Isolation of glycopeptides containing O-linked oligosaccharides by lectin affinity chromatography on jacalin-agarose

Jacalin is a lectin which has high specificity and affinity for the core disaccharide, 1-beta-galactopyranosyl-3-(alpha-2-acetamido-2-deoxygalactopyranoside ), in O-linked oligosaccharides. Here, it is shown that this lectin can be used for isolation of glycopeptides bearing O-linked oligosaccharides. Peptides produced by digestion of reduced and carboxamidomethylated human plasminogen or of bovine protein Z were chromatographed on a column of jacalin-agarose. Reverse-phase high-performance liquid chromatography revealed that two peptides from plasminogen and one from protein Z were eluted from the jacalin-agarose column by alpha-methylgalactopyranoside. Amino acid sequence and compositional analysis showed that both of the peptides from plasminogen consisted of residues 330-357 and that the single peptide from protein Z represented residues 385-396. These sequences contain the single known site of attachment of O-linked oligosaccharides to these proteins. The present analysis suggested that there may be a fraction of plasminogen with two sites of O-linked glycosylation. The two tryptic peptides isolated from plasminogen represented the same segment of the protein but sequence analysis showed that one peptide was modified only at Thr346, the known site of glycosylation, and the other peptide contained a modification of Ser339 as well. Results of the present study indicate that lectin affinity chromatography using jacalin-agarose can be a useful technique for isolating glycopeptides containing O-linked oligosaccharides and thereby localizing sites of attachment of these oligosaccharides.     

Further identification of human plasma glycoproteins interacting with the galactose-specific lectin Jacalin

In this report we show that Jacalin binds the heme-binding protein hemopexin and the C4b-binding protein sgp120 in human plasma. The interaction of Jacalin with hemopexin confirms that a single O-linked oligosaccharide is sufficient to mediate binding of a protein to this lectin. Retention of sgp120 by immobilized Jacalin demonstrated that this protein was O-glycosylated and, therefore, clearly different from another C4b-binding protein, the complement protein C2 which is physicochemically similar but exclusively N-glycosylated. In addition, Jacalin was also shown to bind several proteolytic enzymes which remain to be identified.     

Elucidation of binding specificity of Jacalin toward O-glycosylated peptides: quantitative analysis by frontal affinity chromatography

Jacalin, a lectin from the jackfruit Artocarpus integrifolia, has been known as a valuable tool for specific capturing of O-glycoproteins such as mucins and IgA1. Though its sugar-binding preference for T/Tn-antigens is well established, its detailed specificity has not been elucidated. In this study, we prepared a series of mucin-type glycopeptides using human glycosyltransferases, that is, ST6GalNAc1, Core1Gal-T1 and -T2, beta3Gn-T6, and Core2GnT1, and investigated their binding to immobilized Jacalin by frontal affinity chromatography (FAC). As a result, consistent with the previous observation, Jacalin showed high affinity for T-antigen (Core1) and Tn-antigen (alpha N-acetylgalactosamine)-attached peptides. Furthermore, we here show as novel findings that (1) Jacalin also showed significant affinity for Core3 and sialyl-T (ST)-attached peptides, but (2) Jacalin could not bind to Core2, Core6, and sialyl-Tn (STn)-attached peptides. The results were also confirmed by FAC using p-nitrophenyl (pNP)-derivatized saccharides. In conclusion, Jacalin binds to a GalNAcalpha1-peptide, in which C6-OH of alphaGalNAc is free (i.e., Core1, Tn, Core3, and ST), whereas it cannot recognize a GalNAcalpha1-peptide with a substitution at the C6 position (i.e., Core2, Core6, and STn). These findings provide useful information when applying jacalin for functional analysis of mucin-type glycoproteins and glycopeptides.     

