Comprehensive Profiling of Phosphopeptides Based on Anion Exchange Followed by Flow-Through Enrichment with Titanium Dioxide (AFET)

For large-scale analysis of phosphorylation at proteome-wide scale, a variety of affinity-based strategies have been developed to enrich phosphopeptide. Because each method differed in their specificity of isolation, the global and unbiased enrichment of phosphopeptides remains a major technical challenge in phosphoproteomics. In the present work, we demostrate that the phosphopeptide enrichment method based on an online continuous pH gradient in a strong anion exchange column (SAX method) is highly complementary to the method based on titanium dioxide (TiO2) affinity enrichment. Moreover, we found that the flow-through fraction of either SAX or SCX is very phosphopeptide-rich, which necessitates further analysis by complementary method. Here, we developed a comprehensive phosphopeptides profiling strategy based on anion exchange followed by flow-through enrichment by TiO2 (AFET). In this strategy, SAX method was used as the first separation/enrichment step, which was online coupled with LC-MS/MS. The phosphopeptides in the SAX flow-through fraction were further enriched with TiO2. As a result, a more comprehensive, less biased phosphoproteome was aquired. Careful comparison of four different combination strategies reveal that the AFET method showed the advantages of more identified phosphopeptides, less mass spectrometry analysis time, as well as simple and automatic process step. It is well-suited for robust and reproducible phosphoproteomics, especially in the case of small amounts of sample.