N-Glycan Profile of Cerebrospinal Fluids from Alzheimer’s Disease Patients Using Liquid Chromatography with Mass Spectrometry

Abstract

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.

Implications of Glycosylation in Alzheimer’s Disease

“…One issue with this approach has been the fact that the CSF contains a rather low concentration of proteins, and a large volume is often required. However, recent studies have shown the possibility of performing CSF glycoanalysis with a very small amount of fluid ( Cho et al, 2019 ). Increased levels of fucosylated and bisecting GlcNAc in AD CSF has been observed in several studies, which has also been confirmed in postmortem brains.…”

Glycomics and glycoproteomics: Approaches to address isomeric separation of glycans and glycopeptides

“…The native glycan is made less hydrophilic by the substitution of the hydrogen atoms in all hydroxyl, N ‐acetyl, and carboxylic acid moieties for methyl groups. Glycan permethylation is particularly efficient for the separation of isomers and, therefore, it is the most used derivatization technique for glycome analysis [3,7,11,16,20,82,84,85,95,96,138–142]. Otherwise, the use of permethylation in glycopeptides is not an easy task; to date, there is no reliable proof of its application [143].…”

N-Glycomics of Cerebrospinal Fluid: Method Comparison

“…Genomics [ 3 ], transcriptomics [ 4 ], proteomics [ 5 ], glycoproteomics [ 6 ], metabolomics [ 7 ], lipidomics [ 8 ], and glycomics [ 9 ] are some of the omics analysis more often investigated by the researchers in the field. Recently, glycomic profiles of different human fluids such as blood, urine, saliva, and cerebrospinal fluid (CSF) have been associated with the progression of multiple diseases [ 10 , 11 , 12 , 13 , 14 ]. Both serum and plasma are the fluids of choice to perform glycomic analysis since unlike CSF, blood samples are easy to acquire.…”

“…Unlike the other above-mentioned human biofluids, CSF need a more invasive extraction procedure. Its direct contact with the brain and spinal cord makes this fluid a reliable analytical sample for the identification of biochemical changes that occur during the progression of neurodegenerative diseases [ 10 , 16 , 17 ]. Currently, CSF is a source of biomarkers for diagnostic workups of Alzheimer’s disease (AD), mild cognitive impairment (MCI), multiple sclerosis (MS), Parkinson’s, and other related diseases [ 18 , 19 , 20 , 21 ].…”

“…A number of neurodegenerative studies based on CSF glycomics have described significant changes in abundances for the bisecting N -acetylglucosamine (GlcNAc) and core fucose glycans, which are structures known as “brain type N -glycans” [ 22 , 23 , 24 , 25 , 26 , 27 , 28 ]. Thus, glycans are valuable biomolecules that may serve as potential biomarkers [ 10 , 22 , 29 ]. Although CSF is a useful source of information for neurodegenerative diseases, its complex matrix and low glycoprotein concentration present arduous challenges in glycomic studies [ 30 , 31 ].…”