O-Linked fucose in glycoproteins from Chinese hamster ovary cells

We report our discovery that many glycoproteins synthesizedby Chinese hamster ovary (CHO) cells contain fucose in O-glycosidiclinkage to polypeptide. To enrich for the possible presenceof O-linked fucose, we studied the lectin-resistant mutant ofCHO cells known as Lec1. Lec1 cells lack N-acetylglucosaminyltransferaseI and are therefore unable to synthesize complex-type N-linkedoligosaccharides. Lec1 cells were metabolically radiolabelledwith [6-³H]fucose and total glycoproteins were isolated. Glycopeptideswere prepared by proteolysis and fractionated by chromatographyon a column of concanavalin A (Con A)— Sepharose. Thesets of fractionated glycopeptides were treated with mild base/borohydrideto effect the ß-elimination reaction and release potentialO-linked fucosyl residues. The ß-elimination produced[³H]fucitol quantitatively from [³H]fucose-labelled glycopeptidesnot bound by Con A-Sepharose, whereas none was generated bytreatment of glycopeptides bound by the lectin. The total [³H]fucose-labelledglycoproteins from Lec1 cells were separated by SDS—PAGEand detected by fluorography. Treatment of selected bands ofdetectable glycoproteins with mild base/borohydride quantitativelygenerated [³H]fucitol. Pretreatment of the glycoproteins withN-glycanase prior to the SDS—PAGE method of analysis causedan enrichment in the percentage of radioactivity recovered as[³H]fucitol. Trypsin treatment of [³H]fucose-labelled intactCHO cells released glycopeptides that contained O-linked fucose,indicating that it is present in surface glycoproteins. Thesefindings demonstrate that many glycoproteins from CHO cellscontain O-linked fucosyl residues and raise new questions aboutits biosynthesis and possible function. fucose glycoproteins monosaccharide O-linked     

Presence of O-linked oligosaccharide on a threonine residue in the human transferrin receptor.

We have previously demonstrated that the human transferrin receptor (TfR) of approximately 90 kDa contains Ser/Thr-linked (O-linked) oligosaccharides. In the present study, we report our identification of the site of attachment of the O-linked oligosaccharides in the receptor. A 70 kDa fragment from the external domain of the TfR was generated by trypsin treatment of the [3H]glucosamine-labelled receptor purified from human K562 cells. The beta-elimination of the intact TfR, but not the 70 kDa fragment, released Gal-[3H]Gal-NAcitol, indicating that the 70 kDa fragment lacks O-linked oligosaccharides. In the remaining 20 kDa fragment there are three potential sites (Thr96, Thr104 and Ser106) for O-glycosylation in the extracellular domain. To identify which of these residues are O-glycosylated, both the [3H]Thr- and [3H]Ser-labelled TfR were directly treated with mild base to effect beta-elimination, and the radiolabelled amino acids and their derivatives were analysed. Approximately 2% of the total radiolabelled Thr, but no radiolabelled Ser, was converted to expected beta-elimination products by this treatment. These and other results demonstrate that only one O-linked oligosaccharide is present in the TfR and that it occurs on either Thr96 or Thr104. From human serum we purified the cleaved, soluble form of the TfR (s-TfR), which contains Thr104, but lacks Thr96. The s-TfR was sensitive to O-glycanase and bound to Jacalin lectin, indicating that the s-TfR contains an O-linked oligosaccharide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Schistosoma mansoni synthesizes glycoproteins containing terminal O-linked N-acetylglucosamine residues

In this report, we describe our studies on the structures of the O-linked oligosaccharides in glycoproteins synthesized by the human blood fluke Schistosoma mansoni. Adult male schistosomes were incubated with either [2-3H]mannose, [6-3H]glucosamine, or [6-3H]galactose to metabolically radiolabel newly synthesized glycoproteins. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis and fluorographic analyses indicated that many glycoproteins were labeled by each of the radioactive precursors. Glycopeptides were prepared from radiolabeled glycoproteins by pronase treatment and fractionated on columns of concanavalin A-Sepharose and pea lectin-agarose. The O-linked oligosaccharides were released from glycopeptides by treatment with mild base/borohydride. All O-linked material was found in glycopeptides not bound by either of the immobilized lectins. The structures of the released chains were then analyzed by a variety of techniques. Our results demonstrate that the schistosomes synthesize glycoproteins containing two major types of simple O-linked sugar chains. One type, which represents a minor fraction of the O-linked oligosaccharides, contains N-acetylgalactosamine linked to peptide. These O-linked chains occur as terminal O-linked N-acetylgalactosamine and the O-linked disaccharide, galactose----N-acetylgalactosamine. Sialic acid was not present in either of these O-linked chains or in any other glycopeptides derived from adult male schistosomes. However, the major type of O-linked chain in glycoproteins synthesized by adult schistosomes is an unusual terminal O-linked N-acetylglucosamine linked to peptide. This latter structure represents approximately 10% of the total radioactive N-acetylglucosamine recovered in all glycopeptides. Our results also suggest the possibility that the O-linked oligosaccharides are highly clustered on the glycopeptides.     

