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. (c) 2006 Elsevier B.V. All rights reserved

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) MSⁿ 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 MS² spectrum of N-glycopeptide was acquired so that a glycoside-bond cleavage occurs in the chitobiose residue (i.e., GlcNAcβ1-4GlcNAc, 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 Bn-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 MS³ spectra derived from both the sodiated Bn-type fragment ions of N-glycopeptide and the PA (2-aminopyridine) N-glycan standard (i.e., MSⁿ spectral matching). In a similar manner, the structural assignment of sialylated N-glycan was performed by employing the negative-ion CID MSⁿ spectra of deprotonated Bn-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 MSⁿ spectral matching is useful for the direct and simple structural assignment of neutral and sialylated N-glycans of glycopeptides. Copyright © 2006 John Wiley & Sons, Ltd

Structural assignment of isomeric 2-aminopyridine derivatized monosialylated biantennary N-linked oligosaccharides using negative-ion MSn spectral matching

To investigate the possibility of structural assignment based on negative-ion MSn spectral matching, four isomers of 2-aminopyridine-derivatized (PA) monosialylated oligosaccharides (i.e., complex type N-glycans with an α2-3 or α2-6 linked sialic acid on α1-6 or α1-3 antennae) were analyzed by using high-performance liquid chromatography/electrospray ion trap time-of-flight mass spectrometry(HPLC/ESI-IT-TOF MS). The negative ion [M-2H]2- is observed dominantly in the MS1 spectra without the loss of a sialic acid. The MS2 spectra derived from it are sufficiently reproducible that MS2 spectral matching based on correlation coefficients can be applied to the assignment of these isomers. The isomers containing a sialic acid on α1-6 or α1-3 antennae can be distinguished by MS2 spectral matching, but the α2-3 and α2-6 linkage types of sialic acid cannot be distinguished by their MS2 spectra. However, MS3 spectra derived from fragment ions containing a sialic acid (i.e., C4- and D-type ions) clearly differentiate the α2-3 and α2-6 linkage types of sialic acid in their MS3 spectral patterns. This difference might be rationalized in terms of a proton transfer from the reducing-end mannose to the negatively-charged sialic acid. These two moieties are very close in the structural conformations of the precursor C4-type fragment ions, as predicted by molecular mechanics calculations. Thus, negative-ion MSn (n=2, 3) spectral matching was proven to be useful for the structural assignment of these four monosialylated PA N-glycan isomers

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 α1-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

Total cellular glycomics allows characterizing cells and streamlining the discovery process for cellular biomarkers.

Although many of the frequently used pluripotency biomarkers are glycoconjugates, a glycoconjugate-based exploration of novel cellular biomarkers has proven difficult due to technical difficulties. This study reports a unique approach for the systematic overview of all major classes of oligosaccharides in the cellular glycome. The proposed method enabled mass spectrometry-based structurally intensive analyses, both qualitatively and quantitatively, of cellular N- and O-linked glycans derived from glycoproteins, glycosaminoglycans, and glycosphingolipids, as well as free oligosaccharides of human embryonic stem cells (hESCs), induced pluripotent stem cells (hiPSCs), and various human cells derived from normal and carcinoma cells. Cellular total glycomes were found to be highly cell specific, demonstrating their utility as unique cellular descriptors. Structures of glycans of all classes specifically observed in hESCs and hiPSCs tended to be immature in general, suggesting the presence of stem cell-specific glycosylation spectra. The current analysis revealed the high similarity of the total cellular glycome between hESCs and hiPSCs, although it was suggested that hESCs are more homogeneous than hiPSCs from a glycomic standpoint. Notably, this study enabled a priori identification of known pluripotency biomarkers such as SSEA-3, -4, and -5 and Tra-1-60/81, as well as a panel of glycans specifically expressed by hESCs and hiPSCs

Sialic acid-dependent attachment of mucins from three mouse strains to Entamoeba histolytica

Mouse strain-specific differences in the carbohydrate composition of intestinal mucins were hypothesized to account for strain-dependent susceptibility to Entamoeba histolytica. To test this hypothesis, intestinal mucins from susceptible and resistant inbred strains of mice were analyzed for their O-glycan content and for their ability to inhibit amoebic adherence to (GalNAc)12-27-HSA neo-glycoproteins. The results showed that the colorectal mucin O-glycan of susceptible CBA mice was lower in sialic acid content than that of resistant C57BL/6 and BALB/c mice. Mucins from CBA mice were more potent inhibitors of E. histolytica adherence to neo-glycoproteins than were mucins from C57BL/6 or BALB/c mice. Consistent with the role of terminal Gal/GalNAc as a receptor for amoebic adherence, sialidase treatment of C57BL/6 and BALB/c colorectal mucins increased their ability to inhibit E. histolytica adherence to the neo-glycoproteins. These results provide evidence of mouse strain-specific differences in the sialic acids content of mucin O-glycans. These dissimilarities likely contribute to the differential susceptibility of the three mouse strains to E. histolytica infection