Journal
JOURNAL OF PROTEOME RESEARCH
Volume 10, Issue 8, Pages 3474-3483Publisher
AMER CHEMICAL SOC
DOI: 10.1021/pr200092z
Keywords
SCX; ERLIC; HILIC; TiO2; LC-MS; mass spectrometry; chromatography; phosphoproteomics
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Funding
- Excellence Initiative of the German Federal and State Governments through FRIAS and BIOSS
- Federal Ministry of Education and Research through GerontoSys II - NephAge [031 5896 A]
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Reversible phosphorylations play a critical role in most biological pathways. Hence, in signaling studies great effort has been put into identification of a maximum number of phosphosites per experiment. Mass spectrometry (MS)-based phosphoproteomics approaches have been proven to be an ideal analytical method for mapping of phosphosites. However, because of sample complexity, fractionation of phosphopeptides prior to MS analysis is a crucial step. In the current study, we compare the chromatographic strategies electrostatic repulsion-hydrophilic interaction chromatography (ERLIC), hydrophilic interaction liquid chromatography (HILIC), and strong cation exchange chromatography (SCX) for their fractionation behavior of phosphopeptides. In addition, we investigate the use of repetitive TiO2-based enrichment steps for a maximum identification of phosphopeptides. On the basis of our results, SCX yields the highest number of identified phosphopeptides, whereas ERLIC is optimal for the identification of multiphosphorylated peptides. Consecutive incubations of fractions and flow-through by TiO2 beads enrich qualitatively different sets of phosphopeptides, increasing the number of identified phosphopeptides per analysis.
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