The Development and Implementation of a TMT-SRM Assay for the Validation of Candidate Biomarkers of Alzheimer’s Disease

Student thesis: Doctoral ThesisDoctor of Philosophy


Biomarker discovery studies in plasma have revealed several proteins which may have clinical utility in the diagnosis and prognosis of Alzheimer’s disease. The qualification of these proteins as true biomarkers, sensitive and specific to the disease, requires rapid, high quality, quantitative assays. Selected reaction monitoring (SRM) has emerged as an accurate and precise method for targeted protein selection through signature peptide measurement in highly complex mixtures such as plasma. Quantitative SRM-based assays are an attractive alternative to immuno-based approaches where costs can be high and requisite antibodies unavailable. Furthermore, the highly multiplexed nature of SRM allows the quantitation of multiple peptides/proteins in one rapid analysis step. Herein, we combine SRM with isotopic versions of tandem mass tags (TMT) to provide an internal reference for quantitation, an approach termed TMT-SRM, for the validation of prognostic candidate biomarkers of Alzheimer’s disease.
The panel of proteins included clusterin, complement C3, alpha-2- macroglobulin, gelsolin, fibrinogen gamma-chain, serum amyloid p-component and complement factor H. These proteins were previously found to be differentially expressed between plasma of Alzheimer’s disease subjects and non-demented controls. Additionally, apolipoprotein E4 was included in the panel as possession of the apolipoprotein e4 allele is the only unequivocal genetic risk factor for late-onset Alzheimer’s disease and the protein expression of this genetic association may translate as an Alzheimer’s disease biomarker.
For technology validation, TMT-SRM was shown to strongly correlate with western blot measurements for one of the proteins, gelsolin, in a sample cohort. Furthermore, the performance characteristics were determined for each target analyte in the assay. For biomarker validation, a cohort of 90 subjects was selected as having Alzheimer’s disease (n = 60) or as non-demented controls (n = 30). To monitor disease progression, the Alzheimer’s disease group was sub-divided into two groups (n = 30 per group), rapid cognitive decliners and slow cognitive decliners, based on decline in Mini Mental State Examiniation score per year.

Plasma alpha-2-macroglobulin expression was found to significantly increase in Alzheimer’s disease and the difference correlated with disease progression. No significant differences were observed for the remaining proteins in the panel. Western blot analysis was performed on the same cohort for alpha-2-macroglobulin and gelsolin. In accordance with the mass spectrometry results, gelsolin showed no significant differences between disease and controls groups, providing confidence that the mass spectrometry-based assay was reflecting the real expression of the protein in the cohort. However, in contrast to the mass spectrometry results, alpha-2- macroglobulin did not show a significant difference between disease and control groups, illustrating the fact that the TMT-SRM approach was able to detect more subtle changes in peptide/protein expression than western blot. Results were replicated on an analogous mass spectrometer with strong correlation, demonstrating the robustness and ease-of-transfer of the TMT-SRM assay. Here, we have demonstrated TMT-SRM to be a rapid, precise and selective strategy for the quantitation of candidate biomarkers of Alzheimer’s disease in plasma.
Date of Award1 Dec 2011
Original languageEnglish
Awarding Institution
  • King's College London
SupervisorSimon Lovestone (Supervisor) & H Byers (Supervisor)

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