Site-Specific Characterization of Glycoprotein Carbohydrates by Exoglycosidase Digestion and Laser-Desorption Mass Spectrometry

Sutton, Chris W.; O’Neill, Jacqui A.; Cottrell, John S.

doi:10.1006/abio.1994.1138

Cited by 165 publications

(118 citation statements)

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“…Lacking ␤4GalT-1, ␤4GalT-6, or Both-MALDI-TOF-MS has been used for both structural characterization (33) and relative quantitation (34) of neutral and sialylated N-glycans in a mixture. To examine in vivo galactosylation of the spectrum of N-glycans in CHO glycoproteins, N-glycans were released from parental and mutant CHO cellular glycoproteins by PNGase F and analyzed by MALDI-TOF-MS. Because most N-glycans derived from mammalian glycoproteins are composed of only a few monosaccharides and generate structures with unique masses that are of the oligomannosyl or bi-, tri-, or tetraantennary complex type, the nature of the species released by PNGase F may be deduced from their molecular mass in the context of known N-glycan structures (25)(26)(27).…”

Section: Maldi-tof-ms Analysis Of N-glycans In Cho Cellsmentioning

confidence: 99%

Chinese Hamster Ovary (CHO) Cells May Express Six β4-Galactosyltransferases (β4GalTs)

Lee

Sundaram

Shaper

et al. 2001

Journal of Biological Chemistry

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Six ␤4-galactosyltransferase (␤4GalT) genes have been cloned from mammalian sources. We show that all six genes are expressed in the Gat ؊ 2 line of Chinese hamster ovary cells (Gat ؊ 2 CHO). Two independent mutants termed Pro ؊ 5Lec20 and Gat ؊ 2Lec20, previously selected for lectin resistance, were found to have a galactosylation defect. Radiolabeled biantennary Nglycans synthesized by Pro ؊ 5Lec20 were proportionately less ricin-bound than similar species from parental CHO cells, and Lec20 cell extracts had a markedly reduced ability to transfer Gal to GlcNAc-terminating acceptors. Northern blot analysis revealed a severe reduction in ␤4GalT-1 transcripts in Pro ؊ 5Lec20 cells. The Gat ؊ 2Lec20 mutant expressed ␤4GalT-1 transcripts of reduced size due to a 311-base pair deletion in the ␤4GalT-1 gene coding region. Northern analysis with probes from the remaining five ␤4GalT genes revealed that Gat ؊ 2 CHO and Gat ؊ 2Lec20 cells express all six ␤4GalT genes. Unexpectedly, the ␤4GalT-6 gene is not expressed in either Pro ؊ 5 or Pro ؊ 5Lec20 cells. Thus, in addition to a deficiency in ␤4GalT-1, Pro ؊ 5Lec20 cells lack ␤4GalT-6. Nevertheless, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry data of N-glycans released from cellular glycoproteins showed that both the ␤4GalT-1 ؊ (Gat ؊ 2Lec20) and ␤4GalT-1 ؊ /␤4GalT-6 ؊ (Pro ؊ 5Lec20) mutants have a similar Gal deficiency, affecting neutral and sialylated bi-, tri-, and tetraantennary N-glycans. By contrast, glycolipid synthesis was normal in both mutants. Therefore, ␤4GalT-1 is a key enzyme in the galactosylation of Nglycans, but is not involved in glycolipid synthesis in CHO cells. ␤4GalT-6 contributes only slightly to the galactosylation of N-glycans and is also not involved in CHO cell glycolipid synthesis. These CHO glycosylation mutants provide insight into the variety of in vivo substrates of different ␤4GalTs. They may be used in glycosylation engineering and in investigating roles for ␤4GalT-1 and ␤4GalT-6 in generating specific glycan ligands.

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Section: Maldi-tof-ms Analysis Of N-glycans In Cho Cellsmentioning

confidence: 99%

Chinese Hamster Ovary (CHO) Cells May Express Six β4-Galactosyltransferases (β4GalTs)

Lee

Sundaram

Shaper

et al. 2001

Journal of Biological Chemistry

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“…. Structural determination of these compounds has classically been performed by chemical or enzymatic release followed by exoglycosidase digestion, with products monitored by gel filtration chromatography [3] or, more recently, high-performance liquid chromatography (HPLC) [4 -6] or matrix-assisted laser desorption/ionization (MALDI) mass spectrometry [7,8]. More recently, in response to the introduction of mass spectrometers, such as the tandem quadrupoletime-of-flight instruments and problems with the commercial availability of exoglycosidases, an increasing number of investigators are using wholly mass spectrometric-based approaches for the analysis of these compounds (see for example the recent review by Zaia [9]).…”

mentioning

confidence: 99%

Fragmentation of negative ions from carbohydrates: Part 3. Fragmentation of hybrid and complex N-linked glycans

