPA) contains blood group A, B or H antigens, relevant to the ABO phenotype of the donor. The structures of these minor O-glycans have been established (Podbielska et al. (2004) [20]). By the use of immunochemical methods we obtained results indicating that ABH blood group epitopes are also present in N-glycan of human GPA (Podbielska and Krotkiewski (2000) [22]). In the present paper we report a detailed analysis of GPA N-glycans using nanoflow electrospray ionization tandem mass spectrometry. N-glycans containing A-, B- and H-related sequences were identified in GPA preparations obtained from erythrocytes of blood group A, B and O donors, respectively. The ABH blood group epitopes are present on one antenna of the N-glycan, whereas a known sialylated sequence NeuAcα2-6Galβ1-4GlcNAc- occurs on the other antenna and other details are in agreement with the known major structure of the GPA N-glycan. In the bulk of the biantennary sialylated N-glycans released from GPA preparations, the blood group ABH epitopes-containing N-glycans, similarly O-glycans, constituted only a minor part. The amount relative to other N-glycans was estimated to 2-6% of blood group H epitope-containing glycans released from GPA-O preparations and 1-2% of blood group A and B epitope-containing glycans, released from GPA-A and GPA-B, respectively.     

The use of solvatochromic mixed copper(II) chelates with N,N,N′,N′-tetramethylethylenediamine and tropolonato or hinokitiolato ligands, [Cu(trop/hino)(tmen)]B(C6H5)4, as indicator for Lewis basicity of solvents and low-coordinating anions

Mixed copper(II) chelates, [Cu(trop/hino)(tmen)]B(C₆H₅)₄, were prepared with a tropolonato or hinokitiolato ligands (trop/hino) and N,N,N′,N′-tetramethylethylenediamine (tmen). These chelates were, as expected, quite similar to the corresponding acetylacetonato analog [Cu(acac)(tmen)]B(C₆H₅)₄, being fairly soluble in a large number of solvents and remarkably solvatochromic in them. They were also useful as excellent Lewis basicity indicators in solution because their d-d bands continuously shift to red in wider ranges with increasing DN (donor number) of solvent. The examination on addition of various anions to these solvatochromic systems led to a quantitative view of the competition between solvent molecule and anion for coordination to metal center.     

Preparation of Novel Monoclonal Antibodies Against Chelated Cadmium Ions

The detection of cadmium ions using enzyme-linked immunosorbent assays (ELISA) has been reported by several research groups. Because cadmium ions are too small to stimulate the immune system, high molecular weight immunogens of cadmium are constructed using bifunctional chelators. At present, the most commonly used bifunctional chelator for the preparation of antigens for heavy metal ions is 1-(4-isothiocyanobenzyl) ethylenediamine N,N,N′,N′-tetraacetic acid (ITCBE). However, the price of ITCBE is high. So we are interested in a cheaper bifunctional chelator, 1-(4-aminobenzyl) ethylenediamine N,N,N′,N′-tetraacetic acid (aminobenzyl-EDTA). Here, cadmium ions were conjugated to carrier proteins using aminobenzyl-EDTA to make artificial antigens. Then, several mice were immunized with the antigen. And monoclonal antibodies (MAbs) against cadmium were produced. Spleen cells of immunized mice were fused with myeloma cells. The resulting hybridomas were screened using protein conjugates which were covalently bound to metal-free EDTA or cadmium. Three hybridoma cell lines (A3, E4 and B5) that produced MAbs with high selectivity and sensitivity were expanded for further study. Cross-reactivities with other metals were below 1 %. These antibodies were used to construct competitive ELISAs. The IC₅₀ for A3 was 8.4 μg/l. The detection range and the lowest detection limit using the antibody A3 was 0.394–64.39 and 0.051 μg/l, respectively. Spike–recovery studies in tap water showed that the antibody A3 could be used for cadmium detection in drinking water.     

