Structural assignment of isomeric 2-aminopyridine-derivatized oligosaccharides using MSn spectral matching

Two isomeric pairs of 2-aminopyridine (PA)-derivatized fucosylated and non-fucosylated oligosaccharides (complex-type N-glycans of IgG) were analyzed using liquid chromatography/ion trap mass spectrometry (LC/ITMS) with a sonic spray ionization source and by varying the collision-induced dissociation voltage. Reproducibility of MS(n) (n = 2) spectra obtained by LC/ITMS was tested considering both fragment ions (m/z) and intensities. A comparison of their MS(n) spectra and evaluation of similarities (or matching), based on correlation coefficients between MS(n) spectra, was investigated as a possibility for structural assignment of the isomers. It is shown that such MS(n) spectral matching is useful and applicable to the structural assignment of relatively large fucosylated and sialylated PA-oligosaccharides released from IgG based on Bn- and Yn-type fragmentations of the corresponding [M+H+Na](2+) ions.     

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

To investigate the possibility of structural assignment based on negative-ion tandem multistage (MSn) mass spectral matching, four isomers of 2-aminopyridine (PA)-derivatized monosialylated oligosaccharides (i.e., complex-type N-glycans with an alpha2-3- or alpha2-6-linked sialic acid on alpha1-6 or alpha1-3 antennae) were analyzed using high-performance liquid chromatography/electrospray ion trap time-of-flight mass spectrometry (HPLC/ESI-IT-TOFMS). The negative ion [M-2H]2- is observed predominantly 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 alpha1-6 or alpha1-3 antennae can be distinguished by MS2 spectral matching, but the alpha2-3 and alpha2-6 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 alpha2-3 and alpha2-6 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 of alpha2-6 linkage type, as predicted by molecular mechanics calculations. Thus, negative-ion MSn (n = 2, 3) spectral matching was demonstrated to be useful for the structural assignment of these four monosialylated PA N-glycan isomers.     

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.     

Structural assignment of isomeric 2‐aminopyridine‐derivatized monosialylated biantennary N‐linked oligosaccharides using negative‐ion multistage tandem mass spectral matching

The original article to which this Erratum refers was published in Rapid Commun. Mass Spectrom. 2006; 20 : 412–418     

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.     

Structural analysis of isomeric chondroitin sulfate oligosaccharides using regioselective 6-O-desulfation method and tandem mass spectrometry

A strategy based on a regioselective 6-O-desulfation reaction and negative ion electrospray ionization tandem mass spectrometry (ESI-MS(n)) was developed for the structural delineation of isomeric chondroitin sulfate oligosaccharides. Product ions resulting from the glycosidic cleavage provided information about the number of sulfate groups in each sugar residue. After the regioselective 6-O-desulfation reaction, the number of sulfate groups on each residue was obtained using a tandem mass spectrometry analysis of the reaction product. The sulfation pattern could be obtained based on the product ions of analytes before and after the desulfation reaction. The strategy was demonstrated using a series of tetrasaccharides prepared from shark cartilage chondroitin sulfate D. Among the 12 identified tetrasaccharides, six structures had not been reported before.     

Characteristic pH and electrolyte concentration dependences of 2-aminopyridine-derivatized oligosaccharides (N-glycans) in sonic-spray ionization mass spectrometry

The intensities of ion signals from neutral oligosaccharides (N-glycans) derivatized with 2-aminopyridine (PA) were analyzed by ion trap mass spectrometry with a sonic-spray ionization (SSI) source, in both positive- and negative-ion modes, while varying the pH and concentration of ammonium acetate buffer solution. Two characteristic results are reported and discussed. The first characteristic is the pH dependence of the ion intensities; on increasing the solution pH from 4.3 to 8.6, positive ion intensities increase and negative ion intensities decrease. The second characteristic concerns the dependence of ion intensities on electrolyte concentration; on increasing the electrolyte concentration, the SSI efficiency for the PA N-glycans first increases and then decreases. Assuming that the SSI mechanism essentially conforms to the statistical charging model and the charge residue model, a new model that focuses a great deal of attention on the counter (electrolyte) ion distribution surrounding the solvated analyte (PA N-glycan) is proposed, in particular to rationalize the characteristic pH dependence.     