凝集素法与酰肼法富集糖肽的方法学比较

蛋白质糖基化(glycosylation)是最常见和最重要的翻译后修饰之一.大规模N-连接糖基化位点鉴定是糖蛋白质组学研究的重要组成部分,而N-连接糖肽富集是高通量N-连接糖基化位点鉴定的关键步骤.凝集素富集法和酰肼化学法是目前被广泛应用的N-连接糖肽富集技术,有报道认为两种方法具有很强的互补性,联合使用能提高糖基化位点的鉴定数目.本文以Hep G2细胞系为模型,系统比较了这两种方法的富集效率和糖蛋白鉴定数目.结果表明,虽然酰肼法的糖肽富集效率为76.6%,远高于凝集素法的54.6%,但是凝集素法却能鉴定到825个糖蛋白和1 959个N-连接糖基化位点,显著多于酰肼法富集到的522个糖蛋白和1 014个糖基化位点.并且,两种方法并未显示出显著的互补性,仅28个糖蛋白和80个糖基化位点未在凝集素法中鉴定到.     

Jacalin: an IgA-binding lectin

We previously reported that seeds of Artocarpus integrifolia (jackfruit) contain a lectin, which we call jacalin, that is both a potent T cell mitogen and an apparently T cell-independent activator of human B cells for the secretion of immunoglobulins. During the above experiments we noted a massive precipitation in cell cultures stimulated with greater than or equal to 100 micrograms of lectin. In this paper, we show that the precipitate is formed after the interaction of jacalin and the serum protein added to the culture medium. More importantly, we demonstrate that IgA is probably the major serum constituent precipitated by the lectin and that no IgG or IgM can be detected in the precipitates. In secretions such as colostrum, IgA is the only protein precipitated by jacalin. On the basis of this specificity we describe a simple and reliable affinity chromatography procedure for the purification of both human serum and colostrum IgA. Jacalin is a D-Gal binding lectin and should be a useful tool for studying of serum and secretory IgA.     

Galactose-binding lectin from the seeds of champedak (Artocarpus integer): sequences of its subunits and interactions with human serum O-glycosylated glycoproteins

Our group has previously reported the isolation, partial characterisation, and application of a Galbeta1-3GalNAc- and IgA1-reactive lectin from the seeds of champedak (Artocarpus integer). In the present study, we have subjected the purified lectin to reverse-phase high performance liquid chromatography and sequenced its subunits. Determination of the N-terminal sequence of the first 47 residues of the large subunit demonstrated at least 95% homology to the N-terminal sequence of the alpha chains of a few other galactose-binding Artocarpus lectins. The two smaller subunits of the lectin, each comprised of 21 amino acid residues, demonstrated minor sequence variability. Their sequences were generally comparable to the beta chains of the other galactose-binding Artocarpus lectins. When used to probe human serum glycopeptides that were separated by two-dimensional gel electrophoresis, the lectin demonstrated strong apparent interactions with glycopeptides of IgA1, hemopexin, alpha2-HS glycoprotein, alpha1-antichymotrypsin, and a few unknown glycoproteins. Immobilisation of the lectin to Sepharose generated an affinity column that may be used to isolate the O-glycosylated serum glycoproteins.     

Assessment of lectin and HILIC based enrichment protocols for characterization of serum glycoproteins by mass spectrometry

Protein glycosylation is a common post-translational modification that is involved in many biological processes, including cell adhesion, protein-protein and receptor-ligand interactions. The glycoproteome constitutes a source for identification of disease biomarkers since altered protein glycosylation profiles are associated with certain human ailments. Glycoprotein analysis by mass spectrometry of biological samples, such as blood serum, is hampered by sample complexity and the low concentration of the potentially informative glycopeptides and -proteins. We assessed the utility of lectin-based and HILIC-based affinity enrichment techniques, alone or in combination, for preparation of glycoproteins and glycopeptides for subsequent analysis by MALDI and ESI mass spectrometry. The methods were successfully applied to human serum samples and a total of 86 N-glycosylation sites in 45 proteins were identified using a mixture of three immobilized lectins for consecutive glycoprotein enrichment and glycopeptide enrichment. The combination of lectin affinity enrichment of glycoproteins and subsequent HILIC enrichment of tryptic glycopeptides identified 81 N-glycosylation sites in 44 proteins. A total of 63 glycosylation sites in 38 proteins were identified by both methods, demonstrating distinct differences and complementarity. Serial application of custom-made microcolumns of mixed, immobilized lectins proved efficient for recovery and analysis of glycopeptides from serum samples of breast cancer patients and healthy individuals to assess glycosylation site frequencies.     

