Low-energy collision-induced dissociation (CID) Has explored for the purpose of differentiating between isomeric pertrideuteroacetylated aldopentosides, 6-deoxyaldohexosides and their respective furanosidic and pyranosidic analogues. The 1-O-methylated derivatives were analyzed by gas chromatography/chemical ionization (CPI,) tandem mass spectrometry. B-1 ions (formally C(1) carbenium ions), which are expected to reflect the core structure of the parent sugar and which were produced by the elimination of methanol from the protonated glycoside derivatives, were used as precursor ions to obtain the corresponding CID spectra. In general, strong similarities which impede an unepuivocal differentiation of the individual constituents were found in the spectral patterns of the different isomeric pairs However, examination of the pseudo-breakdown behavior of the B-1 ions over the range 5-35 eV revealed improved distinction windows which provide for a fuller differentiating capability between five-and six-membered ring isomers including even the most problematic ribose derivatives, The reliability of the analyses is enhanced by the high reproducibility (s < 10%) of these ratios. The considerable similarity between the tandem mass spectra of certain furanosidic and pyranosidic B-1 ions is highly suggestive of a partial loss of structural integrity, mainly due to ring contraction, which occurs to different extents in the different isomers, Copyright (C) 1999 John Wiley & Sons, Ltd.

In proteome analysis, the determination of the phosphorylation status of proteins and protein isoforms, which have been separated by two-dimensional polyacrylamide gel electrophoresis (2D PAGE), is of prime importance in addition to their identification. In this study, the extent to which such information can be directly extracted from the mass spectrometric data used for identification was evaluated. By searching for metastable peaks which are characteristic for loss of phosphoric acid, the Ser-phosphorylated peptides were identified with a high success rate in reflector matrix-assisted laser desorption/ionization (MALDI) mass maps of in-gel digested proteins. Furthermore, by employing a double enzymatic strategy using trypsin and Glu-C in parallel, improved sequence coverage and additional separation of the potential phosphorylation sites of the isoforms were achieved. The precise location of the modified sites within an identified phosphopeptide was obtained by submitting the corresponding molecular ions directly to nano-electrospray tandem mass spectrometric analysis. In this way the detailed phosphorylation status of six isomers of stathmin separated by 2D PAGE was determined. Two of these six isomers were phosphorylated at all four known sites (serines 15, 24, 37 and 62) and were probably derived from the previously reported alpha and beta forms, which differ by a yet unknown modification. In addition, isomers phosphorylated at serines 15, 24 and 37, serines 24, 37 and 62, serines 24 and 37 and serine 37 only were characterized.

Formation of “false” sugar sequence ions from branched tetrasaccharides of the sialyl-Lewis-type by migration of fucose towards sialic acid residues is shown to occur in [M + H]+ and [M + NH4]+ ions produced by electrospray ionization and subjected to low energy collision induced dissociation (CID). For the verification of their composition and sequence, such irregular ions were produced in the orifice region of the ion source, mass selected in Q1, and subjected to a second CID step in Q2 of a triple quadrupole analyser. When produced and analysed in the same “double CID” fashion, the branched B3 ions still containing all four sugar subunits show such migration to only a minor extent. The analysis of Bn fragment ions with high numbers for n may thus have advantages over the analysis of M-like species

Recently, the C-mannosylation of a specific tryptophan residue in RNase 2 from human urine has been reported [Hofsteenge, J., et al. (1994) Biochemistry 33, 13524-13530; de Beer, T., et al. (1995) Biochemistry 34, 11785-11789]. In those studies, identification of this unusual modification was accomplished by mass spectrometric and NMR spectroscopic analysis of peptide fragments. The evidence for the occurrence of C2-alpha-mannosyltryptophan [(C2-Man-)Trp] in the intact protein relied exclusively on the detection of the same phenylthiohydantoin derivatives during Edman degradation. In this paper, we have (1) excluded the possibility that (C2-Man-)Trp arose artificially under the acidic conditions previously employed for protein and peptide isolation and analysis, by maintaining the pH > 5 throughout these procedures, (2) demonstrated the occurrence of (C2-Man-)Trp in the intact protein, by NMR spectroscopy, (3) showed that (C2-Man-)Trp is not unique for RNase 2 from urine but that it is also present in the enzyme isolated from erythrocytes, and (4) found also that high-molecular mass isoforms of urinary RNase 2 are C-mannosylated. These observations firmly establish C-mannosylation as a novel way of post-translationally attaching carbohydrate to protein, in addition to the well-known N- and O-glycosylations. Furthermore, the NMR data, in combination with molecular dynamics calculations, indicate that in the native protein the mannopyranosyl residue is in a different conformation than in the glycopeptide or denatured protein, due to protein-carbohydrate interactions.

