Effect of peptide-to-TiO2 beads ratio on phosphopeptide enrichment selectivity.
Titanium dioxide (TiO(2)) 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-TiO(2) 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-TiO(2) 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.
Li
Qing-run
QR
Key Laboratory of Systems Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 200031, China.
Ning
Zhi-bin
ZB
Tang
Jia-shu
JS
Nie
Song
S
Zeng
Rong
R
D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods; D006367:HeLa Cells; D006801:Humans; D010455:Peptides / Q000737:chemistry*; D010748:Phosphopeptides / Q000737:chemistry / Q000302:isolation & purification*; D010766:Phosphorylation; D011485:Protein Binding; D053719:Tandem Mass Spectrometry / Q000379:methods; D014025:Titanium / Q000737:chemistry*
D010455:Peptides; D010748:Phosphopeptides; C009495:titanium dioxide; D014025:Titanium