Monitoring of glycoprotein products in cell culture lysates using lectin affinity chromatography and capillary HPLC coupled to electrospray linear ion trap-Fourier transform mass spectrometry (LTQ/FTMS)

In this paper, we describe the combination of lectin chromatography with capillary LC coupled to a linear ion trap-Fourier transform mass spectrometer (LTQ/FTMS) to enrich and characterize overexpressed glycoproteins from a cell culture lysate. A well-characterized glycoprotein, recombinant tissue plasminogen activator (rt-PA), was used as a standard, and we demonstrated that the three N-linked glycopeptides (including glycan structures) present in a tryptic digest of the rt-PA standard could be characterized in the new hybrid MS platform. A feature of this approach is that a significant amount of information can be obtained about the carbohydrate structures by direct analysis of the tryptic digest without the need for additional time-consuming sample preparation protocols. A combination of lectins was then studied for improved recovery of captured glycopeptides and was related to the selectivity of different lectins for specific glycosylation motifs. This approach was then extended to the lysate of a cell line routinely used in biotechnology manufacture (Chinese hamster ovary, CHO). This study showed that the combinations of lectins could enrich glycoproteins significantly from a CHO cell lysate. We also demonstrated that with this level of enrichment and with the new hybrid mass spectrometer, we could study the structures of N-linked glycopeptides of rt-PA present in a crude CHO cell lysate, at a ratio of 1:200 (rtPA:total cell lysate protein, w/w) by accurate mass measurement in the FTMS and tandem MSn in the linear ion trap. The generic and high throughput nature of the lectin approach combined with the ability to directly analyze the glycan structures in the tryptic digest suggest that this platform has the potential to routinely monitor glycoprotein products at early stage manufacturing in the biotech industry.     

Structure of the oligosaccharides of three glycopeptides from calf thymocyte plasma membranes.

The carbohydrate composition and oligosaccharide structure of three glycopeptides isolated from delipidated calf thymocyte plasma membranes following Pronase digestion have been determined. Five major glycopeptide fractions were separated using Bio-Gel P-6 gel filtration and diethylaminoethylcellulose chromatography. The structure of the oligosaccharide chains of three of these glycopeptides was determined by a combination of sequential degradation with glycosidases and methylation analysis. These oligosaccharide structures consist of complex, highly branched N-linked chains containing at their nonreducing termini the unusual sequence Gal(beta1 leads to 3)Gal(beta1 leads to 4)GlcNAc leads to as well as the more usual sequence SA(alpha2 leads to 3)Gal(beta1 leads to 4)GlcNAc leads to. In addition, one glycopeptide also contains short O-linked chains with the structure Gal(beta leads to 3)GalNAc leads to Ser(Thr) which have receptor activity for the lectin from the mushroom Agaricus bisporus.     

Human urinary glycoproteomics; attachment site specific analysis of N- and O-linked glycosylations by CID and ECD

