Vascular RAGE transports oxytocin into the brain to elicit its maternal bonding behaviour in mice.

Oxytocin sets the stage for childbirth by initiating uterine contractions, lactation and maternal bonding behaviours. Mice lacking secreted oxcytocin (Oxt -/-, Cd38 -/-) or its receptor (Oxtr -/-) fail to nurture. Normal maternal behaviour is restored by peripheral oxcytocin replacement in Oxt -/- and Cd38 -/-, but not Oxtr -/- mice, implying that circulating oxcytocin crosses the blood-brain barrier. Exogenous oxcytocin also has behavioural effects in humans. However, circulating polypeptides are typically excluded from the brain. We show that oxcytocin is transported into the brain by receptor for advanced glycation end-products (RAGE) on brain capillary endothelial cells. The increases in oxcytocin in the brain which follow exogenous administration are lost in Ager -/- male mice lacking RAGE, and behaviours characteristic to abnormalities in oxcytocin signalling are recapitulated in Ager -/- mice, including deficits in maternal bonding and hyperactivity. Our findings show that RAGE-mediated transport is critical to the behavioural actions of oxcytocin associated with parenting and social bonding.

Intestinal transepithelial permeability of oxytocin into the blood is dependent on the receptor for advanced glycation end products in mice.

Plasma oxytocin (OT) originates from secretion from the pituitary gland into the circulation and from absorption of OT in mother's milk into the blood via intestinal permeability. However, the molecular mechanism underlying the absorption of OT remains unclear. Here, we report that plasma OT concentrations increased within 10 min after oral delivery in postnatal day 1-7 mice. However, in Receptors for Advanced Glycation End Products (RAGE) knockout mice after postnatal day 3, an identical OT increase was not observed. In adult mice, plasma OT was also increased in a RAGE-dependent manner after oral delivery or direct administration into the intestinal tract. Mass spectrometry evaluated that OT was absorbed intact. RAGE was abundant in the intestinal epithelial cells in both suckling pups and adults. These data highlight that OT is transmitted via a receptor-mediated process with RAGE and suggest that oral OT supplementation may be advantageous in OT drug development.

Absorption and Urinary Excretion of Peptides after Collagen Tripeptide Ingestion in Humans.

Collagen tripeptide (CTP) is a collagen hydrolysate containing a high concentration of tripeptides with a Gly-X-Y sequence, such as Gly-Pro-Hyp. To test the effects of this preparation, we compared the absorption of peptides in humans after ingestion of a tripeptide fraction of CTP (CTP-100), a CTP preparation containing ca. 50% Gly-X-Y tripeptides (CTP-50), and a collagen peptide that did not contain tripeptides (CP). The postprandial levels of Gly-Pro-Hyp and Pro-Hyp in the plasma increased in those subjects who ingested CTP-100 and CTP-50, and were higher with greater Gly-Pro-Hyp ingestion. This demonstrated that collagen hydrolysates were efficiently absorbed when the collagen was ingested in the tripeptide form. Gly-Pro-Hyp and Pro-Hyp were also found in the urine after ingestion of CTP-100 or CTP-50. Similar to the results for the plasma concentration, the urinary excretion of Gly-Pro-Hyp and Pro-Hyp was also dependent on the amount of Gly-Pro-Hyp ingested. This indicates that ingested Gly-Pro-Hyp and generated Pro-Hyp were relatively stable in the body and were transported to the urine in the peptide form. The concentration of Hyp-Gly in the plasma was low after the ingestion of CP and CTP-100 but higher after the ingestion of CTP-50. Overall, our results suggest that tripeptides derived from collagen are absorbed efficiently by the body.

Absorption and plasma kinetics of collagen tripeptide after peroral or intraperitoneal administration in rats.