Semiquantitative analysis of isomeric oligosaccharides by negative-ion mode UV-MALDI TOF postsource decay mass spectrometry and their fragmentation mechanism study at N-acetyl hexosamine moiety

Postsource decay (PSD) spectra of isomeric neutral lactooligosaccharide mixtures were measured from the chlorinated molecules [M + Cl]- by negative-ion mode ultraviolet matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (UV-MALDI TOF MS) to estimate quantitatively the mixing ratios in their mixtures. The PSD ions specific to each isomeric structure were used to distinguish the linkage and branching isomers, and the molar ratios of the isomers were estimated from their ion abundances. The relative ion abundances changed linearly in the PSD spectra of the mixtures of the isomers as their molar ratio was varied in the analyte solutions. Therefore, the molar ratios of the isomers in the analyte mixtures could be estimated semiquantitatively. In addition, we studied their fragmentation mechanisms in N-acetyl hexosamines such as GlcNAc, which enabled us to quantitatively analyze the structures of the isomers of lactooligosaccharides. The conjugated systems elongate in the chemical species of the Z-type fragmentation on the 3-linked GlcNAc owing to the acetoamido groups at the C-2 positions, which made the chemical species of the Z-type ions stable. The glycosyl bonds of the front of GlcNAc cleaved easily as a C-type fragmentation because the negative charge at the anomeric position could be delocalized to the carbonyl oxygen atom at the acetoamido group of GlcNAc. These factors caused the stabilization of the chemical species of the C/Z fragment ions produced by the double cleavage around GlcNAc.     

Sequence analysis of alginate-derived oligosaccharides by negative-ion electrospray tandem mass spectrometry

Negative-ion electrospray tandem mass spectrometry (ES-MS/MS) with collision-induced dissociation (CID) is attempted for sequence determination of alginate oligosaccharides, derived from polyanionic alginic acid, polymannuronate, and polyguluronate by partial depolymerization using either alginate lyase or mild acid hydrolysis. Sixteen homo- and hetero-oligomeric fragments were obtained after fractionation by gel-filtration and strong anion exchange high performance liquid chromatography. The product-ion spectra of these alginate oligosaccharides were dominated by intense B-, C-, Y-, and Z-type ions together with (0,2)A- and (2,5)A-ions of lower intensities. Internal mannuronate residues (M) produce weak but specific decarboxylated Z(int)-ions (Z(int) - 44 Da; int: denotes internal), which can be used for distinction of M and a guluronate residue (G) at an internal position. A reducing terminal M or G, although neither gives rise to a specific ion, can be identified by differences in the intensity ratio of fragment ions of the reducing terminal residue [(2,5)A(red)]/[(0,4)A(red)] (red: denotes reducing terminal).     

Configurational assignment and 1H NMR spectral parameters of isomeric 1,2-diacetoxy-4-alkyl-3-methylcyclopentanes

The ¹H NMR spectral parameters of all eight 1,2-diacetoxy-3,4-dimethylcyclopentanes and 1,2-diacetoxy-4-t-butyl-3-methylcyclopentanes are discussed.     

1H NMR spectral parameters and configurational assignment of the isomeric 1,4-diacetoxy-2- t-butyl-3-methylcyclopentanes

The isomeric 1,4-diacetoxy-2-t-butyl-3-methylcyclopentanes have been prepared starting from 2-t-butyl-3-methyl-4-hydroxy-2-cyclopentenone and 3-t-butyl-2-methyl-4-hydroxy-2-cyclopentenone. Configurational assignments are made on the basis of ¹H NMR data.     

Complementary structural information of positive- and negative-ion MSn spectra of glycopeptides with neutral and sialylated N-glycans

Positive- and negative-ion MSn spectra of chicken egg yolk glycopeptides binding a neutral and a sialylated N-glycan were acquired by using electrospray ionization linear ion trap time-of-flight mass spectrometry (ESI-LIT-TOFMS) and collision-induced dissociation (CID) with helium as collision gas. Several characteristic differences were observed between the positive- and negative-ion CID MSn (n = 2, 3) spectra. In the positive-ion MS2 spectra, the peptide moiety was presumably stable, but the neutral N-glycan moiety caused several B-type fragmentations and the sialylated N-glycan almost lost sialic acid(s). In contrast, in the negative-ion MS2 spectra, the peptide moiety caused several side-chain and N-glycan residue (e.g., N-acetylglucosamine (GlcNAc) residue) fragmentations in addition to backbone cleavages, but the N-glycan moieties were relatively stable. The positive-ion MS3 spectra derived from the protonated peptide ion containing a GlcNAc residue (203.1 Da) provided enough information to determine the peptide amino-acid sequence including the glycosylation site, while the negative-ion MS3 spectra derived from the deprotonated peptide containing a 0,2X1-type cross-ring cleavage (83.1 Da) complicated the peptide sequence analysis due to side-chain and 0,2X1 residue related fragmentations. However, for the structural information of the N-glycan moiety of the glycopeptides, the negative-ion CID MS3 spectra derived from the deprotonated 2,4A6-type cross-ring cleavage ion (neutral N-glycan) or the doubly deprotonated B6-type fragment ion (sialylated N-glycan) are more informative than are those of the corresponding positive-ion CID MS3 spectra. Thus, the positive-ion mode of CID is useful for the analyses of peptide amino-acid sequences including the glycosylation site. The negative-ion mode of CID is especially useful for sialylated N-glycan structural analysis. Therefore, in the structural analysis of N-glycopeptides, their roles are complementary.     

