Site-specific carbohydrate identification in recombinant proteins using MALD-TOF MS

Huberty, Michael C.; Vath, James E.; Yao, Wen; Martin, Stephen A.

doi:10.1021/ac00068a015

Cited by 165 publications

(98 citation statements)

References 8 publications

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“…Recently, high-energy CID fragmentation, including X-type cross-ring cleavage ions, has been observed for a variety of nonacidic oligosaccharides using matrix-assisted laser desorption/ionization (MALDI) tandem time-of-flight analyzers [4,5]. However, since fragile oligosaccharides, particularly polysialylated [6], and polysulfated molecules [7,8] have been observed to undergo prompt fragmentation in the vacuum MALDI source and metastable decomposition in the analyzer, they are more amenable to electrospray ionization. Negatively charged neutral oligosaccharides undergo fragmentation at very low collision energies [9 -11], and such ions are also more amenable to electrospray ionization than MALDI.…”

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confidence: 99%

The influence of sialylation on glycan negative ion dissociation and energetics

Seymour

Costello

Zaia

2006

J. Am. Soc. Mass Spectrom.

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For the analysis of native glycans using tandem mass spectrometry (MS), it is desirable to choose conditions whereby abundances of cross-ring cleavages indicative of branch positions are maximized. Recently, negative ion tandem mass spectrometry has been shown to produce significantly higher abundances of such ions in glycans compared to the positive ion mode. Much of this prior work has concerned fragmentation patterns in asialo glycans. The present work compares the abundances of critical cross-ring cleavage ions using negative mode tandem mass spectrometry for milk oligosaccharides and N-linked glycans. For comparison, product ion formation was studied for deprotonated and nitrated ions formed from asialo glycans and deprotonated ions from sialylated glycans. Breakdown profiles demonstrate clearly that more energy was required to fragment sialylated compounds to the same extent as either their asialo or nitrate adducted counterparts. The extraction of a proton from a ring hydroxyl group during the ionization process may be viewed, qualitatively, as imparting significantly more energy to the ion than would that from a molecule bearing an acidic group, so that acidic glycans are more stable in the gas phase, as the negative charge resides on the carboxyl group. These results have strong practical implications because a major portion of glycans released from mammalian proteins will be sialylated. T he most information-rich tandem mass spectra of oligosaccharides are those in which a combination of abundant glycosidic bond and cross-ring cleavages is observed. Cross-ring cleavages at branching residues and those where linkages are known to vary in the given oligosaccharide or glycoconjugate class provide particularly valuable information. Although abundant A-type and X-type cross-ring cleavages are observed using high-energy collisionalinduced dissociation (CID) [1,2], most modern mass analyzers operate in the low-energy regime. Thus, positive ion CID MS using triple quadrupole, ion trap, quadrupole orthogonal time-of-flight, and FT-ICR analyzers results in low-energy fragmentation in which Band Y-type ions and only those cross-ring cleavage ions that result from particularly facile processes are abundant [3]. Recently, high-energy CID fragmentation, including X-type cross-ring cleavage ions, has been observed for a variety of nonacidic oligosaccharides using matrix-assisted laser desorption/ionization (MALDI) tandem time-of-flight analyzers [4,5]. However, since fragile oligosaccharides, particularly polysialylated [6], and polysulfated molecules [7,8] have been observed to undergo prompt fragmentation in the vacuum MALDI source and metastable decomposition in the analyzer, they are more amenable to electrospray ionization. Negatively charged neutral oligosaccharides undergo fragmentation at very low collision energies [9 -11], and such ions are also more amenable to electrospray ionization than MALDI. Now, there remains no commercial instrument option for producing high-energy CID for fragile molecules. Introdu...

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confidence: 99%

The influence of sialylation on glycan negative ion dissociation and energetics

Seymour

Costello

Zaia

2006

J. Am. Soc. Mass Spectrom.

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“…M atrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) developed by Karas and Hillenkamp [1][2][3] has become a versatile and important tool for the determination of molecular masses of various fragile and nonvolatile samples such as proteins and carbohydrates [2,4,5]. Some success in the use of MALDI-TOF MS for the characterization of many low molecular weight compounds has been reported [6 -11].…”

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confidence: 99%

Immobilized carbon nanotubes as matrix for MALDI-TOF-MS analysis: Applications to neutral small carbohydrates