Collagen tripeptide (CTP) is a collagen-derived compound containing a high concentration of tripeptides with a Gly-X-Y sequence. In this study, the concentrations and metabolites of CTP were monitored in rat plasma after its administration. We performed a quantitative analysis using high-performance liquid chromatography tandem mass spectrometry according to the isotopic dilution method with stable isotopes. We confirmed that the tripeptides Gly-Pro-Hyp, Gly-Pro-Ala, and Gly-Ala-Hyp were transported into the plasma. Dipeptides, which are generated by degradation of the N- or C-terminus of the tripeptides Gly-Pro-Hyp, Gly-Pro-Ala, and Gly-Ala-Hyp, were also present in plasma. The plasma kinetics for peroral and intraperitoneal administration was similar. In addition, tripeptides and dipeptides were detected in no-administration rat blood. The pharmacokinetics were monitored in rats perorally administered with Gly-[(3)H]Pro-Hyp. Furthermore, CTP was incorporated into tissues including skin, bone, and joint tissue. Thus, administering collagen as tripeptides enables efficient absorption of tripeptides and dipeptides.

Lipo-oxytocin-1, a Novel Oxytocin Analog Conjugated with Two Palmitoyl Groups, Has Long-Lasting Effects on Anxiety-Related Behavior and Social Avoidance in CD157 Knockout Mice.

Oxytocin (OT) is a nonapeptide hormone that is secreted into the brain and blood circulation. OT has not only classical neurohormonal roles in uterine contraction and milk ejection during the reproductive phase in females, but has also been shown to have new pivotal neuromodulatory roles in social recognition and interaction in both genders. A single administration of OT through nasal spray increases mutual recognition and trust in healthy subjects and psychiatric patients, suggesting that OT is a potential therapeutic drug for autism spectrum disorders, schizophrenia, and some other psychiatric disorders. Although the mechanism is not well understood, it is likely that OT can be transported into the brain where it activates OT receptors to exert its function in the brain. However, the amount transported into the brain may be low. To ensure equivalent effects, an OT analog with long-lasting and effective blood-brain barrier penetration properties would be beneficial for use as a therapeutic drug. Here, we designed and synthesized a new oxytocin analog, lipo-oxytocin-1 (LOT-1), in which two palmitoyl groups are conjugated at the amino group of the cysteine9 residue and the phenolic hydroxyl group of the tyrosine8 residue of the OT molecule. To determine whether LOT-1 actually has an effect on the central nervous system, we examined its effects in a CD157 knockout model mouse of the non-motor psychiatric symptoms of Parkinson's disease. Similar to OT, this analog rescued anxiety-like behavior and social avoidance in the open field test with the social target in a central arena 30 min after intraperitoneal injection in CD157 knockout mice. When examined 24 h after injection, the mice treated with LOT-1 displayed more recovery than those given OT. The results suggest that LOT-1 has a functional advantage in recovery of social behavioral impairment, such as those caused by neurodegenerative diseases, autism spectrum disorders, and schizophrenia.

Profiling of N- and O-glycopeptides of erythropoietin by capillary zwitterionic type of hydrophilic interaction chromatography/electrospray ionization mass spectrometry.

Capillary zwitterionic-type hydrophilic interaction chromatography (ZIC-HILIC)/ESI-MS has been applied to the Glu-C digest of recombinant human erythropoietin (rhEPO) expressed in Chinese hamster ovary (CHO) cells. N-Glycopeptides (105) and O-glycopeptides (8) were detected in a single run of the capillary ZIC-HILIC/ESI-MS analysis. Among them, N-acetyl-neuraminic acids (Neu5Ac) of N- and O-glycans were partially acetylated and some were replaced with N-glycoyl-neuraminic acid (Neu5Gc). Their retentions in the ZIC-HILIC separation can be explained to some extent with the degree of acetylation and N-glycoylation, both of which influence the hydrophilicity/hydrophobicity of the N- and O-glycan moieties of glycopeptides.

Chromatographic deuterium isotope effects of derivatized N-glycans and N-glycopeptides in a zwitterionic type of hydrophilic interaction chromatography.