Occurrence in high concentrations of N1-acetylspermidine and sym-homospermidine in the hamster epididymis

In mature hamster epididymis several unknown peaks were observed on our high-performance liquid chromatograms in addition to the common polyamines, putrescine, spermidine and spermine. Three of the peaks were identified as N¹-acetylspermidine, N¹-acetylspermine and sym-homospermidine by means of thin-layer chromatography, gas chromatography-mass spectrometry and acid hydrolysis. The concentrations of N¹-acetylspermidine and sym-homospermidine were highest in the distal caput epididymidis among epididymal regions studied. This is the first report to show that sym-homospermidine occurs in mammalian tissues.     

High-performance liquid chromatographic analysis of neutral glycosphingolipids as their per-O-benzoyl derivatives

Previous reports from this laboratory (1–4) described the perbenzoylation of neutral glycosphingolipids (GSL)¹ with benzoyl chloride in pyridine and analysis of the perbenzoylated derivatives by high performance liquid chromatography (hplc). A disadvantage of this procedure is that N-benzoylation occurs as well as the desired O-benzoylation. This does not permit recovery of the parent GSL after mild alkaline hydrolysis due to formation of a mixture of N-acylated and N-benzoylated GSLs(1). It has also been demonstrated that the benzoylation with benzoic anhydride in pyridine does not lead to the formation of N-benzoylated products. However, the anhydride reaction is sluggish and the benzoyl chloride method has been the preferred procedure.Gupta et al. (5) used N,N-dimethyl-4 amino pyridine (DMAP) as a catalyst in the acylation of phospholipids by the anhydrides of fatty acids. F. B. Jungalwala (private communication) has shown that this catalyst greatly accelerates the reaction of benzoic anhydride with sulfatides.In this communication we report the preparation and hplc analysis of per-O-benzoyl derivatives of GSLs by reaction with benzoic acid anhydride in the presence of DMAP as a catalyst. Reaction with these reagents avoids amide acylation, forms single products with satisfactory chromatographic properties and parent GSLs can be regenerated by mild alkaline hydrolysis.     

N-(1-Carboxyethyl)alanine (alanopine), a new non-sugar component of lipopolysaccharides of Providencia and Proteus

O-Polysaccharides were released by mild acid degradation of lipopolysaccharides of Providencia alcalifaciens O35 and Proteus vulgaris O76 and were studied by 1D and 2D ¹H and ¹³C NMR spectroscopies, including HMBC and NOESY (ROESY) experiments. Both polysaccharides were found to contain N-(1-carboxyethyl)alanine (alanopine) that is N-linked to 4-amino-4,6-dideoxyglucose. Analysis of published data [Vinogradov, E.; Perry, M. B. Eur. J. Biochem.2000, 267, 2439-2446] shows that alanopine is present also on the same sugar in the lipopolysaccharide core of Proteus mirabilis O6 and O57.     

Hantupeptins B and C,cytotoxic cyclodepsipeptides from the marine cyanobacterium Lyngbya majuscula

Hantupeptins B (2) and C (3) were isolated, along with the previously reported hantupeptin A (1), from the marine cyanobacterium, Lyngbya majuscula, collected from Pulau Hantu Besar, Singapore. Their structures were elucidated by interpretation of extensive 1D and 2D NMR spectroscopic data. Compounds 2 and 3 are cyclic depsipeptides consisting of five α-amino/hydroxy acid residues, including phenyllactic acid, proline, N-methyl-valine, valine, N-methyl-isoleucine, and a β-hydroxy acid unit with different degrees of unsaturation at the terminal end of each molecule. The absolute configurations of the common amino acids and phenyllactic acid were determined by the advanced Marfey’s and chiral HPLC analyses, respectively. The complete stereochemistry of 3-hydroxy-2-methyl-7-octynoic acid moiety in hantupeptin A was elucidated by a combination of homonuclear J-resolved 2D NMR experiments and by Mosher’s method. Hantupeptins B and C showed moderate in vitro cytotoxicity when tested against MOLT-4 (leukemic) and MCF-7 (breast cancer) cell lines.     

