Advances in fluorescence derivatization methods for high-performance liquid chromatographic analysis of glycoprotein carbohydrates

Anumula, Kalyan R.

doi:10.1016/j.ab.2005.09.037

Cited by 243 publications

(169 citation statements)

References 119 publications

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“…Another critical point is the derivatization procedure with 2-AB. This label was preferred over the more sensitive 2-aminobenzoic acid [34] as it does not introduce an additional charge (i.e., polarity) to all glycans that complicates charge analysis by WAX and is more difficult to ionise in negative ion mode MS. The introduction of the fluorophore at the reducing-end sugar is carried out in a slightly acidic environment.…”

Section: Discussionmentioning

confidence: 99%

Evaluation of protein N‐glycosylation in 2‐DE: Erythropoietin as a study case

Llop¹,

Gallego²,

Belalcazar³

et al. 2007

Proteomics

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The structure, function, and physico-chemical properties of many proteins are determined by PTM, being glycosylation the most complex. This study describes how a combination of typical proteomics methods (2-DE) combines with glycomics strategies (HPLC, MALDI-TOF-MS, exoglycosidases sequencing) to yield comprehensive data about single spot-microheterogeneity, providing meaningful information for the detection of disease markers, pharmaceutical industry, antidoping control, etc. Recombinant erythropoietin and its hyperglycosylated analogue darbepoetin-a were chosen as showcases because of their relevance in these fields and the analytical challenge they represent. The combined approach yielded good results in terms of sample complexity (mixture glycoforms), reproducibility, sensitivity (,25 pmoles of glycoprotein/spot), and identification of the underlying protein. Heterogeneity was present in all spots but with a clear tendency; spots proximal to the anode contained the highest amount of tetra-antennary tetrasialylated glycans, whereas the opposite occurred for spots proximal to the cathode with the majority of the structures being undersialylated. Spot microheterogeneity proved a consequence of the multiple glycosylation sites as they contributed directly to the number of possibilities to account for a discrete charge in a single spot. The interest of this combined glycoproteomics method resides in the efficiency for detecting and quantifying subtle dissimilarities originated from altered ratios of identical glycans including N-acetyl-lactosamine repeats, acetylation, or antigenic epitopes, that do not significantly contribute to the electrophoretic mobility, but affect the glycan microheterogeneity and the potential underlying related functionality.

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Section: Discussionmentioning

confidence: 99%

Evaluation of protein N‐glycosylation in 2‐DE: Erythropoietin as a study case

Llop¹,

Gallego²,

Belalcazar³

et al. 2007

Proteomics

View full text Add to dashboard Cite

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“…The reaction speed and yield of this acidcatalyzed labeling reaction is greatly influenced by the amount and type of acid used. Organic acids with low pK a , such as acetic acid (pK a = 4.75), malonic acid (pK a1 = 2.83) or citric acid (pK a1 = 3.15), are more commonly used to accelerate glycan labeling [27,28]. Studies have shown that stronger acids were able to increase derivatization yield [29], but their use was associated with higher sialic acid loss [30].…”

Section: Introductionmentioning

confidence: 99%

Evaporative fluorophore labeling of carbohydrates via reductive amination

Reider

Szigeti

Guttman

2018

Talanta

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As analytical glycomics became to prominence, newer and more efficient sample preparation methods are being developed. Albeit, numerous reductive amination based carbohydrate labeling protocols have been reported in the literature, the preferred way to conduct the reaction is in closed vials. Here we report on a novel evaporative labeling protocol with the great advantage of continuously concentrating the reagents during the tagging reaction, therefore accommodating to reach the optimal reagent concentrations for a wide range of glycan structures in a complex mixture. The optimized conditions of the evaporative labeling process minimized sialylation loss, otherwise representing a major issue in reductive amination based carbohydrate tagging. In addition, complete and uniform dispersion of dry samples was obtained by supplementing the low volume labeling mixtures (several microliters) with the addition of extra solvent (e.g., THF).Evaporative labeling is an automation-friendly glycan labeling method, suitable for standard open 96 well plate format operation. Graphical abstract 2/13

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“…In this method, N-linked glycans are labeled with 2AB [50]. Fluorescence derivatization with 2AB is a common approach in carbohydrate analysis [51], including structural characterization by MS (see, for example, Reference [52]). Glycans labeled with 2AB demonstrate good on-line MS signal in both positive and negative ion modes.…”

mentioning

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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Advances in fluorescence derivatization methods for high-performance liquid chromatographic analysis of glycoprotein carbohydrates

Cited by 243 publications

References 119 publications

Evaluation of protein N‐glycosylation in 2‐DE: Erythropoietin as a study case

Evaluation of protein N‐glycosylation in 2‐DE: Erythropoietin as a study case

Evaporative fluorophore labeling of carbohydrates via reductive amination

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

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