HPLC/MS is now an essential method for the analysis of glycans and glycopeptides. A technique using ICATs is also becoming popular for their comparative/quantitative analysis based on MS signal intensities. However, the RP HPLC most often used causes "doublet" peaks of deuterium-labeled and nonlabeled peptides, which are well known for causing the chromatographic deuterium isotope effect. Such doublet peaks are undesirable for precise comparison of MS signal intensities. This report shows that, in the zwitterionic type of hydrophilic interaction chromatography, the chromatographic deuterium isotope effect is negligibly small for N-glycans derivatized with deuterium-labeled 2-aminopyridine and N-glycopeptides derivatized with deuterium-labeled succinic anhydride.

Two-dimensional hydrophilic interaction chromatography coupling anion-exchange and hydrophilic interaction columns for separation of 2-pyridylamino derivatives of neutral and sialylated N-glycans.

A novel chromatographic approach coupling anion-exchange (diethylaminoethylene) and hydrophilic-interaction (amide or zwitterionic type) columns was developed for the separating of 2-pyridylamino derivatives of N-glycans (PA-N-glycans). This is a kind of on-line, two-dimensional (2D) separation approach in hydrophilic-interaction chromatography (called the 2D-HILIC method), analogous to that of coupling cation- (or anion-, or mixed ion-) exchange and reversed-phase columns in hydrophobic interaction (reversed-phase) chromatography. The efficiency of the 2D-HILIC method was tested with biantennary neutral and sialylated PA-N-glycan standards by properly combining linear gradient elutions of water-acetonitrile and spiked-salt (ammonium acetate) elutions. The retention time RSDs of all the peaks in three sequential runs of a 100 min cycle are less than 0.52%, which indicates a reasonably good repeatability of the 2D-HILIC method. Then, the method was applied to a complex mixture of PA-N-glycans from human serum proteins. It was demonstrated that the neutral PA-N-glycans and mono-, di-, tri-, and tetrasialylated PA-N-glycans are able to be eluted in turn according to the number of sialic acids in an automated (programmed) single run.

Swainsonine reduces 5-fluorouracil tolerance in the multistage resistance of colorectal cancer cell lines.

BACKGROUND: Drug resistance is a major problem in cancer chemotherapy. Acquisition of chemo-resistance not only reduces the effectiveness of drugs, but also promotes side effects and markedly reduces the patient's quality of life. However, a number of resistance mechanisms have been reported and are thought to be the reason for the difficulties in solving drug-resistance problems. RESULT: To investigate the mechanisms of drug resistance, a set of cell lines with different levels of sensitivity and possessing different mechanisms of resistance to 5-fluorouracil (5-FU) was established from a colorectal cancer cell line. The expression of thymidylate synthase, orotic acid phosphoribosyltransferase and dihydropyrimidine dehydrogenase, which are well known to be related to drug resistance, differed among these cell lines, indicating that these cell lines acquired different resistance mechanisms. However, swainsonine, an inhibitor of N-glycan biosynthesis, reduced 5-FU-tolerance in all resistant cells, whereas the sensitivity of the parental cells was unchanged. Further analysis of the N-glycan profiles of all cell lines showed partial inhibition of biosynthesis and no cytotoxicity at the swainsonine dosage tested. CONCLUSION: These observations suggest that N-linked oligosaccharides affect 5-FU resistance more widely than do drug-resistance related enzymes in colorectal cancer cells, and that the N-glycan could be a universal target for chemotherapy. Further, swainsonine may enhance the performance of chemotherapy by reducing tolerance.

Analysis of N-glycan in serum glycoproteins from db/db mice and humans with type 2 diabetes.

