Quantitation of Permethylated <b><i>N</i></b>-Glycans through Multiple-Reaction Monitoring (MRM) LC-MS/MS

Zhou, Shiyue; Hu, Yunli; Desantos-Garcia, Janie L.; Mechref, Yehia

doi:10.1007/s13361-014-1054-1

Cited by 41 publications

(54 citation statements)

References 60 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…7,10,20 Glycans were purified by ice-cold ethanol precipitation. In the case of the cell line samples, dialysis was also utilized to remove sodium deoxycholate (SDC), a detergent used in cell lysis.…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2017

Self Cite

View full text Add to dashboard Cite

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.

show abstract

Section: Methodsmentioning

confidence: 99%

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

et al. 2017

Self Cite

View full text Add to dashboard Cite

show abstract

“…With this work, we have clearly demonstrated the utility of the methodology for profiling the N-glycans of monoclonal antibodies, the most frequently exploited modality for therapeutic applications. Nevertheless, it is interesting to consider the use of these sample preparation techniques for the analysis of N-glycans from complex matrices, such as human blood serum, 53 though additional work will be required to investigate their applicability to such studies. …”

Section: ■ Conclusionmentioning

confidence: 99%

Rapid Preparation of Released N-Glycans for HILIC Analysis Using a Labeling Reagent that Facilitates Sensitive Fluorescence and ESI-MS Detection

et al. 2015

View full text Add to dashboard Cite

N-glycosylation of proteins is now routinely characterized and monitored because of its significance to the detection of disease states and the manufacturing of biopharmaceuticals. At the same time, hydrophilic interaction chromatography (HILIC) has emerged as a powerful technology for N-glycan profiling. Sample preparation techniques for N-glycan HILIC analyses have however tended to be laborious or require compromises in sensitivity. To address these shortcomings, we have developed an N-glycan labeling reagent that provides enhanced fluorescence response and MS sensitivity for glycan detection and have also simplified the process of preparing a sample for analysis. The developed labeling reagent rapidly reacts with glycosylamines upon their release from glycoproteins. Within a 5 min reaction, enzymatically released N-glycans are labeled with this reagent comprised of an NHS-carbamate reactive group, a quinoline fluorophore, and a tertiary amine for enhancing ESI+ MS ionization. To further expedite the released N-glycan sample preparation, rapid tagging has been integrated with a fast PNGase F deglycosylation procedure that achieves complete deglycosylation of a diverse set of glycoproteins in approximately 10 min. Moreover, a technique for HILIC-SPE of the labeled glycans has been developed to provide quantitative recovery and facilitate immediate HILIC analysis of the prepared samples. The described approach makes it possible to quickly prepare N-glycan samples and to incorporate the use of a fluorescence and MS sensitivity enhancing labeling reagent. In demonstration of these new capabilities, we have combined the developed sample preparation techniques with UHPLC HILIC chromatography and high sensitivity mass spectrometry to thoroughly detail the N-glycan profile of a monoclonal antibody.

show abstract

“…The combination of precursor and product ion transitions can be utilized with triple quadrupole MS operated in multiple reaction monitor (MRM) mode for reliable quantitative analysis. 11, 12 Although native glycans can be analyzed by MS, 13 low sensitivity in positive mode MS and the potential in-source fragmentation always impede intact glycan analysis.…”

Section: Introductionmentioning

confidence: 99%

Direct comparison of derivatization strategies for LC-MS/MS analysis ofN-glycans

Zhou

Veillon

Dong

et al. 2017

Analyst

Self Cite

106

105

View full text Add to dashboard Cite

Protein glycosylation is a common post-translational modification that has significant impacts on protein folding, lifespan, conformation, distribution and function. N-glycans, which are attached to asparagine residues of proteins, are studied most often due to their compatibility with enzymatic release. Despite the ease of N-glycan release, compositional and structural complexity coupled with poor ionization efficiency during liquid chromatography mass spectrometry (LC-MS) make quantitative glycomic studies a significant challenge. To overcome these challenges, glycans are almost always derivatized prior to LC-MS analyses to impart favorable characteristics, such as improved ionization efficiency, increased LC separation efficiency and the production of more informative fragments during tandem MS. There are a number of derivatization methods available for LC-MS analysis of glycans, each of which imparts different properties that affect both glycan retention on LC columns and MS analyses. To provide guidance for the proper selection of derivatizing reagents and LC columns, herein, we describe a comprehensive assessment of 2-aminobenzamide, procainamide, aminoxyTMT, RapiFluor-MS (RFMS) labeling, reduction and reduction with permethylation for N-glycan analysis. Of the derivatization strategies examined, RFMS provided the highest MS signal enhancement for neutral glycans, while permethylation significantly enhanced the MS intensity and structural stability of sialylated glycans.

show abstract

Quantitation of Permethylated N-Glycans through Multiple-Reaction Monitoring (MRM) LC-MS/MS

Cited by 41 publications

References 60 publications

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

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

Rapid Preparation of Released N-Glycans for HILIC Analysis Using a Labeling Reagent that Facilitates Sensitive Fluorescence and ESI-MS Detection

Direct comparison of derivatization strategies for LC-MS/MS analysis ofN-glycans

Contact Info

Product

Resources

About