Urine is a complex mixture of proteins and waste products and a challenging biological fluid for biomarker discovery. Previous proteomic studies have identified more than 2800 urinary proteins but analyses aimed at unraveling glycan structures and glycosylation sites of urinary glycoproteins are lacking. Glycoproteomic characterization remains difficult because of the complexity of glycan structures found mainly on asparagine (N-linked) or serine/threonine (O-linked) residues. We have developed a glycoproteomic approach that combines efficient purification of urinary glycoproteins/glycopeptides with complementary MS-fragmentation techniques for glycopeptide analysis. Starting from clinical sample size, we eliminated interfering urinary compounds by dialysis and concentrated the purified urinary proteins by lyophilization. Sialylated urinary glycoproteins were conjugated to a solid support by hydrazide chemistry and trypsin digested. Desialylated glycopeptides, released through mild acid hydrolysis, were characterized by tandem MS experiments utilizing collision induced dissociation (CID) and electron capture dissociation fragmentation techniques. In CID-MS(2), Hex(5)HexNAc(4)-N-Asn and HexHexNAc-O-Ser/Thr were typically observed, in agreement with known N-linked biantennary complex-type and O-linked core 1-like structures, respectively. Additional glycoforms for specific N- and O-linked glycopeptides were also identified, e.g. tetra-antennary N-glycans and fucosylated core 2-like O-glycans. Subsequent CID-MS(3), of selected fragment-ions from the CID-MS(2) analysis, generated peptide specific b- and y-ions that were used for peptide identification. In total, 58 N- and 63 O-linked glycopeptides from 53 glycoproteins were characterized with respect to glycan- and peptide sequences. The combination of CID and electron capture dissociation techniques allowed for the exact identification of Ser/Thr attachment site(s) for 40 of 57 putative O-glycosylation sites. We defined 29 O-glycosylation sites which have, to our knowledge, not been previously reported. This is the first study of human urinary glycoproteins where "intact" glycopeptides were studied, i.e. the presence of glycans and their attachment sites were proven without doubt.     

Identification of protein-bound oligosaccharides on the surface of growth cones that bind to muscle cells

In the accompanying paper (Gabel, Den, and Ambron, in press) it was shown that eight populations of glycopeptides are synthesized by single neurons of Aplysia californica. To see which glycopeptides might mediate interactions with target cells, we first identified glycopeptides that are transported selectively to synapses and growth cones. The giant neuron R2 was injected intrasomatically with 3H-glucosamine. Twenty-four hours later, 3H-glycopeptides in the axon and cell body were isolated and resolved by serial lectin affinity chromatography. Of the eight populations, the biantennary-type glycopeptides (GPbi) and those that bind to WGA (GPwga) were preferentially associated with rapidly transported glycoproteins. In contrast, the glycopeptide that consists of N-acetylglucosamine O-linked to ser/thr was mostly retained in the cell body. GPbi and GPwga were also preferentially transported to growth cones. Analyses of RUQ cells, exposed to 3H-glucosamine in vitro for 36 h showed an enrichment of GPbi and GPwga at the growth cone relative to the cell body. The disposition of the various glycopeptides in growing neurons was also examined using FITC lectins. FITC-coupled WGA, Vicia vellosa, and lentil lectin showed extensive staining of the cell body, but only WGA stained the growth cones. To investigate if GPwga interacts specifically with target cells, these glycopeptides were isolated from the neurons of 180 abdominal ganglia. GPwga, other Aplysia glycopeptides, and glycopeptides prepared from ovalbumin were coupled separately to fluorescent spheres. The spheres were then added to muscle cells isolated from the auricle of the heart, which is innervated by many neurons from the ganglion. While spheres coupled to GPwga bound to the muscle cell surface, the other glycopeptides did not. These results indicate that glycopeptides class GPwga, found among rapidly transported glycoproteins and on the growth cone surface, is able to bind to muscle cells and may therefore play some role in neuron-target interactions.     

Quantitative site-specific analysis of protein glycosylation by LC-MS using different glycopeptide-enrichment strategies

A common technique for analysis of protein glycosylation is HPLC coupled to mass spectrometry (LC-MS). However, analysis is challenging due to a low abundance of glycopeptides in complex protein digests, microheterogeneity at the glycosylation site, ion suppression effects, and competition for ionization by coeluting peptides. Specific sample preparation is necessary for a comprehensive and site-specific glycosylation analysis by MS. In this study we qualitatively compared hydrophilic interaction chromatography (HILIC) and hydrazine chemistry for the enrichment of all N-linked glycopeptides and titanium dioxide for capturing sialylated glycopeptides from a complex peptide mixture. Bare silica, microcrystalline cellulose, amino-, amide- (TSKgel Amide-80), and sulfobetaine-(ZIC-HILIC) bonded phases were evaluated for HILIC enrichment. The experiments revealed that ZIC-HILIC and TSKgel Amide-80 are very specific for capturing glycopeptides under optimized conditions. Quantitative analysis of N-glycosidase F-released and 2-aminobenzamide-labeled glycans of a ZIC-HILIC-enriched monoclonal antibody demonstrated that glycopeptides could be enriched without bias for particular glycan structures and without significant losses. Sialylated glycopeptides could be efficiently enriched by titanium dioxide and in addition to HILIC both methods enable a comprehensive analysis of protein glycosylation by MS. Enrichment of N-linked glycopeptides by hydrazine chemistry resulted in lower peptide recovery using a more complex enrichment scheme.     