Glycosylation has an important role in regulating properties of proteins and is associated with many diseases. To examine the alteration of serum N-glycans in type 2 diabetes, we used the db/db mouse model. Serum N-glycans were fluorescence labeled and applied to HPLC. There were reproducible differences in N-glycan profiles between the db/db mouse model and the db/+ control. The structures of the oligosaccharides, which had changed in their amounts, were analyzed by a two-dimensional mapping method, matrix-assisted laser desorption ionization-time-of-flight mass spectrometry, and exoglycosidase digestion. Those analyses revealed an increase in the N-glycans possessing alpha1,6-fucose in the serum of db/db mice. The level of alpha1,6-fucosyltransferase mRNA was increased in the liver of the db/db mice. The ratio of a biantennary N-glycan with alpha1,6-fucose to that without alpha1,6-fucose in the liver tissue of the db/db mouse was increased relative to the db/+ control. Next, we analyzed the serum N-glycan profile in human subjects with type 2 diabetes and found an increased amount of a biantennary N-glycan that had an alpha1,6-fucose with a bisecting N-acetylglucosamine. In conclusion, the increase in alpha1,6-fucosylation is a striking change in the serum N-glycans of the db/db mice, whereas the change in the fucosylation in humans with type 2 diabetes was small, albeit statistically significant. It is likely that the change is caused, at least partially, by the increase in the alpha1,6-fucosyltransferase mRNA level in the liver. The increased alpha1,6-fucosylation may affect protein properties associated with the pathophysiology of type 2 diabetes.

N-glycan alterations are associated with drug resistance in human hepatocellular carcinoma.

BACKGROUND: Correlations of disease phenotypes with glycosylation changes have been analysed intensively in the tumor biology field. Glycoforms potentially associated with carcinogenesis, tumor progression and cancer metastasis have been identified. In cancer therapy, drug resistance is a severe problem, reducing therapeutic effect of drugs and adding to patient suffering. Although multiple mechanisms likely underlie resistance of cancer cells to anticancer drugs, including overexpression of transporters, the relationship of glycans to drug resistance is not well understood. RESULTS: We established epirubicin (EPI)--and mitoxantrone (MIT)--resistant cell lines (HLE-EPI and HLE-MIT) from the human hepatocellular carcinoma cell line (HLE). HLE-EPI and HLE-MIT overexpressed transporters MDR1/ABCB1 and BCRP/ABCG2, respectively. Here we compared the glycomics of HLE-EPI and HLE-MIT cells with the parental HLE line. Core fucosylated triantennary oligosaccharides were increased in the two resistant lines. We investigated mRNA levels of glycosyltransferases synthesizing this oligosaccharide, namely, N-acetylglucosaminyltransferase (GnT)-IVa, GnT-IVb and alpha1,6-fucosyltransferase (alpha1,6-FucT), and found that alpha1,6-FucT was particularly overexpressed in HLE-MIT cells. In HLE-EPI cells, GnT-IVa expression was decreased, while GnT-IVb was increased. Both GnT-IVs were downregulated in HLE-MIT cells. HLE-MIT cells also showed decreases in fucosylated tetraantennary oligosaccharide, the product of GnT-V. GnT-V expression was decreased in both lines, but particularly so in HLE-MIT cells. Thus both N-glycan and glycosyltransferase expression was altered as cells acquired tolerance, suggesting novel mechanisms of drug resistance. CONCLUSION: N-glycan and glycosyltransferase expression in HLE-EPI and HLE-MIT were analysed and presented that glycans altered according with acquired tolerance. These results suggested novel mechanisms of drug resistance.

Tumor suppressor p16INK4a--modulator of glycomic profile and galectin-1 expression to increase susceptibility to carbohydrate-dependent induction of anoikis in pancreatic carcinoma cells.

