Abstract
Background: Alzheimer’s disease (AD) is a devastating neurodegenerative disease without guidelines for early diagnosis or personalized treatment. Previous studies have highlighted a crucial role of increasing phosphorylation levels of the amyloid precursor protein (APP) Tyr682 residue in predicting neuronal deficits in AD patients. However, the lack of a method for the identification and quantification of Tyr682 phosphorylation levels prevents its potential clinical applications. Methods: Here we report a method to identify and quantify APP Tyr682 phosphorylation levels in blood mononuclear cells of AD patients by tandem mass spectrometry (tMS). Results: This method showed excellent sensitivity with detection and quantification limits set respectively at 0.035 and 0.082 ng injected for the phosphorylated peptide and at 0.02 and 0.215 ng injected for the non-phosphorylated peptide. The average levels of both peptides were quantified in transfected HELA cells (2.48 and 3.53 ng/μg of protein, respectively). Preliminary data on 3 AD patients showed quantifiable levels of phosphorylated peptide (0.10–0.15 ng/μg of protein) and below the LOQ level of non-phosphorylated peptide (0.13 ng/μg of protein). Conclusion: This method could allow the identification of patients with increased APP Tyr682 phosphorylation and allow early characterization of molecular changes prior to the appearance of clinical signs.
Highlights
We recently found that amyloid precursor protein (APP) Tyr682 phosphorylation increases in fibroblasts of Alzheimer’s disease (AD) patients [16], opening up to the possibility to use APP Tyr682 phosphorylation level as a potential powerful tool in detecting early signs of AD-related cognitive disorders in peripheral cells
We recently suggested that changes related to APP Tyr682 phosphorylation in fibroblasts may reflect Aβ-related abnormalities in the brain [16], emphasizing the promising potential of using APP Tyr682 phosphorylation levels to develop new diagnostic strategies and to optimize therapeutic approaches with better outcomes in patients who are included in clinical trials [5,6,16]
This is the first time that the MQQNGYpENPTYK has been detected in the blood mononuclear cells of patients with AD, placing the basis for further analysis to verify the potential role of APP Tyr682 phosphorylation as biomarker in AD. This approach is useful to calculate the ratio between phosphorylated and unphosphorylated peptides. These findings highlight the utility of this novel tandem mass spectrometry (tMS) approach to selectively identify and analyze APP Tyr682 phosphorylation levels in human mononuclear cells, as well as other cells
Summary
Despite the latest progresses in the knowledge of the mechanisms responsible for AD, the biggest challenge of the AD research still consists of finding new biomarkers for the early detection and the accurate diagnosis in the preclinical stages of AD. Previous studies have highlighted a crucial role of increasing phosphorylation levels of the amyloid precursor protein (APP) Tyr682 residue in predicting neuronal deficits in AD patients. The lack of a method for the identification and quantification of Tyr682 phosphorylation levels prevents its potential clinical applications. Methods: Here we report a method to identify and quantify APP Tyr682 phosphorylation levels in blood mononuclear cells of AD patients by tandem mass spectrometry (tMS). The average levels of both peptides were quantified in transfected HELA cells
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