Oligosaccharide structural specificity of the soluble agglutinin released from guinea pig colonic epithelial cells

Abstract Guinea pig colonic epithelial cells release a soluble lectin capable of agglutinating numerous strains of Shigella and Escherichia coli as well as other bacteria. Using pure oligosaccharides and glycopeptides with well-defined structures to inhibit the agglutination of Shigella flexneri 1b by the soluble intestinal lectin, we have been able to demonstrate that the latter recognises different structural types. Inhibition by human milk glucoprotein glycopeptides with biantennary glycans of the N -acetyllactosamine type was dependent on the simultaneous presence of unsubstituted terminal non-reducing galactose residues and of a fucose residue α-1,6-linked to the asparagine-conjugated N -acetylglucosamine residue. Unsubstituted terminal non-reducing galactose was also determinant for inhibition by human milk oligosaccharides. Finally oligosaccharides possessing the Man (α1–2) Man structure inhibited more effectively than those with a Man(α1–3)Man sequence. The fact that these different structural motifs were all inhibitory raises the problem of the possible existence of a multispecific lectin or of several different lectins in the guinea pig colonic mucosa mediating bacterial adherence.     

Site-specific detection and structural characterization of the glycosylation of human plasma proteins lecithin:cholesterol acyltransferase and apolipoprotein D using HPLC/electrospray mass spectrometry and sequential glycosidase digestion.

Site-specific structural characterization of the glycosylation of human lecithin:cholesterol acyltransferase (LCAT) was carried out using microbore reversed-phase high performance liquid chromatography coupled with electrospray ionization mass spectrometry (HPLC/ESIMS). A recently described mass spectrometric technique involving monitoring of carbohydrate-specific fragment ions during HPLC/ESIMS was employed to locate eight different groups of glycopeptides in a digest of a human LCAT protein preparation. In addition to the four expected N-linked glycopeptides of LCAT, a di-O-linked glycopeptide was detected, as well as three additional glycopeptides. Structural information on the oligosaccharides from all eight glycopeptides was obtained by sequential glycosidase digestion of the glycopeptides followed by HPLC/ESIMS. All four potential N-linked glycosylation sites (Asn20, Asn84, Asn272, and Asn384) of LCAT were determined to contain sialylated triantennary and/or biantennary complex structures. Two unanticipated O-linked glycosylation sites were identified at Thr407 and Ser409 of the LCAT O-linked glycopeptide, each of which contain sialylated galactose beta 1-->3N-acetylgalactosamine structures. The three additional glycopeptides were determined to be from a copurifying protein, apolipoprotein D, which contains potential N-linked glycosylation sites at Asn45 and Asn78. These glycopeptides were determined to bear sialylated triantennary oligosaccharides or fucosylated sialylated biantennary oligosaccharides. Previous studies of LCAT indicated that removal of the glycosylation site at Asn272 converts this protein to a phospholipase (Francone OL, Evangelista L, Fielding CJ, 1993, Biochim Biophys Acta 1166:301-304). Our results indicate that the carbohydrate structures themselves are not the source of this functional discrimination; rather, it must be mediated by the structural environment around Asn272.     

Glycoform composition profiling of O-glycopeptides derived from human serum IgA1 by matrix-assisted laser desorption ionization-time of flight-mass spectrometry

Pools of O-glycopeptides prepared from trypsin-digested reduced and alkylated human serum IgA1 have been analyzed using matrix-assisted laser desorption ionization-time of flight-mass spectrometry (MALDI-ToF-MS) in the positive-ion mode, using 2,4,6-trihydroxy acetophenone-ammonium citrate matrix. Dozens of such pools prepared from normal serum IgA1 and from serum of patients with a number of different medical conditions have been routinely analyzed in this manner. The glycopeptides present in these pools possess identical amino acid sequences but are substituted with a variety of neutral and sialylated glycans and the spectra obtained were such that individual compositional glycoforms were baseline resolved. In addition, the spectra were reproducible, exhibiting a relative peak intensity and area variation of around 11-16%, enabling the technique to be used for the relative quantitation of the different compositional glycoforms present. This could be achieved manually or by applying a Java program especially developed for this purpose. The MS analysis described here is a major improvement over present MALDI methods used for profiling the O-glycosylation of IgA1. The MS methodology together with the Java data analysis are expected to be generally applicable for profiling O-linked glycopeptides derived from other glycoproteins and probably for N-linked glycopeptide pools.     

