Isomeric separation of permethylated glycans by extra-long reversed-phase liquid chromatography (RPLC)-MS/MS

Wang, Junyao; Dong, Xue; Yu, Aiying; Huang, Yifan; Peng, Wenjing; Mechref, Yehia

doi:10.1039/d2an00010e

Cited by 13 publications

(17 citation statements)

References 68 publications

(95 reference statements)

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“…Fragmentation differences as well as the advantages of using a quadrupole-orbitrap instrument for permethylated Nglycan analysis, were highlighted. While most of the fragment ions shown in this work are b-and y-ions, permethylated Nglycans can undergo multiple cross-ring fragmentations with metal adducts such as lithium, as shown by Kurz et al 55 For our future study, we believe that PRM-based analysis in conjunction with the permethylated glycan isomeric separation technique, such as a mesoporous graphitized carbon column 53 or extra-long C18 column 56 may produce more detailed characterization of glycan isomers without the use of…”

Section: ■ Conclusionmentioning

confidence: 67%

Targeted N-Glycan Analysis with Parallel Reaction Monitoring Using a Quadrupole-Orbitrap Hybrid Mass Spectrometer

et al. 2022

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Targeted mass spectrometric analysis is widely employed across various omics fields. The approach has been successfully employed for the structural analysis of proteins, glycans, lipids, and small molecules. Selected reaction monitoring and multiple reaction monitoring (MRM) have been a method of choice for targeted structural studies of biomolecules. However, innovations in instrument designs have led to the development of parallel reaction monitoring (PRM). PRM detects all product ions simultaneously rather than optimizing/preselecting the target glycan transitions, simplifying the analytical workflow. By reducing background interference, increasing selectivity/specificity, and improving data quality, PRM allows reliable quantification of target glycans in complex matrices. PRM can also improve sensitivity for detecting low-abundance target glycans and reduce low-level limit of quantification values with an improved S/N ratio. PRM’s advantages are attributed to the development of sensitive and highly selective mass analyzers, orbitrap, and time of flight. In this study, we developed a sensitive PRM method for the quantitative analysis of permethylated N-glycans, an important class of disease biomarkers, using a quadrupole-orbitrap hybrid mass spectrometer. Pooled human cerebrospinal fluid was used for the study as a source of permethylated N-glycans. The method illustrates the fragmentation of N-glycans at different collision energies as well as the optimization of collision energy. The method also detects low-abundance N-glycans more efficiently than MRM. This study is the first attempt to develop a sensitive PRM-based method to analyze permethylated N-glycans.

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Section: ■ Conclusionmentioning

confidence: 67%

Targeted N-Glycan Analysis with Parallel Reaction Monitoring Using a Quadrupole-Orbitrap Hybrid Mass Spectrometer

et al. 2022

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“…Two previously developed in-house strategies capable of separating an important number of isomeric N -glycans were used to complete the isomeric N -glycan profile of low-abundant serum glycoproteins in MCI samples. The MGC-LC-MS that uses a home-made 10 mm mesoporous graphitized carbon (MGC) column [ 30 ] and the C18(50 cm)-LC-MS that takes advantage of the path length to increase the interactions between analytes and the stationary phase were both used and resulted in isomeric separation [ 31 ].…”

Section: Methodsmentioning

confidence: 99%

“…Later, it was increased to 9% in 3 min and kept constant for 17 min. Finally, it was decreased to 20% in 3 min and maintained to equilibrate the column [ 31 ].…”

Section: Methodsmentioning

confidence: 99%

“…The comprehensive characterization of Isomeric glycans in human serum requires the application of a strategy that combines a sensitive mass spectrometry [ 29 ] detector with a powerful separation technique. To meet this challenge, our research group recently introduced an in-house developed mesoporous graphitized carbon (MGC) column [ 30 ] and a C18 column with a length of 500 mm [ 31 ], which can achieve separation of isomeric glycans. With recent advances in mass spectrometry [ 29 ] and separation methods, LC-MS/MS has thus been proven to be a reliable and sensitive analytical tool for various omics studies, including glycomics [ 13 , 32 , 33 , 34 ].…”

Section: Introductionmentioning

confidence: 99%

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LC-MS/MS Isomeric Profiling of N-Glycans Derived from Low-Abundant Serum Glycoproteins in Mild Cognitive Impairment Patients

et al. 2022

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Mild cognitive impairment (MCI) is an early stage of memory loss that affects cognitive abilities, such as language or virtual/spatial comprehension. This cognitive decline is mostly observed with the aging of individuals. Recently, MCI has been considered as a prodromal phase of Alzheimer’s disease (AD), with a 10–15% conversion rate. However, the existing diagnostic methods fail to provide precise and well-timed diagnoses, and the pathophysiology of MCI is not fully understood. Alterations of serum N-glycan expression could represent essential contributors to the overall pathophysiology of neurodegenerative diseases and be used as a potential marker to assess MCI diagnosis using non-invasive procedures. Herein, we undertook an LC-MS/MS glycomics approach to determine and characterize potential N-glycan markers in depleted blood serum samples from MCI patients. For the first time, we profiled the isomeric glycome of the low abundant serum glycoproteins extracted from serum samples of control and MCI patients using an LC-MS/MS analytical strategy. Additionally, the MRM validation of the identified data showed five isomeric N-glycans with the ability to discriminate between healthy and MCI patients: the sialylated N-glycans GlcNAc5,Hex6,Neu5Ac3 and GlcNAc6,Hex7,Neu5Ac4 with single AUCs of 0.92 and 0.87, respectively, and a combined AUC of 0.96; and the sialylated-fucosylated N-glycans GlcNAc4,Hex5,Fuc,Neu5Ac, GlcNAc5,Hex6,Fuc,Neu5Ac2, and GlcNAc6,Hex7,Fuc,Neu5Ac3 with single AUCs of 0.94, 0.67, and 0.88, respectively, and a combined AUC of 0.98. According to the ingenuity pathway analysis (IPA) and in line with recent publications, the identified N-glycans may play an important role in neuroinflammation. It is a process that plays a fundamental role in neuroinflammation, an important process in the progression of neurodegenerative diseases.

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“…1–4 A glycan is a chain-like molecule of glucose, galactose and other monosaccharide molecules formed in a specific sequence. 5–7 They, as bioinformational molecules, not only determine the functions and metabolic pathways of glycoproteins, but are also involved in almost all life processes of cellular biology. 8–10 It has been identified that the aberrant alterations of N -glycans including increased branching, sialylation and fucosylation can induce various diseases and even accelerate cancer progression.…”

Section: Introductionmentioning

confidence: 99%

Facile preparation of a novel chitosan-derived porous graphitized carbon biomaterial for highly efficient capture of N-glycans

Weng

Liu

Zhang

et al. 2022

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Isomeric separation of permethylated glycans by extra-long reversed-phase liquid chromatography (RPLC)-MS/MS

Abstract: Glycosylation is known as a critical biological process that can largely affect the properties and the functions of proteins. Glycan isomers have been shown to be involved in a variety...

Cited by 13 publications

References 68 publications

Targeted N-Glycan Analysis with Parallel Reaction Monitoring Using a Quadrupole-Orbitrap Hybrid Mass Spectrometer

Targeted N-Glycan Analysis with Parallel Reaction Monitoring Using a Quadrupole-Orbitrap Hybrid Mass Spectrometer

LC-MS/MS Isomeric Profiling of N-Glycans Derived from Low-Abundant Serum Glycoproteins in Mild Cognitive Impairment Patients

Facile preparation of a novel chitosan-derived porous graphitized carbon biomaterial for highly efficient capture of N-glycans

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