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Effect of Peptide-to-TiO 2 Beads Ratio on Phosphopeptide Enrichment Selectivity

2009; American Chemical Society; Volume: 8; Issue: 11 Linguagem: Inglês

10.1021/pr900659n

1535-3907

Qingrun Li, Zhibin Ning, Jia-shu Tang, Song Nie, Rong Zeng,

Advanced Biosensing Techniques and Applications

Titanium dioxide (TiO2) has been proven to be a highly efficient strategy and widely used for phosphopeptide enrichment. Many advances have been made recently, including online/offline mode and optimization of sample loading/elution buffer; however, beads usage has rarely been explored. In the current study, we found that the peptide-to-TiO2 beads ratio was a significant factor for phosphopeptide enrichment, and insufficient or excessive beads could decrease the selectivity. Specifically, for HeLa total cell lysate, the optimum peptide-to-beads ratio is about 1:2−1:8 (mass/mass) to obtain the highest enrichment selectivity and the maximum phosphopeptides identification with single incubation. Pre-experiments are recommended to decide an optimum peptide-to-TiO2 beads ratio when it comes to different samples. Interestingly, deficient beads can help identify much more multiphosphorylated peptides than the optimum peptide-to-beads ratio by consecutive incubations. Therefore, if multiphosphorylated peptides identification is desired, deficient beads amount is preferred. In addition, consecutive incubation using deficient beads could be used as a fractionation of phosphopeptides besides as an enrichment method.