CD BioGlyco has established a powerful one-step analysis platform to meet clients' requirements. Nowadays numerous methods have been developed for the study of HMOs. We have the ability to use multiple assays alone or in combination, we aim to be the best choice for HMO analysis.

Introduction

Numerous methods have been developed for the study of human milk oligosaccharides (HMOs). Advances in mass spectrometry (MS) made it possible to quickly determine an exact mass and, as a result, the makeup of HMOs. The generation of gas-phase ions is the most crucial stage in any mass spectrometer study. Electrospray ionization (ESI), electron ionization (EI), and matrix-assisted laser desorption/ionization are the top three techniques for producing gas-phase ions (MALDI). Additionally, the combo of mass spectrometry and separation methods yields the best analytical instrument for detecting complicated HMO combinations. And without the need for chromatography, fourier transform ion cyclotron resonance mass spectrometry (FTICR/MS) can commonly identify complicated metabolite combinations.

Fig.1 Diagram of different ionization systems. (Wikipedia)

Analysis Methods

MS/MS

Low molecular weight HMO structural assignment is generally straightforward. For the differentiation of low molecular weight HMOs by MS² analysis, some studies have described isomer-specific collision-induced dissociation (CID) fragments; however, MS² fragmentation alone cannot provide enough unambiguous information to completely determine entire glycan structures, especially for HMOs of high molecular weight.

Fig.2 Schematic of tandem mass spectrometry. (Wikipedia)

LC/MS

The structural identification of HMOs is made simpler by using MS in conjunction with chromatographic or electromigration separation techniques. Picomolar concentrations can be used for MS-based fragmentation procedures in situations with low levels of HMO, and with the right LC coupling, such investigations may even be carried out in mixes.

Fig.3 Overview of a typical LC/MS metabolomic workflow. (Bowen & Northen, 2010)

CE/MS

Modern techniques include CE and CE-MS coupling. The excellent sensitivity and resolving power of CE are well recognized in the investigation of oligosaccharide combinations. One of the most advantageous and often employed routine techniques for the separation of carbohydrates by CE is LIF analysis. To define HMOs in the feces of breastfeeding and breastfed babies, ESI-MS was combined with CE and an online LIF detector.

Fig.4 A capillary electrophoresis–mass spectrometry. (Wikipedia)

MALDI-FTICR-MS

More recently, a method for tracking bacterial consumption of HMOs utilizing matrix-assisted laser desorption ionization-Fourier transform ion cyclotron resonance-mass spectrometry (MALDI-FTICR-MS) was devised. The MALDI matrix used for the observation of neutral oligosaccharides was 2,5-dihydroxybenzoic acid (DHB). Relative quantitation was demonstrated to benefit from the use of deuterium-labeled internal standards, which was made possible by the high resolution of the fourier transform ion cyclotron resonance-mass spectrometry (FTICR-MS).

Fig.5 A working principle of MALDI-FTICR-MS. (Wang, et al., 2011)

MALDI-TOF-MS

Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) analysis of HMOs was first described by Stahl and colleagues who were able to examine neutral oligosaccharides in positive mode as monosodium adducts as well as acidic oligosaccharides in both the positive and negative modes.

Fig.6 Principle of MALDI-TOF-MS-based identification of proteins and peptides. (Schubert & Wieser, 2011)

HILIC-ESI-MS

An approach to discover unknown reduced (or nonreduced) HMOs as well as, possibly, permethylated HMOs based on spectrum matching was proposed in recent work from a NIST group that established a searchable, reference MS library of annotated oligosaccharides. This library, which is an attractive alternative for future investigation, presently has 469 positive and negative ion spectra that were obtained using Hydrophilic interaction liquid chromatography-electrospray ionization-mass spectrometry (HILIC-ESI-MS) analysis.

Fig.7 Analysis of polar organic water contaminants with HILIC-ESI-MS. (Müller, et al., 2020)

nLC-PGC-TOF-MS

Glycan structures produced from glycoproteins may be separated with great resolution using porous graphitized carbon (PGC) based chromatography. For the purpose of identifying and measuring two significant free disaccharides, LNB and LacNAc, from human milk, a PGC phase-based LC-MS/MS approach was created. The device uses porous graphitized carbon in a column and microchip-based nano LC technology (PGC). The detector is an orthogonal time-of-flight (TOF) mass spectrometer, and the nano-LC is connected to it via nanoelectrospray ionization (nano-ESI). Additionally, MALDI-FTICR-MS with collision-induced dissociation (CID) and infrared multiphoton dissociation (IRMPD) performs the structural analysis.

Fig.8 Examples of "branched" and "linear" HMO structures. (Wu, et al., 2010)

MS-based HMO Analysis Service at CD BioGlyco

Based on the current development of MS-based analysis methods for HMOs (such as MS, LC/MS, CE/MS, MALDI-FTICR-MS, MALDI-TOF-MS, HILIC-ESI-MS, and so on), CD BioGlyco provides several MS-based analysis services. Not only use single methods but also combinate using of these analysis technologies according to clients' requirements.

Advantages of Us

• We have an advanced lab and research teams for analysis services.
• We have custom analysis services such as MS, LC/MC, CE/MS, HPAEC-PAD, HILIC-ESI-MS, MALDI-FTICR-MS, and so on.
• According to customers' needs, we can provide a combination of multiple analysis method services.
• We also have experienced researchers and well-trained technicians.

CD BioGlyco aims to help customers to study the compounds, structure, and content of HMOs, we are a great choice for HMO analysis. If you are interested in our analysis services, please feel free to contact us for more detailed information. We look forward to inquiries from around the world and our research scientists will do their best to help our clients solve their problems and meet their needs in the field of HMO research.

References:

1. Bowen, B.P.; and Northen, T.R. Dealing with the unknown: Metabolomics and Metabolite Atlases. Journal of the American Society for Mass Spectrometry. 2010, 21(9): 1471–1476.
2. Müller, K.; et al. Matrix effects in the analysis of polar organic water contaminants with HILIC-ESI-MS. Analytical and Bioanalytical Chemistry. 2020, 412(20): 4867–4879.
3. Schubert, S.; and Wieser, A. Einsatz der matrix-assisted laser desorption/ionisation-time of Flight Massenspektrometrie (MALDI-Tof MS) in der Mikrobiologischen routinediagnostik/matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) in clinical microbiological routine diagnostics. LaboratoriumsMedizin. 2011, 35(4): 195–203.
4. Wang, H.Y.; et al. Analysis of low molecular weight compounds by MALDI-FTICR-MS. Journal of Chromatography B. 2011, 879(17-18): 1166–1179.
5. Wu, S.; et al. Development of an annotated library of neutral human milk oligosaccharides. Journal of Proteome Research. 2010, 9(8): 4138–4151.

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