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.

The characterization of glycosylation is critical for obtaining a comprehensive view of the regulation and functions of glycoproteins of interest. Due to the complex nature of oligosaccharides, due to variable compositions and linkages, and ion suppression effects, the chromatographic separation of glycans, including isomeric structures, is necessary for exhaustive characterization by mass spectrometry (MS). This review introduces the fundamental principles underlying the techniques in liquid chromatography (LC) utilized by modern day glycomics researchers. Recent advances in porous graphitized carbon, reverse phase, ion exchange and HILIC LC utilized in conjunction with MS, for the characterization of protein glycosylation, are described with an emphasis on methods capable of resolving isomeric glycan structures.

Motor imagery (MI) requires subjects to visualize the requested motor behaviors, which involves a large-scale network that spans multiple brain areas. The corresponding cortical activity reflected on the scalp is characterized by event-related desynchronization (ERD) and then by event-related synchronization (ERS). However, the network mechanisms that account for the dynamic information processing of MI during the ERD and ERS periods remain unknown. Here, we combined ERD/ERS analysis with the dynamic networks in different MI stages (i.e. motor preparation, ERD and ERS) to probe the dynamic processing of MI information. Our results show that specific dynamic network structures correspond to the ERD/ERS evolution patterns. Specifically, ERD mainly shows the contralateral networks, while ERS has the symmetric networks. Moreover, different dynamic network patterns are also revealed between the two types of MIs, in which the left-hand MIs exhibit a relatively less sustained contralateral network, which may be the network mechanism that accounts for the bilateral ERD/ERS observed for the left-hand MIs. Similar to the network topologies, the three MI stages also appear to be characterized by different network properties. The above findings all demonstrate that different MI stages that involve specific brain networks for dynamically processing the MI information.

The diversification of the chemical properties and biological functions of proteins is attained through posttranslational modifications, such as glycosylation. Glycans, which are covalently attached to proteins, play a vital role in cell activities. The microheterogeneity and complexity of glycan structures associated with proteins make comprehensive glycomic analysis challenging. However, recent advancements in mass spectrometry (MS), separation techniques, and sample preparation methods have primarily facilitated structural elucidation and quantitation of glycans. This review focuses on describing recent advances in MS-based techniques used for glycomic analysis (2012-2018), including ionization, tandem MS, and separation techniques coupled with MS. Progress in glycomics workflow involving glycan release, purification, derivatization, and separation will also be highlighted here. Additionally, the recent development of quantitative glycomics through comparative and multiplex approaches will also be described.

Protein glycosylation, an important PTM, plays an essential role in a wide range of biological processes such as immune response, intercellular signaling, inflammation, and host–pathogen interaction. Aberrant glycosylation has been correlated with various diseases. However, studying protein glycosylation remains challenging because of low abundance, microheterogeneities of glycosylation sites, and poor ionization efficiency of glycopeptides. Therefore, the development of sensitive and accurate approaches to characterize protein glycosylation is crucial. The identification and characterization of protein glycosylation by MS is referred to as the field of glycoproteomics. Methods such as enrichment, metabolic labeling, and derivatization of glycopeptides in conjunction with different MS techniques and bioinformatics tools, have been developed to achieve an unequivocal quantitative and qualitative characterization of glycoproteins. This review summarizes the recent developments in the field of glycoproteomics over the past 6 years (2012 to 2018).