Large-scale preparation and characterization of N-linked glycopeptides from bovine fetuin

Rice, Kevin G.; Rao, Narasinga B.N.; Lee, Yuan C.

doi:10.1016/0003-2697(90)90676-z

Cited by 44 publications

(19 citation statements)

References 21 publications

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“…The site-specific composition and heterogeneity data for the N-linked carbohydrates reported here are consistent with previous reports (Green et al, 1988;Townsend et al, 1988;Cumming et al, 1989;Rice et al, 1990). Compositions consistent with triantennary Asn-linked carbohydrates with the general structure (Ne~Ac)~(HexH~x N A c )~H~x , H~x N A c~ were identified at Ams1, and AsnI5'.…”

Section: Discussionsupporting

confidence: 91%

“…of fetuin was different than that used in the present study. Our findings that biantennary structures are absent or Chromatography present at only very low levels at Asn" and Asn'" but are present at significant levels at are consistent The gradient HPLC system is a Brownlee Labs (Santa with the reports by Lee and coworkers (Rice et al, 1990) Clara, California) Microgradient'" with dual syringe…”

Section: Discussionsupporting

confidence: 82%

“…Tetrasialylated carbohydrates, if present, gave signals below our detection limit (Green et al, 1988;Cumming et al, 1989). These data are consistent with the presence of triantennary complex carbohydrates at Am", in agreement with the literature (Townsend et al, 1988;Rice et al, 1990). It is important to note that the 2+ charge state of the glycopeptide (m/z 1,610. l), which gave a strong signal in the LC-ESMS data (Fig.…”

Section: Selective Detecfion Of Glycopeptidessupporting

confidence: 86%

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Selective identification and differentiation of N‐and O‐linked oligosaccharides in glycoproteins by liquid chromatography‐mass spectrometry

1993

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A mass spectrometry method has been developed for selective detection of glycopeptides at the low (525) picomole level during chromatography of glycoprotein digests and for differentiation of 0-linked from N-linked oligosaccharides. The technique involves observation of diagnostic sugar oxonium-ion fragments, particularly the HexNAc+ fragment at m/z 204, from collisionally excited glycopeptides. Collision-induced fragmentation can be accomplished in either of two regions of a triple quadrupole mass spectrometer equipped with an atmospheric pressure, electrospray (ES) ionization source. If collisions before the first quadrupole are chosen, it is possible to enhance formation of carbohydrate-related fragment ions without distorting the distribution of peptide and glycopeptide signals by increasing the collisional excitation potential only during that portion of each scan in which the low mass carbohydrate-related ions are being detected. This procedure, requiring only a single quadrupole instrument, identifies putative glycopeptide-containing fractions in the chromatogram but suffers from a lack of specificity in the case of co-eluting peptides. Increased specificity is obtained by selectively detecting only those parent ions that fragment in 4 2 , the second collision region of the triple quadrupole, to produce an ion at m/z 204 (HexNAc'). Only (M + H)+ ions of glycopeptides are observed in these liquid chromatography-electrospray tandem mass spectrometry (LC-ESMS/MS) "parent-scan" spectra. N-linked carbohydrates are differentiated from 0-linked by LC-ESMS/MS analysis of the digested glycoprotein prior to and after selective removal of N-linked carbohydrates by peptide N:glycosidase E These methods, which constitute the first liquid chromatography-mass spectrometry (LC-MS)-based strategies for selective identification of glycopeptides in complex mixtures, facilitate location and preparative fractionation of glycopeptides for further structural characterization. In addition, these techniques may be used to assess the compositional heterogeneity at specific attachment sites, and to define the sequence context of the attachment site in proteins of known sequence. The strategy is demonstrated for bovine fetuin, a 42-kDa glycoprotein containing three N-linked, and at least three 0-linked carbohydrates. Over 90% of the fetuin protein sequence was also corroborated by these LC-ESMS studies.