Harvey

2005

J. Am. Soc. Mass Spectrom.

224

247

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Hybrid and complex N-linked glycans were ionized by electrospray in the presence of ammonium nitrate to give [M ϩ NO 3 ] Ϫ and [M ϩ (NO 3 ) 2 ] 2Ϫ ions. Low energy collisioninduced decomposition (CID) spectra of both types of ions were almost identical and were dominated by C-type glycosidic and cross-ring fragments, unlike the corresponding spectra of the positive ions that contained mainly B-and Y-type glycosidic fragments. Also, in contrast to fragments in the positive ion spectra, many of these ions appeared to be produced by single pathways following proton abstraction from specific hydroxy groups. Consequently, many ions were diagnostic for specific structural features. Such features included the composition of each of the two antennas, the presence or absence of a bisecting GlcNAc residue, and the location of fucose residues on the core GlcNAc residues and on the antennas. C-ions defined the sequence of the constituent monosaccharide residues. . Structural determination of these compounds has classically been performed by chemical or enzymatic release followed by exoglycosidase digestion, with products monitored by gel filtration chromatography [3] or, more recently, high-performance liquid chromatography (HPLC) [4 -6] or matrix-assisted laser desorption/ionization (MALDI) mass spectrometry [7,8]. More recently, in response to the introduction of mass spectrometers, such as the tandem quadrupoletime-of-flight instruments and problems with the commercial availability of exoglycosidases, an increasing number of investigators are using wholly mass spectrometric-based approaches for the analysis of these compounds (see for example the recent review by Zaia [9]).Most of this work has concentrated on the production and fragmentation of positive ions although it has become apparent in recent years [10 -16] that negative ion fragmentation produces complementary information and can, in many cases, yield less ambiguous spectra than those provided by positive ion spectra. We have recently investigated methods for producing stable negative ions from N-linked glycans and found that nitrate adducts, formed by electrospray from solvents containing small amounts of ammonium nitrate, yield intense negative ions that, on fragmentation, give several characteristic and diagnostic ions that are not present in the corresponding positive ion spectra [17]. Details of the fragmentation of high-mannose N-linked glycans, the early compounds in the biosynthesis of these compounds, were reported in an earlier paper [18] and this paper concentrates on the fragmentation of hybrid and complex glycans. Materials and Methods MaterialsHybrid and complex glycans were released with hydrazine [19,20] Methylation of the Carboxy Group of Sialic AcidsIn order to stabilize the sialic acids, the carboxy groups of°glycans°from°bovine°fetuin,°as°a°representative°ex-ample, were converted into their methyl esters by exchanging the protons for sodium and reacting the product with methyl iodide as described by Powell and Harvey°[27].

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“…Carbohydrates, on the other hand, are usually branched structures with individual monosaccharide constituents able to link together at different positions. Structural determination of N-linked glycans (those attached to asparagine in glycoproteins) has classically been performed by exoglycosidase digestion, with products monitored by techniques such as gel filtration chromatography [3] or, more recently, highperformance liquid chromatography (HPLC) [4 -6] or matrix-assisted laser desorption/ionization (MALDI) mass spectrometry [7,8]. Knowledge of the enzyme's specificity and the number of monosaccharide units removed during incubation with the glycan leads directly to the determination of both the nature of the monosaccharides removed and their linkage.…”

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confidence: 99%

Fragmentation of negative ions from carbohydrates: Part 1. Use of nitrate and other anionic adducts for the production of negative ion electrospray spectra from N-linked carbohydrates

Harvey

2005

J. Am. Soc. Mass Spectrom.

244

189

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Negative ion spectra of N-linked glycans were produced by electrospray from a dilute solution of the glycans and various salts in methanol:water using a Waters-Micromass Q-TOF Ultima Global tandem quadrupole/time-of-flight (Q-TOF) mass spectrometer. Stable anionic adducts were formed with chloride, bromide, iodide, nitrate, sulphate, and phosphate. Unstable adducts that fragmented by a cross-ring cleavage of the reducing N-acetylglucosamine (GlcNAc) residue, were formed with fluoride, nitride, sulphide, carbonate, bicarbonate, hydroxide, and acetate. Nitrate adducts prepared from ammonium nitrate produced the most satisfactory spectra as they were relatively free from in-source fragmentation products and gave signals that were about ten times as strong as those from corresponding [M Ϫ H] Ϫ ions prepared from solutions containing ammonium hydroxide. Detection limits were in the region of 20 fmol. Neutral glycans gave both singly-and doubly-charged ions with the larger glycans preferring the formation of doubly-charged ions. Acidic glycans with several acidic groups gave ions in higher charge states as the result of ionization of the anionic groups. Low energy collision-induced decomposition (CID) spectra of the singly-charged ions were dominated by cross-ring and C-type fragments, unlike the corresponding spectra of the positive ions that contained mainly B-and Y-type glycosidic fragments. Formation of these ions could be rationalized by proton abstraction from various hydroxy groups by an initially-formed anionic adduct. Prominent glycosidic and cross-ring cleavage ions defined structural features such as the specific composition of each of the two antennae, presence of a bisecting GlcNAc residue and location of fucose residues, details that were difficult to determine by conventional techniques. Acidic glycans fragmented differently on account of charge localization on the acid functions rather than the hydroxy groups. (J Am Soc Mass Spectrom 2005, 16, 622-630)

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Site-Specific Characterization of Glycoprotein Carbohydrates by Exoglycosidase Digestion and Laser-Desorption Mass Spectrometry

Cited by 165 publications

References 0 publications

Chinese Hamster Ovary (CHO) Cells May Express Six β4-Galactosyltransferases (β4GalTs)

Chinese Hamster Ovary (CHO) Cells May Express Six β4-Galactosyltransferases (β4GalTs)

Fragmentation of negative ions from carbohydrates: Part 3. Fragmentation of hybrid and complex N-linked glycans

Fragmentation of negative ions from carbohydrates: Part 1. Use of nitrate and other anionic adducts for the production of negative ion electrospray spectra from N-linked carbohydrates

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