Phosphoproteomic analysis of human brain by calcium phosphate precipitation and mass spectrometry.
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 polyacrylamide 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.
Department of Human Genetics, The Center for Neurodegenerative Diseases, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
Qiangwei
Q
Xia
Dongmei
D
Cheng
Duc M
DM
Duong
Marla
M
Gearing
James J
JJ
Lah
Allan I
AI
Levey
Junmin
J
Peng
D000368:Aged; D000544:Alzheimer Disease / Q000378:metabolism; D001921:Brain / Q000378:metabolism*; D002130:Calcium Phosphates; D011232:Chemical Precipitation; D002853:Chromatography, Liquid; D006801:Humans; D007202:Indicators and Reagents; D008297:Male; D010748:Phosphopeptides / Q000378:metabolism*; D020543:Proteome / Q000378:metabolism*; D053719:Tandem Mass Spectrometry
D002130:Calcium Phosphates; D007202:Indicators and Reagents; D010748:Phosphopeptides; D020543:Proteome; C020243:calcium phosphate