Electrospray Ionization-Ion Trap Mass Spectrometry for Structural Analysis of Complex N-Linked Glycoprotein Oligosaccharides

Weiskopf, Andrew; Vouros, Paul; Harvey, David J.

doi:10.1021/ac980289r

Cited by 147 publications

(124 citation statements)

References 21 publications

(34 reference statements)

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“…In these examples, we will use many of the concepts and tools previously described. 7,8,15,[26][27][28] All structures were derived de novo with input data from a single instrument.…”

Section: Resultsmentioning

confidence: 99%

“…6 In both cases, however, the product structures remain to be characterized. A large body of work has shown that spectral comparisons of isomers at different stages of sequential molecular fragmentation (MS n ) [7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24] are clearly different, although in one case, reproducibility has been reported to be "vague". 25 In our experience, spectral reproducibility has been excellent, 26 and this very feature has provided an opportunity to build a searchable fragment library 27 and derive glycan structure with supporting tools.…”

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confidence: 99%

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Carbohydrate Structural Isomers Analyzed by Sequential Mass Spectrometry

et al. 2007

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Consistent with the goals of a comprehensive carbohydrate sequencing strategy, we extend earlier reports to include the characterization of structural (constitutional) isomers. Protocols were developed around ion trap instrumentation providing sequential mass spectrometry (MS n ) and supported with automation and related computational tools. These strategies have been built on the principle that for a single structure all product spectra upon sequential fragmentation are reproducible and each stage represents a rational spectrum of its precursor; i.e., all major fragments should be accounted for. Anomalous ions at any stage are clues indicating the presence of structural isomers. Gas-phase isolation and subsequent fragmentation of such ions provide an opportunity to specifically resolve selected structures for their detailed characterization. Importantly, some isomers were not detected following MS 2 and required multiple (MS n>2 ) stages for their characterization. Derivatization remains critical to position substructures in a glycan array since product ions carry fragmentation "scars" throughout the MS n tree. Equally as important are the pathway relationships between each stage and the greater yield of fragments with the smaller number of oscillators. Applications were directed to the structural isomers in ovalbumin and IgG, where, in the latter case, several previously unreported glycans were detected. Procedures were supported with bioinformatics tools for assimilating structure from the MS n data sets.A comprehensive carbohydrate sequence is particularly challenging when considering the constitutional and stereoisomers that are abundant in glycan arrays. Constitutional isomers, also referred to as structural isomers, are defined as an identical atomic composition arranged in a different structure. Stereoisomers are exemplified with those frequently encountered in the hexoses, mannose, galactose, and glucose, while the two G1 glycans found in IgG are representative of simple structural isomers. In this latter case, an identical monomer may be linked on either of two antennae providing a different topology. In another nomenclature clarification, the term isomer is often confused with isobar. which relates to a different composition of atoms occurring at a nominal (unit) mass resolution. 1 At the MS profile stage, isobars are not usually a significant problem in carbohydrate structural characterization, but structural isomers and stereoisomers are. During fragmentation, however, isobaric fragments may be formed, but identification will require higher mass resolving power and mass accuracy.

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“…In these examples, we will use many of the concepts and tools previously described. 7,8,15,[26][27][28] All structures were derived de novo with input data from a single instrument.…”

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

Carbohydrate Structural Isomers Analyzed by Sequential Mass Spectrometry

et al. 2007

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“…TOF/TOF or Q-TOF mass spectrometers have been widely used in tandem MS characterization of oligosaccharides. In contrast to such tandem-in-space types of instruments, where tandem MS is limited to a MS 2 stage (not including in-source fragmentation), ion storage devices, such as ion trap mass spectrometers, allow multi-stage activations of the parent oligosaccharide ions into successive fragment ions [13,[47][48][49]. Sequential fragmentation, analogous to successive enzyme treatment in the classic sequencing technique, is thus possible with ion trap instruments.…”

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confidence: 99%

Gas-phase oligosaccharide nonreducing end (GONE) sequencing and structural analysis by reversed phase hplc/mass spectrometry with polarity switching

