Refined phosphopeptide enrichment by phosphate additive and the analysis of human brain phosphoproteome
Alzheimer's disease (AD) is the most common form of dementia, characterized by progressive loss of cognitive function. One of the pathological hallmarks of AD is the formation of neurofibrillary tangles composed of abnormally hyperphosphorylated tau protein, but global deregulation of protein p...
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Published in | Proteomics (Weinheim) Vol. 15; no. 2-3; pp. 500 - 507 |
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Main Authors | , , , , , , , , |
Format | Journal Article |
Language | English |
Published |
Germany
Blackwell Publishing Ltd
01.01.2015
Wiley Subscription Services, Inc |
Subjects | |
Online Access | Get full text |
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Summary: | Alzheimer's disease (AD) is the most common form of dementia, characterized by progressive loss of cognitive function. One of the pathological hallmarks of AD is the formation of neurofibrillary tangles composed of abnormally hyperphosphorylated tau protein, but global deregulation of protein phosphorylation in AD is not well analyzed. Here, we report a pilot investigation of AD phosphoproteome by titanium dioxide enrichment coupled with high resolution LC‐MS/MS. During the optimization of the enrichment method, we found that phosphate ion at a low concentration (e.g. 1 mM) worked efficiently as a nonphosphopeptide competitor to reduce background. The procedure was further tuned with respect to peptide‐to‐bead ratio, phosphopeptide recovery, and purity. Using this refined method and 9 h LC‐MS/MS, we analyzed phosphoproteome in one milligram of digested AD brain lysate, identifying 5243 phosphopeptides containing 3715 nonredundant phosphosites on 1455 proteins, including 31 phosphosites on the tau protein. This modified enrichment method is simple and highly efficient. The AD case study demonstrates its feasibility of dissecting phosphoproteome in a limited amount of postmortem human brain. All MS data have been deposited in the ProteomeXchange with identifier PXD001180 (http://proteomecentral.proteomexchange.org/dataset/PXD001180). |
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Bibliography: | ark:/67375/WNG-HD4V1G0M-0 Arizona Department of Health Services - No. 211002 ArticleID:PMIC7930 Arizona Biomedical Research Commission - No. 4001 Michael J. Fox Foundation NIH Cancer Center Support - No. P30CA021765 istex:9F90F634DEBF2CCCE0E1814A6C7120C5BBF2C8D8 ALSAC (American Lebanese Syrian Associated Charities) NIH - No. R21AG039764 See the article online to view Figs. 1–5 in colour. Colour Online ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 |
ISSN: | 1615-9853 1615-9861 |
DOI: | 10.1002/pmic.201400171 |