Configurational assignment and 1H-NMR spectral parameters of the isomeric 1,4-diacetoxy-2,3-dimethylcyclopentanes

All six 1,4-diacetoxy-2,3-dimethyl-cyclopentanes have been prepared starting from 2, 3-dimethyl-4-hydroxy-2-cyclopentenone. ¹H-NMR spectral parameters, allowing configurational assignment, are discussed.     

Structural and spectral assignment of three forskolin-like diterpenoids isolated from Plectranthus ornatus

Three labdane diterpenoids were isolated from an acetone extract of Plectranthus ornatus. Their structures, closely related to that of forskolin, were determined by NMR studies. Unambiguous and complete assignments of the 1H and 13C NMR chemical shifts for these substances are presented. The assignments are based on 2D shift-correlated [1H, 1H-COSY, 1H, 13C-gHSQC-1J (C,H), 1H, 13C-gHMBC-(n)J (C,H) (n = 2 and 3)] and NOE experiments.     

Mass spectral identification of isomeric fluoronitroanilines

The electron impact and methane chemical ionization mass spectra of 2-fluoro-5-nitroaniline and 4-fluoro-3-nitroaniline have been investigated. Similar fragment ions were observed for both compounds under electron impact conditions, but the difference in the relative ion intensities of two key fragment ions allowed the positive identification of these two isomers. The difference can be attributed to an ortho effect involving fluorine and the amino substituent. A fragmentation pathway consistent with this observation is suggested.     

Structural differentiation of isomeric N-oxides

The recently reported photochemical isomerization of 1-(2-pyridyl)benzotriazole 3-oxide to the 2-oxide created a need for a method of distinguishing between the structural isomers. Nmr experiments using Eu(fod)₃ established the position of the oxygen atoms and also yielded convincing evidence regarding attachment of the lanthanide shift reagent to these multi-functional compounds. Double irradiation experiments were used to resolve the structural problem while shift gradient considerations led to probable sites of coordination.     

Structural characterization of oligosaccharides in recombinant soluble human interferon receptor 2 using fluorophore-assisted carbohydrate electrophoresis

The N-linked oligosaccharide profiles (banding patterns in gels) and structures of recombinant soluble human interferon receptor 2 (r-shIFNAR2) were determined using fluorophore-assisted carbohydrate electrophoresis (FACE, Glyko, Novato, CA). The method involves releasing N-linked oligosaccharide moieties from a glycoprotein by digestion with peptide-N glycanase (PNGase F), labeling the released oligosaccharides with the fluorescent dye 8-aminonaphthalene-1,3,6-trisulfonate (ANTS), and separating the labeled oligosaccharides by gel electrophoresis. The isolated oligosaccharides in the bands from the profiling gels can then be sequenced using exoglycosidases to reveal the oligosaccharide structures. The oligosaccharide profile of r-shIFNAR2 consists of at least nine oligosaccharide bands. The relative amount of oligosaccharide in each band can vary, depending on the culture conditions of the source cells. FACE structural analysis shows that r-shIFNAR2 contains only core-fucosylated N-linked oligosaccharides, most of which are fully sialylated (approximately 92%). The major types and relative amounts of the oligosaccharides from a representative sample are: disialylated, galactosylated, biantennary (15%); trisialylated, galactosylated, triantennary (19%), tetrasialylated, galactosylated, tetraantennary (30%), and N-acetyllactosamine-containing higher-order oligosaccharides including tri-, tetra-, and pentaantennary (28%). The remaining oligosaccharides are not fully sialylated and/or not fully galactosylated di-, tri-, and tetraantennary structures (approximately 5%) and unidentified structures (approximately 3%). A method for determining the types and structures of the N-acetyllactosamine containing oligosaccharides is also reported in this study.     

Configurational assignment of some isomeric 1,4-diacetoxy-2,3-dialkylcyclopentanes from 1H-n.m.r. spectral parameters

From the study of the ¹H n.m.r. spectral parameters of differently functionalized 1,4-diacetoxy-2,3-dialkylcyclopentanes it is shown that the magnitude of those parameters allows an easy configurational assignment within a set of four different configurations.