Conversion of N-acetylglucosamine to CMP-N-acetylneuraminic acid in a cell-free system of rat liver

A soluble fraction of rat liver converts glucosamine and N-acetylglucosamine in the presence of ATP and UTP to N-acetylneuraminic acid. This system, when supplemented with CTP, forms CMP-N-acetylneuraminic acid in high yield. Nicotinamide was found to enhance the synthesis of UDP-N-acetylglucosamine and N-acetylneuraminic acid. Kinetic analysis reveals N-acetylglucosamine 6-phosphate, UDP-N-acetylglucosamine, N-acetylmannosamine, N-acetylmannosamine 6-phosphate and N-acetylneuraminic acid 9-phosphate as intermediates. Under certain experimental conditions, however, an epimerisation of N-acetylglucosamine to N-acetylmannosamine was seen.     

N-acetylneuraminic acid and sialoglycoconjugate metabolism in fibroblasts from a patient with generalized N-acetylneuraminic acid storage disease

Cultured skin fibroblasts from a patient suffering from generalized N-acetylneuraminic acid storage disease were found to accumulate large amounts (approx. 4.0 μmol/g fresh weight) of free N-acetylneuraminic acid in a lysosome-enriched subcellular fraction. However, there were no detectable deficiencies in lysosomal hydrolase activities (including neuraminidase), and the activities of CMP-N-acetylneuraminic acid synthetase and N-acetylneuraminic acid aldolase were within normal limits. The cellular glycoconjugate composition was normal, and pathologic fibroblasts labeled with either [³H]glucosamine-HCl or $\text{N-[}{}^3\text{H]acetylmannosamine}$ showed a marked accumulation of labeled free N-acetylneuraminic acid, along with elevated incorporation into sialoglycoconjugates. Neither normal nor pathologic fibroblasts secreted labeled free N-acetylneuraminic acid into the culture medium. These results are consistent with an inherited defect in N-acetylneuraminic acid reutilization, resulting in the lysosomal accumulation of the free monosaccharide in generalized N-acetylneuraminic acid storage disease.     

Na+/solute symport in membrane vesicles from Bacillus alcalophilus

The characteristics of α-aminoisobutyric acid translocation were examined in membrane vesicles from obligately alkalophilic Bacillus alcalophilus and its non-alkalophilic mutant derivative, KM23. Vesicles from both strains exhibited α-aminoisobutyric acid uptake upon energization with ascorbate and N,N,N′,N′-tetramethyl-p-phenylenediamine. The presence of Na⁺ caused a pronounced reduction in the Km for α-aminoisobutyric acid in wild-type but not KM23 vesicles; the maximum velocity (V) was unaffected in vesicles from both strains. Passive efflux and exchange of α-aminoisobutyric acid from wild-type vesicles were Na⁺-dependent and occurred at comparable rates (with efflux slightly faster than exchange). This latter observation suggests that the return of the unloaded carrier to the inner surface is not rate-limiting for efflux. The rates of α-aminoisobutyric acid efflux and exchange were also comparable in KM23 vesicles, but were Na⁺-independent. Furthermore, in vesicles from the two strains, both efflux and exchange were inhibited by generation of a transmembrane electrochemical gradient of protons, outside positive. This suggests that the ternary complex between solute, carrier, and coupling ion bears a positive charge in both strains even though the coupling ion is changed. Evidence from experiments with an alkalophilic strain that was deficient in l-methionine transport indicated that the porters, i.e., the solute-translocating elements, used by non-alkalophilic mutants are not genetically distinct from those used by the alkalophilic parent; that is, the change in coupling ion cannot be explained by the expression of a completely new set of Na⁺-independent, H⁺-coupled porters upon mutation of B. alcalophilus to non-alkalophily.     

β-N-Acetylhexosaminidases in the spleen of a patient with hairy-cell leukaemia

The spleen from a patient with hairy-cell leukaemia had β-N-acetylhexosaminidase activity that could be resolved into three isoenzymes by chromatography on phenyl boronate agarose. Two of these were the major forms, A and B, found in normal tissues but, in addition, there was an ‘extra’ form that accounted for 15% of total activity. The ‘extra’ form hydrolysed the synthetic substrate 4-methylumbelliferyl-β-N-acetylglucosamine 6-sulphate, indicating the presence of α-subunits. It was more acidic than A, was less heat-stable and showed no generation of B on denaturation under a variety of conditions. These findings and the immunoblot (Western blotting) analysis demonstrate that the ‘extra’ form is entirely composed of α-subunits, and most closely resembles S, the residual activity in Sandhoff's disease.     

