Collision-Induced Dissociation Tandem Mass Spectrometry for Structural Elucidation of Glycans
Department of Chemistry, University of California, Davis, CA, USA. Methods in Molecular Biology
(Impact Factor: 1.29).
02/2009; 534:133-45. DOI: 10.1007/978-1-59745-022-5_10
The complexity of glycans poses a major challenge for structure elucidation. Tandem mass spectrometry is currently an efficient and powerful technique for the structural characterization of glycans. Collision-induced dissociation (CID) is most commonly used, and involves first isolating the glycan ions of interest, translationally exciting them, and then striking them with inert target gas to fragment the precursor ions. The structural information of the glycan can be obtained from the fragment ions of the tandem MS spectra. In this chapter, sustained off-resonance irradiation-collision-induced dissociation (SORI-CID) implemented with matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI FT ICR MS) is demonstrated to be a useful analysis tool for structural elucidation of mucin-type O-glycans released from mucin glycoproteins. The mechanisms by which the glycans undergo fragmentations in the tandem mass analysis are also discussed.
Available from: Jerry L Hedrick
- "Glycoproteins were obtained from the jelly coat by a procedure described in previous publications (Xie et al. 2001, 2004; Zhang, Lindsay, et al. 2004; Zhang, Xie, et al. 2004; Zhang et al. 2005). The O-linked oligosaccharides were released from glycoproteins by a β-elimination reaction (Li et al. 2009a). Briefly, a certain amount of glycoprotein was incubated in 1.0 M NaBH 4 and 0.1 M NaOH at 42°C for 16 h followed by neutralization with pre-cooled 1.0 M hydrochloric in an ice bath. "
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ABSTRACT: Differences in the fertilization behavior of Xenopus borealis from X. laevis and X. tropicalis suggest differences in the glycosylation of the egg jellies. To test this assumption, O-linked glycans were chemically released from the egg jelly coat glycoproteins of X. borealis. Over 50 major neutral glycans were observed, and no anionic glycans were detected from the released O-glycan pool. Preliminary structures of ∼30 neutral oligosaccharides were determined using matrix-assisted laser desorption/ionization
(MALDI) infrared multiphoton dissociation tandem mass spectrometry (MS). The mass fingerprint of a group of peaks for the
core-2 structure of O-glycans was conserved in the tandem mass spectra and was instrumental in rapid and efficient structure determination. Among
the 29 O-glycans, 22 glycans contain the typical core-2 structure, 3 glycans have the core-1 structure and 2 glycans contained a previously
unobserved core structure with hexose at the reducing end. There were seven pairs of structural isomers observed in the major
O-linked oligosaccharides. To further elucidate the structures of a dozen O-linked glycans, specific and targeted exoglycosidase digestions were carried out and the products were monitored with MALDI-MS.
Reported here are the elucidated structures of O-linked oligosaccharides from glycoproteins of X. borealis egg jelly coats. The structural differences in O-glycans from jelly coats of X. borealis and its close relatives may provide a better understanding of the structure–function relationships and the role of glycans
in the fertilization process within Xenopodinae.
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ABSTRACT: Mass spectrometry has emerged as an invaluable technique with a wide array of applications ranging from clinical to biodefense.
With the development of different ionization techniques and mass analyzers, even challenging samples can be analyzed, thereby
making mass spectrometry an important analytical tool in the field of biophysics. Mass spectrometry is the only technique
that offers the combination of high sensitivity (attomole) with structural information. While other analytical techniques
may provide higher sensitivity, these techniques do not provide structural information. Conversely, other techniques may provide
more complete structures but have significantly less sensitivity. The different ionization techniques allow for the examination
of analytes ranging from small metabolites to large macromolecular assemblies. In this chapter the major components are described
rather than the possible applications, which would require volumes. With the major concepts in hand, the student is encouraged
to read specific reviews regarding the kinds of applications of interest to the researcher.
Available from: Katherine Williams
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ABSTRACT: We report covalent attachment via a thiol ester linkage of 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid or SA) to cysteine-containing protein biomarkers from bacterial cell lysates of E. coli analyzed by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry when using SA as the matrix. Evidence to support this conclusion is the appearance of additional peaks in the MS spectra when using SA, which are absent when using alpha-cyano-4-hydroxycinnamic acid (HCCA). The additional peaks appear at a mass-to-charge (m/z) approximately 208 greater to the m/z of a more abundant protein ion peak. Protein biomarkers were identified by tandem mass spectrometry (MS/MS) using a MALDI time-of-flight/time-of-flight (TOF-TOF) mass spectrometer and top-down proteomics. Three protein biomarkers, HdeA, HdeB, and homeobox or YbgS (each containing two cysteine residues) were identified as having reactivity to SA. Non-cysteine-containing protein biomarkers showed no evidence of reactivity to SA. MS ions and MS/MS fragment ions were consistent with covalent attachment of SA via a thiol ester linkage to the side-chain of cysteine residues. MS/MS of a protein biomarker ion with a covalently attached SA revealed fragment ion peaks suggesting dissociative loss SA. We propose dissociative loss of SA is facilitated by a pentacyclic transition-state followed by proton abstraction of the beta-hydrogen of the bound SA by a sulfur lone pair followed by dissociative loss of 3-(4-hydroxy-3,5-dimethoxyphenyl)prop-2-ynal. The apparent reactivity of SA to cysteine/disulfide-containing proteins may complicate identification of such proteins, however the apparent differential reactivity of SA and HCCA toward cysteine/disulfide-containing proteins may be exploited for identification of unknown cysteine-containing proteins.
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