Off-Line High-pH Reversed-Phase Fractionation for In-Depth Phosphoproteomics

Protein phosphorylation is an important post-translational modification (PTM) involved in embryonic development, adult homeostasis, and disease. Over the past decade, several advances have been made in liquid chromatography–tandem mass spectrometry (LC–MS/MS)-based technologies to identify thousands...

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Bibliographic Details
Published in:Journal of proteome research 2014-12, Vol.13 (12), p.6176-6186
Main Authors: Batth, Tanveer S, Francavilla, Chiara, Olsen, Jesper V
Format: Article
Language:English
Subjects:
Analytic Sample Preparation Methods
Animals
Cation Exchange Resins
Chemical Fractionation
Chromatography, Ion Exchange - methods
Chromatography, Liquid - methods
Chromatography, Reverse-Phase - methods
Hydrogen-Ion Concentration
Mice
NIH 3T3 Cells
Phosphopeptides - analysis
Phosphopeptides - metabolism
Phosphoproteins - analysis
Phosphoproteins - metabolism
Phosphorylation
Proteome - analysis
Proteome - metabolism
Proteomics - methods
Reproducibility of Results
Tandem Mass Spectrometry - methods
Titanium
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Summary:Protein phosphorylation is an important post-translational modification (PTM) involved in embryonic development, adult homeostasis, and disease. Over the past decade, several advances have been made in liquid chromatography–tandem mass spectrometry (LC–MS/MS)-based technologies to identify thousands of phosphorylation sites. However, in-depth phosphoproteomics often require off-line enrichment and fractionation techniques. In this study, we provide a detailed analysis of the physicochemical characteristics of phosphopeptides, which have been fractionated by off-line high-pH chromatography (HpH) before subsequent titanium dioxide (TiO2) enrichment and LC–MS/MS analysis. Our results demonstrate that HpH is superior to standard strong-cation exchange (SCX) fractionation in the total number of phosphopeptides detected when analyzing the same number of fractions by identical LC–MS/MS gradients. From 14 HpH fractions, we routinely identified over 30 000 unique phosphopeptide variants, which is more than twice the number of that obtained from SCX fractionation. HpH chromatography displayed an exceptional ability to fractionate singly phosphorylated peptides, with minor benefits for doubly phosphorylated peptides over that with SCX. Further optimizations in the pooling and concatenation strategy increased the total number of multiphosphorylated peptides detected after HpH fractionation. In conclusion, we provide a basic framework and resource for performing in-depth phosphoproteome studies utilizing off-line basic reversed-phased fractionation. Raw data is available at ProteomeXchange (PXD001404).
ISSN:1535-3893
1535-3907
DOI:10.1021/pr500893m