The site-specific distribution of oligosaccharides on murine polymeric immunoglobulin A (pIgA) consisting of two or more immunoglobulin A (IgA) antibodies connected through J-chain was analysed by liquid chromatography/electrospray mass spectrometry. Glycopeptides from pIgA were localized in a reversed-phase tryptic peptide chromatogram by collision excitation scanning and their amino acid sequences determined by electrospray tandem mass spectrometry and Edman degradation, Two glycosylation sites on IgA and a single glycosylation site on J-chain were identified, Using biosynthetic constraints on carbohydrate structures, molecular mass information on the glycan moieties of the glycopeptides was translated into specific carbohydrate structure proposals, The glycopeptide incorporating the single glycosylation site at Asn49 in J-chain carried fucosylated and non-fucosylated di-and triantennary N-acetyllactosamine type carbohydrate chains terminated by N-acetyl- and/or N-glycolylneuraminic acid residues, The glycosylation site in the IgA heavy chain at Asn446 contained two oligomannose-type carbohydrate chains, one carrying five and the other six mannose residues, To the other glycosylation site in the IgA heavy chain at Asn155 one oligomannose structure, hybrid structures with the lactosamine branch terminated by either an additional galactose residue, N-glycolylneuraminic acid or N-acetylneuraminic acid, and non-fucosylated N-acetyllactosamine-type structures carrying the same terminating residues were attached, This glycosylation site was present in two separate glycopeptides differing only in their degree of carboxymethylation and yielding identical oligosaccharide distributions, thus providing additional confidence in the assignment method.

We report a new type of linkage between a carbohydrate and a protein, involving the rarely modified side chain of a tryptophan residue. An aldohexopyranosyl residue was found to be linked via a C-C bond to the indole ring of the tryptophan residue at position 7 of human RNase Us. Mass spectrometric analysis of peptides containing this residue showed a molecular mass 162 Da higher than that expected for tryptophan. The fragmentation pattern of the modified amino acid side chain was reminiscent of that of aromatic C-glycosides, suggesting a direct attachment of a hexose residue to a C-position of the tryptophan indole moiety. 1H and 13C NMR spectroscopic data confirmed this inference and unequivocally demonstrated the substituent to be an aldohexopyranosyl residue, C-glycosidically linked to the C2 atom of the indole. This mode of attachment differs from the ones known so far, in which carbohydrates are linked to an amino acid side chain by N- or O-glycosidic bonds.

Applicability and performance of electrospray ionization mass spectrometry (ESIMS) is demonstrated for protein analysis. ESIMS is applied in conjunction with on-line HPLC (LC-ESlMS) and direct tandem mass spectrometry (positive and negative ion mode ESlMS/MS) to the structural characterization of a recombinant protein (r-hirudin variant 1) and a congener phosphorylated at threonine45 (RP-1).

The potential of tandem mass spectrometry (MS/MS) as a stand-alone technique in the structural analyses of an oviposition-deterring pheromone (ODP, 1) is reviewed. Two facets of the salt-like glycolipid structure of 1 were of major interest in this context: the substitution pattern of the lipid backbone (15-glucosyloxy-8-hydroxypalmitate) and, more specifically, the configurational identity of the sugar portion (glucopyranose). Throughout this study, trideuterioacetyl derivatives of ODP (1 --> pentakis(trideuterioacetyl)-ODP la) and the reference substrates were used. Probing of the sugar moiety by fast atom bombardment (FAB) and both low- and high-energy collision-induced dissociation (CID) of B1-type sugar ions surprisingly failed as a single exception within a larger number of glycosidic substrates subjected to this approach. However, electrospray ionization (ESI) of la with the formation of the sugar ions in the gas phase by 'first-stage' CID before mass selection circumvented this difficulty and provided an unambiguous and sensitive probe for sugar stereochemistry. When studying the ODP molecule as a whole, FAB-generated M-like ions such as [M - H]-, [M + Na]+ and [M - H + 2Na]+ were subjected to high-energy CID using a four-sector tandem mass spectrometer. Analyses of simple model substrates such as the 12-trideuterioacetoxystearate anion facilitated the interpretation of the distinct charge-remote fragmentation (CRF) behaviour of 1a. Whereas all M-like species provided complete records of the lipid portion of 1a and its oxygenation pattern, only the sodiated cations allowed reliable location of the individual substituents. In these latter species complementary series of 'sequence ions' were observed that incorporated either the taurine or the sugar terminus and thus reflected CRF for both alternatives of terminal charge fixation.

