Studying Protein Phosphorylation in Low MW CSF Fractions with capLC−ICPMS and nanoLC−CHIP-ITMS for Identification of Phosphoproteins

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• 作者：Jenny Ellis ; Rudolf Grimm ; Joseph F. Clark ; Gail Pyne-Gaithman ; Steve Wilbur ; Joseph A. Caruso
• 刊名：Journal of Proteome Research
• 出版年：2008
• 出版时间：November 7, 2008
• 年：2008
• 卷：7
• 期：11
• 页码：4736-4742
• 全文大小：441K
• 年卷期：v.7,no.11(November 7, 2008)
• ISSN：1535-3907

文摘

An initial study of protein phosphorylation in human cerebral spinal fluid (CSF) is described. CSF is an important body fluid for study of proteins and metabolites and may lead to the ultimate development of molecular markers to predict neurological diseases or their complications, such as in the case of hemorrhagic stroke. The use of capillary liquid chromatography coupled to inductively coupled plasma mass spectrometry (capLC−ICPMS) for screening using ³¹P as the internal elemental tag atom at ultratrace levels, in combination with molecular mass spectrometry using Spectrum Mill and MASCOT database search engines for peptide identification, is a novel approach in its application to CSF relevant phosphopeptides and phosphorylated proteins. CapLC−ICPMS combined with nano liquid chromatography electrospray ionization, ion trap mass spectrometry (nanoLC−CHIP/ITMS), was utilized for initial experiments with CSF. Specific low-level screening for ³¹P containing compounds is accomplished, and nanoLC−CHIP/ITMS provided the corresponding peptide information and subsequent protein identifications. The fractions containing ³¹P from screening by the capLC−ICPMS were collected offline and analyzed separately with nanoLC−CHIP/ITMS. Synthetic phosphopeptides were used to test the method and to estimate lowest quantifiable limits for phosphorus. Tryptically digested β-casein was then used to demonstrate the viability of the methodology for the complex CSF matrix from hemorrhagic stroke patients while also analyzing for native phosphopeptides in the CSF.