Expression of the tumor suppressor p16(INK4a) after stable transfection can restore the susceptibility of epithelial tumor cells to anoikis. This property is linked to increases in the expression and cell-surface presence of the fibronectin receptor. Considering its glycan chains as pivotal signals, we assumed an effect of p16(INK4a) on glycosylation. To test this hypothesis for human Capan-1 pancreatic carcinoma cells, we combined microarray for selected glycosyltransferase genes with 2D chromatographic glycan profiling and plant lectin binding. Major differences between p16-positive and control cells were detected. They concerned expression of beta1,4-galactosyltransferases (down-regulation of beta1,4-galactosyltransferases-I/V and up-regulation of beta1,4-galactosyltransferase-IV) as well as decreased alpha2,3-sialylation of O-glycans and alpha2,6-sialylation of N-glycans. The changes are compatible with increased beta(1)-integrin maturation, subunit assembly and binding activity of the alpha(5)beta(1)-integrin. Of further functional relevance in line with our hypothesis, we revealed differential reactivity towards endogenous lectins, especially galectin-1. As a result of reduced sialylation, the cells' capacity to bind galectin-1 was enhanced. In parallel, the level of transcription of the galectin-1 gene increased conspicuously in p16(INK4a)-positive cells, and even figured prominently in a microarray on 1996 tumor-associated genes and in proteomic analysis. The cells therefore gain optimal responsiveness. The correlation between genetically modulated galectin-1 levels and anoikis rates in engineered transfectants inferred functional significance. To connect these findings to the fibronectin receptor, galectin-1 was shown to be co-immunoprecipitated. We conclude that p16(INK4a) orchestrates distinct aspects of glycosylation that are relevant for integrin maturation and reactivity to an endogenous effector as well as the effector's expression. This mechanism establishes a new aspect of p16(INK4a) functionality.

Detection of altered N-glycan profiles in whole serum from rheumatoid arthritis patients.

Altered N-glycosylation occurs in many diseases. In rheumatoid arthritis (RA), for example, reduction in galactose residues in IgG and an increase in fucose residues in alpha1-acid glycoprotein have been observed. To further analyse N-glycans in disease, we show N-glycan profiling from whole serum employing reversed phase high performance liquid chromatography/negative-ion mode by sonic spray ionization ion trap mass spectrometry with pyridylamination. Profiles from female 15 RA patients and 18 aged-matched healthy women were compared. The most significant change seen in RA was decreased levels of mono-galactosyl bi-antennary N-glycans, in agreement with the previous reports regarding IgG. We also show previously unreported differences between isomers and increased tri-antennary oligosaccharides. These results indicate that LC-MS analysis of whole serum N-glycans can identify N-glycan alterations in RA and that this is a promising method both for studies of RA mechanisms and diagnosis.

Structural analysis of O-glycopeptides employing negative- and positive-ion multi-stage mass spectra obtained by collision-induced and electron-capture dissociations in linear ion trap time-of-flight mass spectrometry.

Structural analyses of various glycans attached to proteins and peptides are highly desirable for elucidating their biological roles. An approach based on mass spectrometry (MS) combining both collision-induced dissociation (CID) and electron-capture dissociation (ECD) in the positive- and negative-ion modes has been proposed as a simple and direct method of assigning an O-glycan without releasing it from the peptide and of determining the amino acid sequence of the peptide and glycosylation site. The instrument used is an electrospray ionization (ESI) linear ion trap (LIT) time-of-flight (TOF) mass spectrometer with tandem LITs for CID by He gas and ECD. The proposed approach was tested with two synthetic O-glycopeptides binding a sialyl Lewis x (sLe(x)) oligosaccharide and a 3'-sialyl N-acetyllactosamine (3'-SLN) on a serine (S) residue. In the negative-ion mode, the CID MS(2) spectra of O-glycopeptides showed a relatively abundant glycoside-bond cleavage between the core N-acetylglucosamine (GlcNAc) and serine (S) that yields deprotonated C(3)-type fragment ions of O-glycan and deprotonated Z(0)-type peptide ions. The structure of the sLe(x) (3'-SLN) oligosaccharide was simply assigned by comparing the CID MS(3) spectrum derived from the C(3)-type fragment ion with the CID MS(2) spectra of the sLe(x) and sLe(a) (3'- and 6'-SLN) standards (i.e., negative-ion MS(n) spectral matching). The amino acid sequence of the peptide including the glycosylation site was determined from the ECD MS(2) spectrum in the positive-ion mode.