Chen

Flynn

2009

J. Am. Soc. Mass Spectrom.

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Here we describe a technique to obtain all the N-linked oligosaccharide structures from a single reversed-phase (RP) HPLC run using on-line tandem MS in both positive and negative ion modes with polarity switching. Oligosaccharides labeled with 2-aminobenzamide (2AB) were used because they generated good ionization efficiency in both ion polarities. In the positive ion mode, protonated oligosaccharide ions lose sugar residues sequentially from the nonreducing end with each round of MS fragmentation, revealing the oligosaccharide sequence from greatly simplified tandem MS spectra. In the negative ion mode, diagnostic ions, including those from cross-ring cleavages, are readily observed in the MS 2 spectra of deprotonated oligosaccharide ions, providing detailed structural information, such as branch composition and linkage positions. Both positive and negative ion modes can be programmed into the same LC/MS experiment through polarity switching of the MS instrument. The gas-phase oligosaccharide nonreducing end (GONE) sequencing data, in combination with the diagnostic ions generated in negative ion tandem MS, allow both sequence and structural information to be obtained for all eluting species during a single RP-HPLC chromatographic run. This technique generates oligosaccharide analyses at high speed and sensitivity, and reveals structural features that can be difficult to obtain by traditional methods. . Proper structural determination is thus critical to the understanding of the biological role of carbohydrates. Although there are fewer monosaccharide building blocks in N-linked glycans than there are amino acid moieties in proteins, the combination of multiple branchings and linkages add greater complexity to the glycan structures. Such structural diversity also adds to the difficulty of structural characterization.Many analytical tools are now available for oligosaccharide characterization, such as high field NMR, liquid chromatography, capillary electrophoresis, and mass spectrometry. Although unambiguous oligosaccharide structural assignments can be made by NMR [2][3][4][5], this technique often requires large quantities of highly purified material. Typically, more than 50 mg of glycoprotein and extensive preparative fractionation are required to resolve oligosaccharide structures of relatively low abundance. A more common characterization approach utilizes sets of specific exoglycosidases, which cleave terminal monosaccharides from the nonreducing end, followed by chromatographic or MS analysis [6][7][8]. In this approach, the sequence of the oligosaccharide chain is revealed, either through sequential removal of monosaccharides from the chain upon successive enzyme treatment, or from the oligosaccharide ladder generated upon treatment with an array of enzymes. Linkage positional information and anomericity of the terminal monosaccharide can also be obtained, based on the specificity of the exoglycosidases used. Unfortunately, this classic sequencing technique is often labor intensive, time consuming, and...

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“…Due to the poor basicity of glycans, formation of sufficient MH ϩ ion abundance in positive mode ESI is difficult to achieve [13][14][15][16]. Negative mode ESI offers ionization advantages for the analysis of glycans, however, positive mode ESI is also desirable due to its capability to generate different MS/MS glycan ion fragmentation than typically observed in negative mode ESI-MS/MS [17,18]. To improve positive mode ESI ionization of glycans, derivatization of glycans with molecules containing basic sites has been employed [19].…”

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confidence: 99%

Using a nanoelectrospray-differential mobility spectrometer-mass spectrometer system for the analysis of oligosaccharides with solvent selected control over ESI aggregate ion formation

Levin

Vouros

Miller

et al. 2007

J. Am. Soc. Mass Spectrom.

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Differential mobility spectrometry (DMS), also commonly referred to as high field asymmetric waveform ion mobility spectrometry (FAIMS) is a rapidly advancing technology for gas-phase ion separation. The interfacing of DMS with mass spectrometry (MS) offers potential advantages over the use of mass spectrometry alone. Such advantages include improvements to mass spectral signal/noise, orthogonal/complementary ion separation to mass spectrometry, enhanced ion and complexation structural analysis, and the potential for rapid analyte quantitation. In this report, we demonstrate the successful use of our nanoESI-DMS-MS system, with a methanol drift gas modifier, for the separation of oligosaccharides. The tendency for ESI to form oligosaccharide aggregate ions and the negative impact this has on nanoESI-DMS-MS oligosaccharide analysis is described. In addition, we demonstrate the importance of sample solvent selection for controlling nanoESI oligosaccharide aggregate ion formation and its effect on glycan ionization and DMS separation. The successful use of a tetrachloroethane/methanol solvent solution to reduce ESI oligosaccharide aggregate ion formation while efficiently forming a dominant MH ϩ molecular ion is presented. By reducing aggregate ion formation in favor of a dominant MH ϩ ion, DMS selectivity and specificity is improved. In addition to DMS, we would expect the reduction in aggregate ion complexity to be beneficial to the analysis of oligosaccharides for other post-ESI separation techniques such as mass spectrometry and ion mobility. The solvent selected control over MH ϩ molecular ion formation, offered by the use of the tetrachloroethane/methanol solvent, also holds promise for enhancing MS/MS structural characterization analysis of glycans. , also referred to as high field asymmetric waveform ion mobility spectrometry (FAIMS) [2], and field ion spectrometry (FIS) [3], is a rapidly advancing technology for gas-phase ion separation. DMS has the potential to emerge as a major stand-alone separation science technique such as LC or GC. Many researchers have focused on interfacing DMS to mass spectrometry because of its atmospheric pressure, gas-phase, continuous ion separation capabilities, and the detection specificity offered by mass spectrometry. In this study, we investigate the use of a specially designed nanoESI-DMS-MS system for the analysis of oligosaccharides. Glycosylation of proteins has been demonstrated to play a significant role in their biological function [4 -7]. Characterization of glycoprotein glycan groups is essential to understanding how they influence protein function [8 -10]. ESI-MS has become a popular analysis platform for characterizing oligosaccharides because of its compatibility with up-stream separation techniques such as liquid chromatography and capillary electrophoresis, as well as the structure-rich information provided by MS n techniques [8 -10]. In this study, we demonstrate the successful use of nanoESI-DMS-MS with a methanol drift gas modifier for the separation of o...

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Electrospray Ionization-Ion Trap Mass Spectrometry for Structural Analysis of Complex N-Linked Glycoprotein Oligosaccharides

Cited by 147 publications

References 21 publications

Carbohydrate Structural Isomers Analyzed by Sequential Mass Spectrometry

Carbohydrate Structural Isomers Analyzed by Sequential Mass Spectrometry

Gas-phase oligosaccharide nonreducing end (GONE) sequencing and structural analysis by reversed phase hplc/mass spectrometry with polarity switching

Using a nanoelectrospray-differential mobility spectrometer-mass spectrometer system for the analysis of oligosaccharides with solvent selected control over ESI aggregate ion formation

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