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A High Throughput, Multiplexed and Targeted Proteomic CSF Assay to Quantify Neurodegenerative Biomarkers and Apolipoprotein E Isoforms Status

Many neurodegenerative diseases are&#160;still lacking effective treatments. Reliable biomarkers for identifying and classifying these diseases will be ...

University College London

Currently there are no effective treatments for many neurodegenerative diseases. Reliable biomarkers for identifying and stratifying these diseases will be important in the development of future novel therapies. Lewy Body Dementia (LBD) is considered an under diagnosed form of dementia for which markers are needed to discriminate LBD from other forms of dementia such as Alzheimer's Disease (AD). This work describes a Label-Free proteomic profiling analysis of cerebral spinal fluid (CSF) from non-neurodegenerative controls and patients with LBD. Using this technology we identified several potential novel markers for LBD. These were then combined with other biomarkers from previously published studies, to create a 10 min multiplexed targeted and translational MRM-LC-MS/MS assay. This test was used to validate our new assay in a larger cohort of samples including controls and the other neurodegenerative conditions of Alzheimer's and Parkinson's disease (PD).

Identification of novel CSF biomarkers for neurodegeneration and their validation by a high-throughput multiplexed targeted proteomic assay.

Erratum to: Identification of novel CSF biomarkers for neurodegeneration and their validation by a high-throughput multiplexed targeted proteomic assay.

The Congenital Disorders of Glycosylation (CDG) are an expanding group of genetic disorders which encompass a spectrum of glycosylation defects of protein and lipids, including N- & O-linked defects and among the latter are the muscular dystroglycanopathies (MD). Initial screening of CDG is usually based on the investigation of the glycoproteins transferrin, and/or apolipoprotein CIII. These biomarkers do not always detect complex or subtle defects present in older patients, therefore there is a need to investigate additional glycoproteins in some cases. We describe a sensitive 2D-Differential Gel Electrophoresis (DIGE) method that provides a global analysis of the serum glycoproteome. Patient samples from PMM2-CDG (n = 5), CDG-II (n = 7), MD and known complex N- & O-linked glycosylation defects (n = 3) were analysed by 2D DIGE. Using this technique we demonstrated characteristic changes in mass and charge in PMM2-CDG and in charge in CDG-II for α1-antitrypsin, α1-antichymotrypsin, α2-HS-glycoprotein, ceruloplasmin, and α1-acid glycoproteins 1&2. Analysis of the samples with known N- & O-linked defects identified a lower molecular weight glycoform of C1-esterase inhibitor that was not observed in the N-linked glycosylation disorders indicating the change is likely due to affected O-glycosylation. In addition, we could identify abnormal serum glycoproteins in LARGE and B3GALNT2-deficient muscular dystrophies. The results demonstrate that the glycoform pattern is varied for some CDG patients not all glycoproteins are consistently affected and analysis of more than one protein in complex cases is warranted. 2D DIGE is an ideal method to investigate the global glycoproteome and is a potentially powerful tool and secondary test for aiding the complex diagnosis and sub classification of CDG. The technique has further potential in monitoring patients for future treatment strategies. In an era of shifting emphasis from gel- to mass-spectral based proteomics techniques, we demonstrate that 2D-DIGE remains a powerful method for studying global changes in post-translational modifications of proteins.

Global serum glycoform profiling for the investigation of dystroglycanopathies & Congenital Disorders of Glycosylation.

The skin is the largest organ of the human body and is the first line barrier defence against trauma, microbial infiltration and radiation. Skin diseases can be a result of multi-systemic disease or an isolated condition. Due to its proteolysis resistant properties there are relatively few human skin proteomic datasets published compared with other human organs or body fluids. Skin is a challenging tissue to analyse using traditional proteomic techniques due to its high lipid content, insolubility and extensive cross-linking of proteins. This can complicate the isolation and digestion of proteins for analysis using mass spectrometry techniques.

Article	Year
Identification of novel CSF biomarkers for neurodegeneration and their validation by a high-throughput multiplexed targeted proteomic assay. Molecular neurodegeneration\| PubMed ID: 26627638	2015
Erratum to: Identification of novel CSF biomarkers for neurodegeneration and their validation by a high-throughput multiplexed targeted proteomic assay. Molecular neurodegeneration\| PubMed ID: 26907468	2016
Global serum glycoform profiling for the investigation of dystroglycanopathies & Congenital Disorders of Glycosylation. Molecular genetics and metabolism reports\| PubMed ID: 27134828	2016
An optimised method for the proteomic profiling of full thickness human skin. Biological procedures online\| PubMed ID: 27445641	2016

Emily Bliss

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