Comparison of Different Labeling Techniques for the LC-MS Profiling of Human Milk Oligosaccharides

Structural complexity of glycans derived from the diversities in composition, linage, configuration, and branching considerably complicates structural analysis. Nanopore-based single-molecule sensing offers the potential to elucidate glycan structure and even sequence glycan. However, the small molecular size and low charge density of glycans have restricted direct nanopore detection of glycan. Here we show that glycan sensing can be achieved using a wild-type aerolysin nanopore by introducing a facile glycan derivatization strategy. The glycan molecule can induce impressive current blockages when moving through the nanopore after being connected with an aromatic group-containing tag (plus a carrier group for the neutral glycan). The obtained nanopore data permit the identification of glycan regio- and stereoisomers, glycans with variable monosaccharide numbers, and distinct branched glycans, either independently or with the use of machine learning methods. The presented nanopore sensing strategy for glycans paves the way towards nanopore glycan profiling and potentially sequencing.

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“…Glycan derivatization was performed with a typical reductive amination reaction 48 . Unless otherwise stated, 1 equiv.…”

Section: Methodsmentioning

confidence: 99%

Identification of tagged glycans with a protein nanopore

Xiong

Cao

et al. 2023

Nat Commun

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“…As mass spectrometric analysis of glycosylation is generally done in positive mode MS, there have been a couple of studies inspecting effects of different labels on ESI-MS positive mode ionization ( Harvey, 2000 ; Keser et al, 2018 : Lattová et al, 2005 ; Walker et al, 2010 ). Although some of the studies were also done in negative mode, they were done on only a few labels and without a focus on their hydrophobicity ( Lattová et al, 2005 ; Lang et al, 2021 ). Our selection of 11 labels included a range of hydrophobicity of the tags (logP 0.51—2.23) and different ionization groups.…”

Section: Discussionmentioning

confidence: 99%

In a pursuit of optimal glycan fluorescent label for negative MS mode for high-throughput N-glycan analysis

et al. 2022

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Over the past few decades, essential role of glycosylation in protein functioning has become widely recognized, rapidly advancing glycan analysis techniques. Because free glycan’s lack chromophore or fluorophore properties, and do not ionize well, they are often derivatized to facilitate their separation or detection, and to enhance the sensitivity of the analysis. Released glycan’s are usually derivatized using a fluorescent tag, which enables their optical detection in LC profiling. Some fluorescent labels can also promote ionization efficiency, thus facilitating MS detection. For this reason, there is a need to design fluorophores that will contribute more to the fluorescence and ionization of glycan’s and the need to quantify these contributions to improve glycan analysis methods. In this paper we focused on negative MS mode as these methods are more informative than methods involving positive MS mode, allowing for a less ambiguous elucidation of detailed glycan structures. Additionally, traditional glycan labels in negative mode MS usually result with diminished sensitivity compared to positive mode, thus making selection of appropriate label even more important for successful high-throughput analysis. Therefore, eleven fluorescent labels of different chemo-physical properties were chosen to study the influence of label hydrophobicity and presence of a negative charge on glycan ionization in negative MS mode. N-glycans released from IgG sample were labeled with one of the eleven labels, purified with HILIC-SPE and analyzed with HILIC-UPLC-FLR-MS. To make evaluation of studied labels performance more objective, analysis was performed in two laboratories and at two mobile phase pH (4.4 and 7.4). Although there was a notable trend of more hydrophobic labels having bigger signal intensities in one laboratory, we observed no such trend in the other laboratory. The results show that MS parameters and intrinsic configuration of the spectrometer have even bigger effect on the final ESI response of the labeled-glycan ionization in negative MS mode that the labels themselves. With this in mind, further research and development of fluorophores that will be suitable for high-throughput glycan analysis in the negative MS mode are proposed.

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“…47 Alternatively, PGC-LC−MS of reductively aminated glycans was recognized as highly useful. 48 In this work, we demonstrate that de-N-acetylation of free, reduced glycans can be performed under moderate conditions using the readily available, cheap, and safe-to-handle hydrazine hydrate. The introduction of stable isotope-labeled acetyl groups can subsequently be achieved using isotope-labeled acetic anhydride.…”

Section: ■ Introductionmentioning

confidence: 87%

“…Backup of MS/MS-based identification by retention times may not yet be standard, but given the enormous number of HMOs and their isomers, it probably should be both for analysis of underivatized glycans on PGC , or permethylated glycans on reversed phase , or PGC . Alternatively, PGC-LC–MS of reductively aminated glycans was recognized as highly useful …”

Section: Introductionmentioning

confidence: 99%

Simple Routes to Stable Isotope-Coded Native Glycans

Helm,

Grünwald-Gruber,

Urteil

et al. 2023

Anal. Chem.

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Understanding the biological role of protein-linked glycans requires the reliable identification of glycans. Isomer separation and characterization often entail mass spectrometric detection preceded by high-performance chromatography on porous graphitic carbon. To this end, stable isotope-labeled glycans have emerged as powerful tools for retention time normalization. Hitherto, such standards were obtained by chemoenzymatic or purely enzymatic methods, which introduce, e.g., 13 C-containing Nacetyl groups or galactose into native glycans. Glycan release with anhydrous hydrazine opens another route for heavy isotope introduction via concomitant de-N-acetylation. Here, we describe that de-N-acetylation can also be achieved with hydrazine hydrate, which is a more affordable and less hazardous reagent. Despite the slower reaction rate, complete conversion is achievable in 72 h at 100 °C for glycans with biantennary glycans with or without sialic acids. Shorter incubation times allow for the isolation of intermediate products with a defined degree of free amino groups, facilitating introduction of different numbers of heavy isotopes. Mass encoded glycans obtained by this versatile approach can serve a broad range of applications, e.g., as internal standards for isomer-specific studies of N-glycans, O-glycans, and human milk oligosaccharide by LC−MS on either porous graphitic carbon or� following permethylation�on reversed phase.

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Comparison of Different Labeling Techniques for the LC-MS Profiling of Human Milk Oligosaccharides

Cited by 6 publications

References 103 publications

Identification of tagged glycans with a protein nanopore

Identification of tagged glycans with a protein nanopore

In a pursuit of optimal glycan fluorescent label for negative MS mode for high-throughput N-glycan analysis

Simple Routes to Stable Isotope-Coded Native Glycans

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