Biomedical & Environmental Mass SpectrometryVolume 19, Issue 6 p. 390-392 Short Communication High performance tandem mass spectrometry for sequence, branching and interglycosidic linkage analysis of peracetylated oligosaccharides B. Domon, B. Domon Central Function Research, Ciba-Geigy AG, CH-402 Basel, SwitzerlandSearch for more papers by this authorD. R. Müller, D. R. Müller Central Function Research, Ciba-Geigy AG, CH-402 Basel, SwitzerlandSearch for more papers by this authorW. J. Richter, W. J. Richter Central Function Research, Ciba-Geigy AG, CH-402 Basel, SwitzerlandSearch for more papers by this author B. Domon, B. Domon Central Function Research, Ciba-Geigy AG, CH-402 Basel, SwitzerlandSearch for more papers by this authorD. R. Müller, D. R. Müller Central Function Research, Ciba-Geigy AG, CH-402 Basel, SwitzerlandSearch for more papers by this authorW. J. Richter, W. J. Richter Central Function Research, Ciba-Geigy AG, CH-402 Basel, SwitzerlandSearch for more papers by this author First published: June 1990 https://doi.org/10.1002/bms.1200190611Citations: 46AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat No abstract is available for this article. References 1 B. Lindberg and J. Lönngren, in Methods in Enzymology ed. by V. Ginsburg, Vol. 50, p. 3, Academic Press, New York (1978). 2 H. Egge and J. Peter-Katalinic, Mass Spectrom. Rev. 6, 331 (1987). 3 S. A. Carr, V. N. Reinhold, B. N. Green and J. R. Hass, Biomed. Mass Spectrom. 12, 288 (1985). 4 S. Santikarn, V. N. Reinhold, C. E. Costello and C. Warren, Proc. 35th Conf. Mass Spectrom. Allied Topics, Denver, CO, p. 870 (1987). 5 B. L. Gillece-Castro and A. L. Burlingame, Proc. 35th Conf. Mass Spectrom. Allied Topics, Miami Beach, FL, p. 1190 (1989). 6 B. Domon, D. R. Müller and W. J. Richter, Org. Mass Spectrom. 24, 357 (1989). 7 D. R. Müller, B. Domon and W. J. Richter, in Advances Mass Spectrometry, ed. by P. Longevialle, Vol. 11B, p. 1309, Heyden & Son Ltd, London (1989). 8 D. R. Müller, B. Domon and W. J. Richter, in Methods in Enzymology, ed. by J. A. McCloskey, Academic Press, Orlando FL (1990), in press. 9 B. Domon, D. R. Müller and W. J. Richter, submitted for publication. 10 B. Domon and C. E. Costello, Glycoconj. J. 5, 397 (1988). Citing Literature Volume19, Issue6June 1990Pages 390-392 ReferencesRelatedInformation

Publisher Summary This chapter discusses tandem mass spectrometry in structural characterization of oligosaccharide residues in glycoconjugates. In tandem mass spectrometry (MS/MS), two-stage mass analysis is performed. In the first stage (MS-l), a specific ion of interest is selected according to its mass from the set of ions that constitute the conventional mass spectrum. The second stage (MS-2) generates, from this selected precursor ion, a spectrum of product ions that arise from metastable ion (MI) or collision-induced dissociation (CID). The discussion in this chapter is limited to the structural analysis of small carbohydrate subunits (up to 5 sugar units) attached to larger aglycon moieties. At present, MS/MS of sugar ions formed from glycoconjugates appears to fulfill some, but not all, of the basic requirements of structure analysis.

MH+ ions of isomeric methyl ethyl phenylmaleates, 2, and -succinates, 3, obtained by chemical ionization (CI), exhibit preferential collision induced elimination of alcohol involving the 1-alkoxyl group which is adjacent to the phenyl substituent. MD+ ions of 2 and 3 exhibit major elimination of ROD under collisional activation, and little (when generated under CD4-CI) or no (under ND3-CI) hydrogen—deuterium exchange of the external deuteron with the phenyl hydrogen atoms is observed in this process. The favored collision-induced alcohol elimination from MH+ ions of isomeric methyl ethyl phenylfumarates, 4, involves the 4-alkoxyl group. An extensive hydrogen—deuterium exchange and scrambling of the external deuteron with the five phenyl hydrogen atoms precedes the elimination of each of the two alcohols from MD+ ions of 4 independent of the reagent gas (CD4 or ND3). A practically complete exchange takes place in the course of elimination involving the 4-alkoxyl group. These results are explained in terms of proximity of the 4-alkoxycarbonyl group to the phenyl ring in phenyl fumarates, 4.

High performance tandem mass spectrometry employing a four-sector instrument is used for the characterization of disaccharides. In contrast to low energy collisions, high energy collision induced dissociation (CID) of B2 sequence ions (C(1)-carbenium ions) derived from trideuterioacetylated dihexopyranosides permits immediate differentiation of all four linkage types (1–2, 1–3, 1–4 and 1–6) irrespective of the nature of the monosaccharide subunits. Moreover, high energy collisions produce spectra richer in features in that less energy-demanding processes (loss of peripheral residues such as AcOH-d3, Ac2O-d6 and ketene-d2) are supplemented by characteristic charge-remote fragmentations (radical eliminations, cleavages within the sugar rings). In some cases, assignment of the anomeric configuration (α/β) of the interglycosidic linkages (requiring difficult conditions in low energy CID) is also possible.