Abstract
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).
Highlights
Great advances have been made in the field of biomedical science due to the availability of large-scale proteomics data sets arising from high-performance liquid chromatography−tandem mass spectrometry (LC−MS/MS) experiments
To determine the efficacy of basic reversed-phase (HpH) fractionation for large-scale phosphoproteomics, we analyzed tryptic digests of whole-cell lysates from mouse NIH-3T3 cells that were separated by high-pH chromatography (HpH) followed by TiO2 enrichment of phosphopeptides from concatenated fractions
We carefully evaluated the number of the identified phosphopeptides and their properties, and, on the basis of this, we optimized the HpH fractionation and concatenation strategy
Summary
Great advances have been made in the field of biomedical science due to the availability of large-scale proteomics data sets arising from high-performance liquid chromatography−tandem mass spectrometry (LC−MS/MS) experiments. Charged-based separation, such as strong cation exchange (SCX) chromatography, is well-suited for enrichment of phosphopeptides from a population of unmodified tryptic peptides due to the negative charges of the phosphoryl group.[9] For these reasons, SCX (as well has HILIC) chromatography in combination with TiO2/IMAC has, so far, been the most widely used off-line fractionation strategy applied in large-scale phosphoproteomics studies.[10−15] fractionation methods based on hydrophobic interactions, such as reversed-phase chromatography, are more efficient for the separation of peptides compared to that of charge-based separations like SCX. We show a thorough analysis of phosphoproteome coverage with standard off-line high-pH reversed-phase fractionation (HpH) prior to phosphopeptide enrichment and MS analysis
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
