N-Glycomics of Cerebrospinal Fluid: Method Comparison

Abstract

Cerebrospinal fluid (CSF) contains valuable biological and neurological information. However, its glycomics analysis is hampered due to the low amount of protein in the biofluid, as has been demonstrated by other glycomics studies using a substantial amount of CSF. In this work, we investigated different N-glycan sample preparation approaches to develop a more sensitive method. These methods, one with an increased amount of buffer solution during the N-glycan release step with a lower amount of sample volume and the other with Filter-Aided N-Glycan Separation (FANGS), were compared with recent work to demonstrate their effectiveness. It was demonstrated that an increased amount of buffer solution showed higher intensity in comparison to the previously published method and FANGS. This suggested that digestion efficiency during the N-glycan release step was not in an optimal condition from the previously published method, and that there is a substantial loss of sample with FANGS when preparing N-glycans from CSF.

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

“…The N -glycans were released and extracted according to the procedure described previously [ 14 ]. Briefly, 25 μg of protein was diluted to a total volume of 100 μL with 50 mM ABC buffer (pH ~ 7.5) and denatured at 90 °C for 15 min.…”

“…Released and purified N -glycans were reduced and permethylated according to the following protocol [ 14 ]. The N -glycan reduction was accomplished by the addition of 10 μL of 10 μg/μL ammonium-borane solution to the released and purified glycans, followed by incubation at 60 °C for 1 h. After incubation, the residual borane was removed with the addition of methanol, generating methyl borate that was evaporated while drying in the vacuum concentrator.…”

“…The N -glycan reduction was accomplished by the addition of 10 μL of 10 μg/μL ammonium-borane solution to the released and purified glycans, followed by incubation at 60 °C for 1 h. After incubation, the residual borane was removed with the addition of methanol, generating methyl borate that was evaporated while drying in the vacuum concentrator. The reduced and dried N -glycans were then permethylated with a solid-phase permethylation protocol, as previously described [ 14 ]. The dried N -glycans were resuspended in 30 μL of DMSO, 1.2 μL of water, and 20 μL of iodomethane.…”

“…There is now considerable evidence that protein glycosylation plays a key role in numerous biological functions and changes during the development of a disease [ 10 , 11 ]. Recently, researchers have found great potential in employing glycomics to identify diagnostic biomarkers in circulating biofluids for brain-related diseases [ 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 ]. Additionally, it is well known that glycans have a variety of isomeric conformations where some of the most important structures have differences in the sialic acid linkage (e.g., α2,3/α2,6), fucosylation in the glycan core or branch, and the formation of bisecting N -acetylglucosamine in the glycan core [ 13 , 21 , 22 ].…”

Advances in mass spectrometry‐based glycomics—An update covering the period 2017–2021

“…However, due to the microheterogeneity and complexity of the glycan structures, isomeric separation of glycans is still lacking. Several LC-MS techniques have been employed to facilitate the isomeric separation of glycans including RP [80,81], HILIC [82,83], porous graphitic carbon (PGC) [84,85], and mesoporous graphitic carbon (MGC) [86]. This section discusses the most recent LC and CE methods that assist in the isomeric characterization and separation of glycans.…”