Purification and properties of two endonucleases specific for apurinic/apyrimidinic sites in DNA from Saccharomyces cerevisiae

Two distinct endonucleases from Saccharomyces cerevisiae, specific for apurinic/apyrimidinic sites (AP-endonucleases A and B), have been extensively purified and characterized. Both are free from unspecific and ultraviolet-specific endonucleases and exonucleases. The two enzymes are monomeric proteins of around 24 000 daltons. Both are sensitive to ionic strength and most active in the presence of 150 and 100 mM NaCl for AP-endonucleases A and B, respectively. They are not absolutely dependent on divalent cations, since they are insensitive to EDTA, although AP-endonuclease A is activated by Ca²⁺ or Mg²⁺ and AP-endonuclease B by Mg²⁺ only. ATP inhibits the enzymes. AP-endonuclease A reacts optimally between pH 6 and 8, and AP-endonuclease B at pH 8. AP-endonuclease A is more stable at 60°C (half-life of 17 min) than B (half-life of 4 min). AP-endonucleuase A is insensitive to N-ethylmaleimide or ρ-chloromercuribenzoate. AP-endonuclease B is also insensitive to N-ethylmaleimide, but ρ-chloromercuribenzoate inhibits its activity.     

A Comparative Study of Lectin Affinity Based Plant N-Glycoproteome Profiling Using Tomato Fruit as a Model

