Journal
JOURNAL OF PROTEOME RESEARCH
Volume 8, Issue 11, Pages 5375-5381Publisher
AMER CHEMICAL SOC
DOI: 10.1021/pr900659n
Keywords
peptide-to-TiO2 beads ratio; phosphorylation; enrichment; selectivity
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Funding
- National Natural Science Foundation [30425021, 30521005]
- Basic Research Foundation [2006CB910700]
- CAS [KSCX2-YW-R-106, KSCX1-YW-02]
- High-technology Project [2007AA02Z334]
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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. Preexperiments 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.
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