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Section: Discussionsupporting

confidence: 91%

Section: Discussionsupporting

confidence: 82%

Section: Selective Detecfion Of Glycopeptidessupporting

confidence: 86%

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Selective identification and differentiation of N‐and O‐linked oligosaccharides in glycoproteins by liquid chromatography‐mass spectrometry

1993

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“…Although slightly more heterogeneous, they agree well with previously published data [10,19,20]. The N-linked structures have up to three antennae and up to four sialic acid moieties.…”

Section: Fetuin Glycan Characterizationsupporting

confidence: 91%

Precursor ion scanning for detection and structural characterization of heterogeneous glycopeptide mixtures

Ritchie

Gill

Deery

et al. 2002

J. Am. Soc. Mass Spectrom.

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The structure of N-linked glycans is determined by a complex, anabolic, intracellular pathway but the exact role of individual glycans is not always clear. Characterization of carbohydrates attached to glycoproteins is essential to aid understanding of this complex area of biology. Specific mass spectral detection of glycopeptides from protein digests may be achieved by on-line HPLC-MS, with selected ion monitoring (SIM) for diagnostic product ions generated by cone voltage fragmentation, or by precursor ion scanning for terminal saccharide product ions, which can yield the same information more rapidly. When glycosylation is heterogeneous, however, these approaches can result in spectra that are complex and poorly resolved. We have developed methodology, based around precursor ion scanning for ions of high m/z, that allows site specific detection and structural characterization of glycans at high sensitivity and resolution. These methods have been developed using the standard glycoprotein, fetuin, and subsequently applied to the analysis of the N-linked glycans attached to the scrapieassociated prion protein, PrP Sc . These glycans are highly heterogeneous and over 30 structures have been identified and characterized site specifically. Product ion spectra have been obtained on many glycopeptides confirming structure assignments. The glycans are highly fucosylated and carry Lewis X or sialyl Lewis X epitopes and the structures are in-line with previous results. [Abbreviations: Hex-Hexose, C 6 H 12 O 6 carbohydrates, including mannnose and galactose; HexNAc-N-acetylhexosamine, C 8 H 15 NO 6 carbohydrates, including N-acetylglucosamine and N-acetylgalactosamine; GlcNAc-N-acetylglucosamine; GalNAc-N-acetylgalactosamine; Fuc-Fucose; NeuAC-N-acetylneuraminic acid or sialic acid; TSE-Transmissible Spongiform Encephalopathy.] (J Am Soc Mass Spectrom 2002Spectrom , 13, 1065Spectrom -1077

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“…Tetrasialylated glycans reportedly contain an unusual sequence of Galβ1 → 3GlcNAc linkage [29,30]. AMC-labeled oligosaccharides from fetuin appeared from 24 to 28 min in HILIC mode, and from 28 to 33 min in reverse-phase HPLC.…”

Section: Online Cleanup Lc Analysis Of Amc-labeled Oligosaccharides Dmentioning

confidence: 99%

Online Cleanup Liquid Chromatography for the Analysis of Glycoprotein-Derived Oligosaccharides Labeled with 7-Amino-4-methylcoumarin

et al. 2017

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In HPLC analysis of glycoprotein glycans, oligosaccharides released from glycoproteins are often derivatized with fluorescent tags for enhancing sensitivity and selectivity. However, existing analysis methods require multiple steps for removing excess reagents and are time consuming and sometime spoil the reliability in quantitative determination. Here, we developed an online sample cleanup procedure for the analysis of 7-amino-4-methylcoumarin (AMC)-labeled oligosaccharides. An NH 2 -bonded silica packed short column (1 mm I.D. x 15 mm) and a Waters HLB TM -packed column (4 mm I.D. x 5 cm) were used for online cleanup HPLC analysis with Amide80 and ODS columns, respectively. Optimized conditions enabled the direct injection of the diluted solution of the reaction mixture onto the chromatographic system without any prior removal of the excess labeling reagents. The method was successfully applied to the analysis of N-linked glycans released from some glycoprotein samples.

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Large-scale preparation and characterization of N-linked glycopeptides from bovine fetuin

Cited by 44 publications

References 21 publications

Selective identification and differentiation of N‐and O‐linked oligosaccharides in glycoproteins by liquid chromatography‐mass spectrometry

Selective identification and differentiation of N‐and O‐linked oligosaccharides in glycoproteins by liquid chromatography‐mass spectrometry

Precursor ion scanning for detection and structural characterization of heterogeneous glycopeptide mixtures

Online Cleanup Liquid Chromatography for the Analysis of Glycoprotein-Derived Oligosaccharides Labeled with 7-Amino-4-methylcoumarin

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