High‐temperature LC‐MS/MS of permethylated glycans derived from glycoproteins

Zhou, Shiyue; Hu, Yunli; Mechref, Yehia

doi:10.1002/elps.201500568

Cited by 54 publications

(64 citation statements)

References 38 publications

(38 reference statements)

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“…With the help of an established separation technique for permethylated glycans coupled with optimized CID and an HCD MS/MS method, we are able to distinguish between glycan isomers by tandem MS. The described approach enabled the identification of fucose and galactose site isomers as well as galactose linkage isomers and expands upon our previous studies (26, 27, 72) by extending the diversity of isomeric structures characterized by tandem MS to include complex glycans from model glycoproteins as well as human serum.…”

Section: Introductionmentioning

confidence: 67%

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LC-MS/MS analysis of permethylated N-glycans facilitating isomeric characterization

et al. 2016

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The biosynthesis of glycans is a template free process, hence compositionally identical glycans may contain highly heterogeneous structures. Meanwhile, the functions of glycans in biological processes are significantly influenced by the glycan structure. Structural elucidation of glycans is an essential component of glycobiology. Although NMR is considered the most powerful approach for structural glycan studies, it suffers from low sensitivity and requires highly purified glycans. Although mass spectrometry (MS) based methods have been applied in numerous glycan structure studies, there are challenges in preserving glycan structure during ionization. Permethylation is an efficient derivatization method that improves glycan structural stability. In this report, permethylated glycans are isomerically separated, thus facilitating structural analysis of a mixture of glycans by LC-MS/MS. Separation by porous graphitic carbon liquid chromatography at high temperatures in conjunction with tandem mass spectrometry (PGC-LC-MS/MS) was utilized for unequivocal characterization of glycan isomers. Glycan fucosylation sites were confidently determined by eliminating fucose rearrangement and assignment of diagnostic ions achieved by permethylation and PGC-LC at high temperatures, respectively. Assigning monosaccharide residues to specific glycan antennae was also achieved. Galactose linkages were also distinguished from each other by CID/HCD tandem MS. This was attainable because of the different bond energies associated with monosaccharide linkages.

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Section: Introductionmentioning

confidence: 67%

“…In our recent study, we demonstrated that the application of elevated temparatures resulted in the partial isomeric separation of permethylated glycans on C18 (27). Isomeric separation of permethylated glycans using PGC-LC-MS/MS at room temperature was first reported by Costello and co-workers (65).…”

Section: Introductionmentioning

confidence: 98%

LC-MS/MS analysis of permethylated N-glycans facilitating isomeric characterization

et al. 2016

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“…In the case of RPLC separation, this increase in resolution is approximately 3 fold. 33 This is due to the fact that PGC prompts better interactions with closely related structures (e.g., glycan isomers).…”

Section: Resultsmentioning

confidence: 99%

“…6 However, such isomeric separation was not adequate. To improve isomeric separation of permethylated glycans on LC, a more structurally sensitive LC column packing material should be used.…”

Section: Introductionmentioning

confidence: 99%

Isomeric Separation of Permethylated Glycans by Porous Graphitic Carbon (PGC)-LC-MS/MS at High Temperatures

et al. 2017

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Permethylation is a common derivatization method for MS-based glycomic analyses. Permethylation enhances glycan ionization efficiency in positive MS analysis and improves glycan structural stability. Recent biological glycomic studies have added to the growing body of knowledge and suggest the need for complete structural analysis of glycans. However, reverse phase LC analysis of permethylated glycans usually results in poor isomeric separation. To achieve isomeric separation of permethylated glycans, a porous graphitic carbon (PGC) column was used. PGC columns are well known for their isomeric separation capability for hydrophilic analyses. In this study, we have optimized temperature conditions to overcome the issues encountered while separating permethylated glycans on a PGC column and found that the highest temperature examined, 75°C, was optimal. Additionally, we utilized tandem MS to elucidate detailed structural information for the isomers separated. Glycan standards were also utilized to facilitate structural identifications through MS/MS spectra and retention time comparison. The result is an efficient and sensitive method capable of the isomeric separation of permethylated glycans. This method was successfully applied for the isomeric characterization of N-glycans released from the breast cancer cell lines MDA-MB-231 and MDA-MB-231BR (brain seeking). A total of 127 unique glycan structures were identified with 39 isobaric structures, represented as 106 isomers, with 21 non-isomeric glycans. Thirty seven structures exhibited significant differences in isomeric distribution (P < 0.05). Additionally, alterations in the distribution of isomeric sialylated glycans, structures known to be involved in cell attachment to the blood-brain barrier during brain metastasis, were observed.

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“…Coelution of permethylated glycans is observed for the more complex samples, but because of MS detection these overlapping glycan species can still be identified [43, 51, 52]. Ritamo et al [44] and Zhou et al [50] observed separation of isomers, although in some cases the sample complexity was limited. The latter researchers performed the separation of permethylated glycans at higher temperature, which increased the chromatographic resolution of isomers and improved the peak shape and decreased the peak width.…”

Section: Separation Of Permethylated Glycansmentioning

confidence: 99%

Reversed-phase separation methods for glycan analysis

2016

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Reversed-phase chromatography is a method that is often used for glycan separation. For this, glycans are often derivatized with a hydrophobic tag to achieve retention on hydrophobic stationary phases. The separation and elution order of glycans in reversed-phase chromatography is highly dependent on the hydrophobicity of the tag and the contribution of the glycan itself to the retention. The contribution of the different monosaccharides to the retention strongly depends on the position and linkage, and isomer separation may be achieved. The influence of sialic acids and fucoses on the retention of glycans is still incompletely understood and deserves further study. Analysis of complex samples may come with incomplete separation of glycan species, thereby complicating reversed-phase chromatography with fluorescence or UV detection, whereas coupling with mass spectrometry detection allows the resolution of complex mixtures. Depending on the column properties, eluents, and run time, separation of isomeric and isobaric structures can be accomplished with reversed-phase chromatography. Alternatively, porous graphitized carbon chromatography and hydrophilic interaction liquid chromatography are also able to separate isomeric and isobaric structures, generally without the necessity of glycan labeling. Hydrophilic interaction liquid chromatography, porous graphitized carbon chromatography, and reversed-phase chromatography all serve different research purposes and thus can be used for different research questions. A great advantage of reversed-phase chromatography is its broad distribution as it is used in virtually every bioanalytical research laboratory, making it an attracting platform for glycan analysis. Graphical AbstractGlycan isomer separation by reversed phase liquid chromatography

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High‐temperature LC‐MS/MS of permethylated glycans derived from glycoproteins

Cited by 54 publications

References 38 publications

LC-MS/MS analysis of permethylated N-glycans facilitating isomeric characterization

LC-MS/MS analysis of permethylated N-glycans facilitating isomeric characterization

Isomeric Separation of Permethylated Glycans by Porous Graphitic Carbon (PGC)-LC-MS/MS at High Temperatures

Reversed-phase separation methods for glycan analysis

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