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Article

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

Supporting Info

Jenny Ellis^†^‡, Rudolf Grimm^‡^§, Joseph F. Clark^‡, Gail Pyne-Gaithman^‡, Steve Wilbur^‡^§ and Joseph A. Caruso*^†^‡

Department of Chemistry, University of Cincinnati, Cincinnati, Ohio 45221-0172, University of Cincinnati/Agilent Technologies Metallomics Center of the Americas, Cincinnati, Ohio 45221-0172, Agilent Technologies Inc. Life Sciences Solutions Unit, Santa Clara, California 95052-8059, and Department of Neurology, University of Cincinnati, Cincinnati, Ohio 45221

J. Proteome Res., 2008, 7 (11), pp 4736–4742

DOI: 10.1021/pr800294r

Publication Date (Web): September 23, 2008

†

Department of Chemistry, University of Cincinnati.

‡

University of Cincinnati/Agilent Technologies Metallomics Center of the Americas.

Agilent Technologies Inc. Life Sciences Solutions Unit.

Department of Neurology, University of Cincinnati.

* To whom communications should be addressed. E-mail: joseph.caruso@uc.edu.

Abstract

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.

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