Ren

Zhang

Cheng

et al. 2005

J. Am. Soc. Mass Spectrom.

161

132

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In this work, we reported on the advantages of immobilized carbon nanotubes as a novel MALDI-matrix. Recently, carbon nanotubes have been reported to be an effective MALDI matrix for small molecules (Anal. Chem. 2003, 75, 6191), as it can eliminate the interfering matrix peaks as well as form a web morphology to fully disperse the analyte and allow strong ultraviolet absorption for enhanced pulsed laser desorption and ionization. In our study, to overcome the problem that the carbon nanotube matrix may fly off from the target, a type of polyurethane adhesive, NIPPOLAN-DC-205, is introduced to immobilize carbon nanotubes on the target, which enables widespread application of carbon nanotubes as matrix for MALDI-MS analysis. At the same time, the properties of the carbon nanotubes as an efficient matrix remained after immobilization. The presence of NIPPO-LAN-DC-205 increases the time for analysis at a particular desorption spot by minimizing the time-consuming search for "hot spots" and facilitating experiments such as post source decay (PSD) which need longer-lasting signals. Moreover, NIPPOLAN-DC-205 produces no interference peaks and can easily be cleaned with acetone. Fast evaporation technology may be used to enhance signal reproducibility in MALDI analysis using carbon nanotubes as matrix. Consequently, the applicability of the carbon nanotube as matrix for matrixassisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis of low molecular mass analytes is highly improved. The feasibility of the method employing polyurethane is demonstrated by comparison of the results produced from the carbon nanotube matrix with and without immobilization. In addition, neutral small carbohydrates, which are difficult to be ionized normally, can be cationized with high efficiency by MALDI-TOF-MS using the immobilized carbon nanotube matrix. The method was further applied to analyze peptides and detect urine glucose successfully. (J Am Soc Mass Spectrom 2005, 16, 333-339)

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“…In addition, we have observed that in insect cells Am25 appears to be glycosylated cotranslationally, whereas Am97 may be glycosylated later on, probably after dimerization of the monomers has taken place Analysis of the glycans of IFN-y at different sites was performed by means of MALDI-MS combined with digestion with specific proteases and glycosidases. This is a newly developed technique for structural analysis of glycans and in the past few years has been used for site-specific analysis of protein glycosylation [27,[42][43][44]. The advantages of the technique are fast analysis, high sensitivity, and the ability to directly detect the many components caused by glycan heterogeneity.…”

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confidence: 99%

Biosynthesis and N‐glycosylation of Human Interferon‐γ

Sareneva

Mørtz²,

Tölö

et al. 1996

European Journal of Biochemistry

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Interferon-y (IFN-y) is a secretory glycoprotein produced by T cells in response to antigenic or mitogenic stimuli. We studied the kinetics of the synthesis, N-glycosylation, and secretion of IFN-y in human CD8' T lymphocytes stimulated via T-cell receptor. Highly elevated IFN-y mRNA levels were found as early as 1 h after stimulation. Maximal IFN-y protein synthesis was observed 2-4 h after induction and appeared to correlate to steady-state IFN-y mRNA levels. As analyzed by pulse/chase experiments, the secretion of IFN-y from T cells was very rapid, the secretion half-time being approximately 20-25 min. Inhibition of N-glycosylation by tunicamycin dramatically reduced the expression of IFN-y, but did not block its secretion. Natural IFN-y is heterogeneously glycosylated and doubly, singly, and unglycosylated forms exist. Experiments performed in a cell-free translation/glycosylation system with mutated IFN-y constructs lacking either one of the potential glycosylation sites suggested that Am25 is more efficiently glycosylated than Asn97. Site-specific oligosaccharide analysis of natural IFN-y by glycosidase treatment followed by matrix-assisted-laser-desorption-ionization mass spectrometry revealed considerable variation in the carbohydrate structures, with more than 30 different forms. The glycans at Am25 consisted of fucosylated, mainly complex-type oligosaccharides, whereas the glycans at Asn97 were more heterogeneous, with hybrid and high-mannose structures. Our results emphasize the essential role of N-linked glycans in the biology of IFN-y and show that there is a considerable heterogeneity in the individual sugar chains of this important human cytokine.

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Site-specific carbohydrate identification in recombinant proteins using MALD-TOF MS

Cited by 165 publications

References 8 publications

The influence of sialylation on glycan negative ion dissociation and energetics

The influence of sialylation on glycan negative ion dissociation and energetics

Immobilized carbon nanotubes as matrix for MALDI-TOF-MS analysis: Applications to neutral small carbohydrates

Biosynthesis and N‐glycosylation of Human Interferon‐γ

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