N-linked neutral oligosaccharides in the stratum corneum of normal and ichthyotic skin.

N-Glycan oligosaccharides are thought to play multiple, important roles in a variety of biological events. However, N-glycan profiles in the stratum corneum of human skin have not yet been studied in detail. To clarify the N-glycan profiles in the stratum corneum of normal and ichthyotic epidermis, N-glycan profiles were studied by high-performance liquid chromatography using normal human epidermal samples and scales from hyperkeratotic skin of ichthyosis patients. Chromatograms of patient scale samples showed unique alterations in three peaks eluted at 15.8, 18.8 and 26.9 min. The N-glycan profiles were significantly altered in ichthyotic hyperkeratotic skin compared with normal non-hyperkeratotic controls. These findings indicate the reduction of N-acetylglucosaminyltransferase II and fucosyltransferase 8 activities. Alteration of N-glycan structures in hyperkeratotic skin suggests the biological role of N-glycans in keratinization.

Structural assignment of disialylated biantennary N-glycan isomers derivatized with 2-aminopyridine using negative-ion multistage tandem mass spectral matching.

To investigate the possibility of structural assignment based on negative-ion multistage tandem mass (MS(n)) spectral matching, four isomers of disialylated biantennary N-glycans (alpha2-6 and/or alpha2-3 linked sialic acid on alpha1-6 and alpha1-3 antennae) derivatized with 2-aminopyridine (PA) were analyzed by employing high-performance liquid chromatography/electrospray ionization linear ion trap time-of-flight mass spectrometry (HPLC/ESI-LIT-TOFMS), which uses helium gas for ion trapping and collision-induced dissociation (CID). It is shown that the MS(2) spectra derived from each precursor ion [M-2H](2-) are reproducible and useful for distinguishing the four isomers. Thus, they can be assigned by negative-ion MS(2) spectral matching based on correlation coefficients. In addition, MS(3) spectra derived from D-type fragment ions clearly differentiate the alpha2-3- or alpha2-6-linked sialic acid on the alpha1-6 antenna due to their characteristic spectral patterns. The C(4)-type fragment ions, which are produced from both the alpha1-6 and alpha1-3 antennae, show the characteristic MS(3) spectra reflecting alpha2-3- or alpha2-6- linkage type or a mixture of both types. Thus, the differentiation and assignment of these disialylated biantennary N-glycan isomers can also be supported with the MS(3) spectra of C(4)- and D-type ions.

Simple separation of isomeric sialylated N-glycopeptides by a zwitterionic type of hydrophilic interaction chromatography.

Asparagine-linked oligosaccharides (N-glycans) usually show structural heterogeneity, especially in proteins with sialylated N-glycans and, therefore, their structural analysis is still very difficult. A zwitterionic type of hydrophilic interaction chromatography column with sulfobetaine functional groups (called a ZIC-HILIC column) was applied to the separation of tryptic peptides of alpha-1-acid glycoprotein. It was demonstrated that the ZIC-HILIC separation column has a selectivity for sialylated N-glycopeptides and a high capability for separation based on the structural recognition of sialylated N-glycan isomers as well as for the previously reported neutral N-glycans and N-glycopeptides. The retention characteristics of neutral and sialylated N-glycans derivatized with 2-aminopyridine (PA N-glycans) demonstrate that the retentions of the N-glycans are based primarily on hydrophilic interaction with the water-rich liquid layer generated on the surface of the ZIC-HILIC column. In addition, the electrostatic repulsion interaction shielded with counter ions effectively tunes the separation and recognition of sialylated N-glycan isomers.

Structural analysis of sialyl N-glycan using pyridylamination and chromatography followed by multistage tandem mass spectrometry.

