Journal
JOURNAL OF PROTEOME RESEARCH
Volume 7, Issue 7, Pages 2845-2851Publisher
AMER CHEMICAL SOC
DOI: 10.1021/pr8000496
Keywords
Alzheimer's disease; protein phosphorylation; immobilized metal-affinity chromatography; calcium phosphate precipitation; proteomics
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Funding
- NIA NIH HHS [AG025688, P50 AG025688, P50 AG025688-040003] Funding Source: Medline
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Alzheimer's disease (AD), the most common form of dementia, is manifested in the brain by the aggregation of amyloid plaques and neurofibrillary tangles. The tangles are primarily composed of microtubule-associated protein tau that is aberrantly hyperphosphorylated, suggesting that deregulated phosphorylation may contribute to AD pathogenesis. However, systematic analysis of the phosphoproteome in AD brain tissues has not been reported. We used-calcium phosphate precipitation to analyze an AD postmortem brain, followed by liquid chromatography-tandem mass spectrometry. The protein sample was first resolved by: one-dimensional polycrylamide gel electrophoresis and subjected to gel excision and in-gel digestion. Phosphopeptides in the resulting peptide mixtures were enriched in a single step of calcium phosphate precipitation, and then analyzed by the LC-MS/MS approach. After database search, stringent filtering, and manual validation of neutral loss in the MS/MS spectra, a total of 466 phosphorylation sites on 185 proteins including tau were identified. A majority of sites were not described previously. This study demonstrates the feasibility of combining calcium phosphate precipitation with mass spectrometry for phosphoproteome analysis of postmortem human brain tissue.
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