Program

Poster Presentations

Day 4, May 18（Fri.）　　Poster

4P-08　　PDF

A streamlined StageTip-based workflow for deep and sensitive phosphoproteomic profiling

(¹NTU, ²IOC, AS, ³Kyoto University)
^oYun-chien Chang^1,2, Reta Birhanu Kitata², Pei-yi Lin², Chia-feng Tsai³, Yu-ju Chen^1,2

Protein phosphorylation is one of the most widespread protein post-translation modification regulating cellular signaling processes. However, the multi-step sample preparation required for phosphoproteomics is labor-intensive and prone to sample loss and poor reproducibility. Here, we introduced the Stop-and-go extraction tip (StageTip) for complete phosphoproteomic workflow. On the analysis of PC9, StageTip-based digestion outperformed conventional in-solution digestion with better reproducibility, evaluated by quantitative comparison with Pearson correlation coefficient (0.925-0.945 and 0.896-0.92, respectively). The workflow was also incorporated with peptide reversed-phase (RP) fractionation strategy. The number of identified phosphopeptides and phosphoproteins increased from 9261 to 17962 and 2828 to 4204, respectively, which is almost 1.9-fold and 1.4-fold of identification number from single-shot elution. Furthermore, we assembled the RP-StageTip and IMAC-StageTip in tandem with optimized fractionation buffer with IMAC system. More than 20000 phosphopeptides of 5000 phosphoproteins and 17175 phosphosites were identified in 7 fractions, while the single-shot elution from the tip of two replicate only yields 8265 phosphosites. The separation efficiency of StageTip-based fractionation was also evaluated by comparing the number of identical phosphopeptides between adjacent fractions which range from 13.1% to 20.7%. Taken together, we demonstrated a rapid, ease-of-use, and reproducible workflow for phosphoproteomic profiling.