Relationship of the terminal sequences to the length of poly-N-acetyllactosamine chains in asparagine-linked oligosaccharides from the mouse lymphoma cell line BW5147. Immobilized tomato lectin interacts with high affinity with glycopeptides containing long poly-N-acetyllactosamine chains

To investigate the factors regulating the biosynthesis of poly-N-acetyllactosamine chains containing the repeating disaccharide [3Gal beta 1,4GlcNAc beta 1] in animal cell glycoproteins, we have examined the structures and terminal sequences of these chains in the complex-type asparagine-linked oligosaccharides from the mouse lymphoma cell line BW5147. Cells were grown in medium containing [6-3H]galactose, and radiolabeled glycopeptides were prepared and fractionated by serial lectin affinity chromatography. The glycopeptides containing the poly-N-acetyllactosamine chains in these cells were complex-type tri- and tetraantennary asparagine-linked oligosaccharides. The poly-N-acetyllactosamine chains in these glycopeptides had four different terminal sequences with the structures: I, Gal beta 1,4GlcNAc beta 1,3Gal-R; II, Gal alpha 1,3Gal beta 1,4GlcNac beta 1,3Gal-R; III, Sia alpha 2,3Gal beta 1,4GlcNAc beta 1,3Gal-R; and IV, Sia alpha 2,6Gal beta 1,4GlcNAc beta 1,3Gal-R. We have found that immobilized tomato lectin interacts with high affinity with glycopeptides containing three or more linear units of the repeating disaccharide [3Gal beta 1,4GlcNAc beta 1] and thereby allows for a separation of glycopeptides on the basis of the length of the chain. A high percentage of the long poly-N-acetyllactosamine chains bound by immobilized tomato lectin were not sialylated and contained the simple terminal sequence of Structure I. In addition, a high percentage of the sialic acid residues that were present in the long chains were linked alpha 2,3 to penultimate galactose residues (Structure III). In contrast, a high percentage of the shorter poly-N-acetyllactosamine chains not bound by the immobilized lectin were sialylated, and most of the sialic acid residues in these chains were linked alpha 2,6 to galactose (Structure IV). These results indicate that there is a relationship in these cells between poly-N-acetyllactosamine chain length and the degree and type of sialylation of these chains.     

Targeted serum glycoproteomics for the discovery of lung cancer‐associated glycosylation disorders using lectin‐coupled ProteinChip arrays

To screen for glycoproteins showing aberrant sialylation patterns in sera of cancer patients and apply such information for biomarker identification, we performed SELDI‐TOF MS analysis coupled with lectin‐coupled ProteinChip arrays (Jacalin or SNA) using sera obtained from lung cancer patients and control individuals. Our approach consisted of three processes (i) removal of 14 abundant proteins in serum, (ii) enrichment of glycoproteins with lectin‐coupled ProteinChip arrays, and (iii) SELDI‐TOF MS analysis with acidic glycoprotein‐compatible matrix. We identified 41 protein peaks showing significant differences (p<0.05) in the peak levels between the cancer and control groups using the Jacalin‐ and SNA‐ProteinChips. Among them, we identified loss of Neu5Ac (α2,6) Gal/GalNAc structure in apolipoprotein C‐III (apoC‐III) in cancer patients through subsequent MALDI‐QIT‐TOF MS/MS. Furthermore, subsequent validation experiments using an additional set of 60 lung adenocarcinoma patients and 30 normal controls demonstrated that there is a higher frequency of serum apoC‐III with loss of α2,6‐linkage Neu5Ac residues in lung cancer patients compared to controls. Our results have demonstrated that lectin‐coupled ProteinChip technology allows the high‐throughput and specific recognition of cancer‐associated aberrant glycosylations, and implied a possibility of its applicability to studies on other diseases.