Lectin affinity chromatography (LAC) can provide a valuable front-end enrichment strategy for the study of N-glycoproteins and has been used to characterize a broad range eukaryotic N-glycoproteomes. Moreover, studies with mammalian systems have suggested that the use of multiple lectins with different affinities can be particularly effective. A multi-lectin approach has also been reported to provide a significant benefit for the analysis of plant N-glycoproteins; however, it has yet to be determined whether certain lectins, or combinations of lectins are optimal for plant N-glycoproteome profiling; or whether specific lectins show preferential association with particular N-glycosylation sites or N-glycan structures. We describe here a comparative study of three mannose-binding lectins, concanavalin A, snowdrop lectin, and lentil lectin, to profile the N-glycoproteome of mature green stage tomato (Solanum lycopersicum) fruit pericarp. Through coupling lectin affinity chromatography with a shotgun proteomics strategy, we identified 448 putative N-glycoproteins, whereas a parallel lectin affinity chromatography plus hydrophilic interaction chromatography analysis revealed 318 putative N-glycosylation sites on 230 N-glycoproteins, of which 100 overlapped with the shotgun analysis, as well as 17 N-glycan structures. The use of multiple lectins substantially increased N-glycoproteome coverage and although there were no discernible differences in the structures of N-glycans, or the charge, isoelectric point (pI) or hydrophobicity of the glycopeptides that differentially bound to each lectin, differences were observed in the amino acid frequency at the −1 and +1 subsites of the N-glycosylation sites. We also demonstrated an alternative and complementary in planta recombinant expression strategy, followed by affinity MS analysis, to identify the putative N-glycan structures of glycoproteins whose abundance is too low to be readily determined by a shotgun approach, and/or combined with deglycosylation for predicted deamidated sites, using a xyloglucan-specific endoglucanase inhibitor protein as an example.N-glycosylation is one of the most heterogeneous and common post-translational modifications of eukaryotic proteins and one that affects many aspects of protein targeting, enzymatic properties, stability and intermolecular interactions (1–3). There is therefore considerable interest in developing robust and sensitive high throughput analytical pipelines to isolate and structurally characterize N-glycoprotein populations (2, 4–8), allowing glycoprotein identification and analysis of the glycosylation site occupancy and N-glycan structure. To this end, lectin affinity chromatography (LAC)¹ is increasingly popular: specifically, various lectins are known to have different binding affinities for N-glycans and so the selective binding of N-glycoproteins in complex protein extracts to these lectins and their subsequent release allows a critical enrichment step before sequencing and glycan analysis by MS (9).As a refinement of this approach, the use of multiple lectin affinity chromatography (MLAC) in yeast and animal studies (4, 6, 10), using different proteomic platforms, has been shown to increase the numbers of isolated N-glycoproteins or N-glycopeptides. Collectively, these studies of taxonomically diverse eukaryotic N-glycoproteomes suggest a general conservation of the glycosylation site (N-X-S/T, where X can be any amino acid except proline), as well as conserved features of three-dimensional protein structure (4, 6, 10). Although there have been several studies to determine the structural basis of the binding specificity of specific lectins to yeast and animal N-glycoproteins (11), larger scale N-glycoproteomic analyses have typically not attempted to determine whether a particular combination of lectins provides optimal enrichment, or whether specific features, such as N-glycan structure or amino acid sequence at and around the N-glycosylation site, are associated with different lectins. Therefore, systematic comparative studies are essential to determine whether particular lectins can be optimal for specific tissues, organs, and organisms.LAC has also been used in plant N-glycoprotein analyses to enrich for populations of cell wall localized proteins (8, 12, 13) and a recent report (13) described the application of MLAC to map substantial numbers of N-glycosylation sites in a range of key experimental model organisms, included the plant Arabidopsis thaliana. Using a LTQ-Orbitrap Velos mass spectrometer, the authors identified 2186 unique N-glycosylation sites in proteins extracted from five different arabidopsis organs (13), which represents a substantial increase in the number of identified N-glycosylation sites that have resulted from previous plant N-glycoprotein studies. However, the particular analytical platform that was used did not allow the structural characterization of the N-glycans or N-glycopeptides (13). Indeed, certain features of N-glycopeptides, such as poor fragmentation, heterogeneity and a large dynamic range in most complex mixtures often limits the structural analysis of N-glycans in high throughput systematic analyses (2). It is also important to note that the structures of plant N-glycans differ from those of animals and yeast, as exemplified by the presence of β-1, 2-xylose and α-1, 3-fucose and the complete absence of multiantennary N-glycans and sialic acid in plant N-glycoproteins (1, 14). Therefore, assumptions that are made with regard to the lectin binding of animal and yeast proteins do not necessarily apply to those from plants. Consequently, there is a need to investigate the structural basis of lectin binding to plant N-glycoproteins. Moreover, the limitations of typical shotgun based profiling approaches in identifying and characterizing low abundance N-glycoproteins in complex protein extracts need to be addressed to allow more comprehensive plant N-glycoproteome profiling.In the present study we address both these issues using mature green stage tomato (Solanum lycopersicum) fruit pericarp as an experimental model to carry out a comparative analysis of N-glycoproteins associated with each of three mannose-binding lectins: concanavalin A (ConA), snowdrop lectin (GNA), and lentil lectin (LCH). Fruit development is associated with substantial cell wall metabolism and the expression of many wall localized N-glycoproteins (8) and tomato in particular represents an excellent model for studies of fleshy fruits and cell wall N-glycoproteins (15). We established an MLAC analytical pipeline that included shotgun proteomic profiling and deglycosylation and deamidation analysis, to allow the determination of N-glycoprotein protein identity, N-glycosylation site and N-glycan structures. This information was then used to establish whether a combination of lectins is indeed advantageous for the study of plant N-glycoproteomes, and to assess whether any of these structural characteristics result in predictable preferential binding to specific lectins. From these studies it became evident that large dynamic range of N-glycoprotein abundance was a significant limiting factor in the structural determination of the tomato, and that there was a bias toward the detection of highly abundant N-glycopeptides. We therefore evaluated the use of an in planta recombinant expression strategy, combined with affinity purification MS (AP-MS), as a means to characterize the N-glycan structures of glycoproteins whose abundance is too low to be readily determined via the primary shotgun pipeline, using a tomato xyloglucan-specific endoglucanase inhibitor protein (XEGIP) as a test case.