Tandem Mass Spectrometry as Strategy for the Selective Identification and Quantification of the Amyloid Precursor Protein Tyr682 Residue Phosphorylation Status in Human Blood Mononuclear Cells

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.

TNF-α Levels Are Increased in Patients with Subjective Cognitive Impairment and Are Negatively Correlated with β Amyloid-42

“…Notably, TNF-α levels correlated negatively with Aβ42 levels in patients with SCI and MCI but not in AD and HLT subjects. Interestingly, TNF-α exposure activates the inflammatory pathway in human healthy microglial cell cultures but does not affect the amyloidogenic pathway, by promoting amyloid precursor protein (APP) phosphorylation on the tyrosine (Tyr) 682 residue (APPpTyr682), which promotes Aβ42 production in neurons, fibroblasts, and blood mononuclear cells [21,[25][26][27], and APP intracellular processing. In addition, we did not detect changes in Fyn tyrosine kinase phosphorylation, which is considered a key player in AD, triggering either APPpTyr682 [27] or tau protein phosphorylation on the Tyr residue [28].…”