Multiple-dimensional mapping (n-DM) methods consisting of pyridylamination and high-performance liquid chromatography (HPLC) are widely used for oligosaccharide analysis. These methods are quantitative, sensitive, and suitable for separating isomers. Oligosaccharide structures are suggested by elution positions on two or three kinds of columns and then confirmed by enzymatic and chemical treatments or mass spectrometry (MS). Multiple-stage tandem MS (MS(n)) analyses have been used to determine oligosaccharide structures by spectrum matching, comparing standard oligosaccharides, and offering detailed MS(n) fragment analysis. However, oligosaccharides usually exist as a mixture including isomers. The 3-DM method provides quantitative information and well-isolated sialyl- or neutral-oligosaccharides; on the other hand, the MS(n) method saves time by eliminating complicated enzyme degradation steps. The combination of both methods, which support each other, represents a reasonable and efficient strategy. This chapter describes 3-DM on HPLC after negative-ion MS(n) spectral matching for sialyl N-glycans.

Direct structural assignment of neutral and sialylated N-glycans of glycopeptides using collision-induced dissociation MSn spectral matching.

Mass spectrometric analyses of various N-glycans binding to proteins and peptides are highly desirable for elucidating their biological roles. An approach based on collision-induced dissociation (CID) MS(n) spectra acquired by electrospray ionization linear ion trap time-of-flight mass spectrometry (ESI-LIT-TOFMS) in the positive- and negative-ion modes has been proposed as a direct method of assigning N-glycans without releasing them from N-glycopeptides. In the positive-ion mode of this approach, the MS(2) spectrum of N-glycopeptide was acquired so that a glycoside-bond cleavage occurs in the chitobiose residue (i.e., GlcNAcbeta1-4GlcNAc, GlcNAc: N-acetylglucosamine) attached to asparagine (N), and two charges on the [M+H+Na](2+) precursor ion are shared with both of the resulting fragments. These fragments are sodiated B(n)-type fragment ions of oligosaccharide (N-glycan) and a protonated peptide ion retaining one GlcNAc residue on the asparagine (N) residue. The structure of N-glycan was assigned by comparing MS(3) spectra derived from both the sodiated B(n)-type fragment ions of N-glycopeptide and the PA (2-aminopyridine) N-glycan standard (i.e., MS(n) spectral matching). In a similar manner, the structural assignment of sialylated N-glycan was performed by employing the negative-ion CID MS(n) spectra of deprotonated B(n)-type fragment ions of N-glycopeptide and the PA N-glycan standard. The efficacy of this approach was tested with chicken egg yolk glycopeptides with a neutral and a sialylated N-glycan, and human serum IgG glycopeptides with neutral N-glycan isomers. These results suggest that the approach based on MS(n) spectral matching is useful for the direct and simple structural assignment of neutral and sialylated N-glycans of glycopeptides.

Separation of isomeric 2-aminopyridine derivatized N-glycans and N-glycopeptides of human serum immunoglobulin G by using a zwitterionic type of hydrophilic-interaction chromatography.

Isomeric oligosaccharides and isomeric glycopeptides are sometimes difficult to separate on normal-phase (NP) and reversed-phase (RP) columns. A zwitterionic type of hydrophilic-interaction chromatography column with sulfobetaine groups (called ZIC-HILIC column) was first applied to the separation of 2-aminopyridine derivatized (PA) N-glycans and tryptic peptides of human serum immunoglobulin G (IgG). It is shown that the ZIC-HILIC column has high capability for structural recognition of isomeric N-glycans as well as high selectivity for glycopeptides. The former feature (i.e., structural recognition) was proven by sufficient separation of neutral PA N-glycan isomers, which are usually difficult to separate on NP and RP columns. In addition, it is noteworthy that IgG glycopeptides consisting of isomeric N-glycans and the same peptide sequences can be sufficiently separated on a ZIC-HILIC column. The latter feature (i.e., selectivity) was also demonstrated by easily separating two peptide groups with/without N-glycans. Thus, we note that the ZIC-HILIC column is highly promising for a simple analysis of N-glycans and N-glycopeptide samples.