Phosphopeptide Enrichment with TiO2-Modified Membranes and Investigation of Tau Protein Phosphorylation

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• 作者：Yu-Jing Tan ; Dexin Sui ; Wei-Han Wang ; Min-Hao Kuo ; Gavin E. Reid ; Merlin L. Bruening
• 刊名：Analytical Chemistry
• 出版年：2013
• 出版时间：June 18, 2013
• 年：2013
• 卷：85
• 期：12
• 页码：5699-5706
• 全文大小：391K
• 年卷期：v.85,no.12(June 18, 2013)
• ISSN：1520-6882

文摘

Selective enrichment of phosphopeptides prior to their analysis by mass spectrometry (MS) is vital for identifying protein phosphorylation sites involved in cellular regulation. This study describes modification of porous nylon substrates with TiO₂ nanoparticles to create membranes that rapidly enrich phosphopeptides. Membranes with a 22-mm diameter bind 540 nmol of phosphoangiotensin and recover 70% of the phosphopeptides in mixtures with a 15-fold excess of nonphosphorylated proteins. Recovery is 90% for a pure phosphopeptide. Insertion of small membrane disks into HPLC fittings allows rapid enrichment from 5 mL of 1 fmol/渭L phosphoprotein digests and concentration into small-volume (tens of microliters) eluates. The combination of membrane enrichment with tandem mass spectrometry reveals seven phosphorylation sites from in vivo phosphorylated tau (p-tau) protein, which is associated with Alzheimer鈥檚 disease.