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Glycosylation, an essential post-translational protein modification, is known to be altered in a variety of diseases, including neurodegenerative diseases such as Alzheimer's disease (AD), which is one of the most common neurodegenerative disorders that results in cognitive and memory impairments. To investigate the progression of such a condition, cerebrospinal fluid (CSF), a unique biofluid that may possess significant biochemical and neurochemical changes due to the disease, is utilized. However, due to the low concentration of proteins in CSF, a large volume of the biofluid is often required to comprehensively characterize the glycome in CSF. In this work, a glycomic study of CSF was performed using as little as 10 μL of CSF. This approach was executed with permethylation of released N-glycans with minimal sample cleanup, in conjunction with an online purification system attached to liquid chromatography and a high-resolution mass spectrometer. This technique was then applied to clinical samples. Preliminary data suggest that fucosylated and bisecting GlcNAc structures were higher in abundances in females with AD, while both females and males exhibited lower abundances of high-mannose structures. Although there seems to be statistically significant differences between disease state and disease-free CSF, due to the lack of number of samples, further validation study should be conducted.

Glycomics continues to be a highly dynamic and interesting research area due to the need to comprehensively understand the biological attributes of glycosylation in many important biological functions such as the immune response, cell development, cell differentiation/adhesion, and host-pathogen interactions. Although matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) has proven to be suitable for glycomic profiling studies, there is a need for improved sensitivity in the detection of native glycans, which ionize inefficiently. In this study, we investigated the efficiencies of graphene nanosheets (GNs) and carbon nanoparticles (CNPs) as MALDI matrices and co-matrices in glycan profiling. Our results indicated an enhancement of signal intensity by several orders of magnitude upon using GNs and CNPs in MALDI analysis of N-glycans derived from a variety of biological samples. Interestingly, increasing the amounts of CNPs and GNs improved not only the signal intensities but also prompted in-source decay (ISD) fragmentations, which produced extensive glycosidic and cross-ring cleavages. Our results indicated that the extent of ISD fragmentation could be modulated by CNP and GN concentrations, to obtain MS 2 and pseudo-MS 3 spectra. The results for glycan profiling in high salt solutions confirmed high salt-tolerance capacities for both CNPs and GNs. Finally, the results showed that by using CNPs and GNs as co-matrices, DHB crystal formation was more homogeneous which improved shot-to-shot reproducibility and sensitivity. Graphical Abstract ᅟ.

Glycosylation is one of the most common and essential protein modifications. Glycans conjugated to biomolecules modulate the function of such molecules through both direct recognition of glycan structures and indirect mechanisms that involve the control of protein turnover rates, stability, and conformation. The biological attributes of glycans in numerous biological processes and implications in a number of diseases highlight the necessity for comprehensive characterization of protein glycosylation. This chapter reviews cutting-edge methods and tools developed to facilitate quantitative glycomics. This chapter highlights the different methods employed for the release and purification of glycans from biological samples. The most effective labeling methods developed for sensitive quantitative glycomics are also described and discussed. The chromatographic approaches that have been used effectively in glycomics are also highlighted., (© 2017 Elsevier Inc. All rights reserved.)

Protein glycosylation plays an important role in various biological processes, such as modification of protein function, regulation of protein-protein interactions, and control of turnover rates of proteins. Moreover, glycans have been considered as potential biomarkers for many mammalian diseases and development of aberrant glycosylation profiles is an important indicator of the pathology of a disease or cancer. Hence, quantitation is an important aspect of a comprehensive glycomics study. Although numerous MS-based quantitation strategies have been developed in the past several decades, some issues affecting sensitivity and accuracy of quantitation still exist, and the development of more effective quantitation strategies is still required. Aminoxy tandem mass tag (aminoxyTMT) reagents are recently commercialized isobaric tags which enable relative quantitation of up to six different glycan samples simultaneously. In this study, liquid chromatography and mass spectrometry conditions have been optimized to achieve reliable LC-MS/MS quantitative glycomic analysis using aminoxyTMT reagents. Samples were resuspended in 0.2 M sodium chloride solution to promote the formation of sodium adduct precursor ions, which leads to higher MS/MS reporter ion yields. This method was first evaluated with glycans from model glycoproteins and pooled human blood serum samples. The observed variation of reporter ion ratios was generally less than 10% relative to the theoretical ratio. Even for the highly complex minor N-glycans, the variation was still below 15%. This strategy was further applied to the glycomic profiling of N-glycans released from blood serum samples of patients with different esophageal diseases. Our results demonstrate the benefits of utilizing aminoxyTMT reagents for reliable quantitation of biological glycomic samples.