- Phosphoproteome: Sample Preparation 47
Hint: Examine the tip after every centrifuge step to make sure there
is no solution remained in the tip.VII. Equilibrate the tips by loading 20μLsolutionC, centrifuge at
1,500×gfor2minutes.
VIII.Load 50 μL of sample, centrifuge at 1,000×g for 2 minutes. Repeat
the step until all the peptide samples are loaded into the StageTip.
Hint: the loading capacity for each C8 StageTip is 100μgpeptides.IX. Wash tips by loading 50μLsolutionC, centrifuge at 1,500×gfor
2minutes.
X. Wash tips by loading 50μLsolutionB, centrifuge at 1,500×gfor
2 minutes. Prepare new 1.5 mL microcentrifuge tubes with boreholes
for elution (one tube for each StageTip).
XI. Transfer the StageTip to the new 1.5 mL tube.
XII. Elutephosphopeptides by loading 50μL solution D (0.5%), centrifuge
at 1,000×gfor2minutes.
XIII. Repeat step XII. Acidify the eluted peptides by adding 10% TFA to a
final concentration of 0.5% and examine the pH value by test paper.
Hint 1: Peptides are more stable in the environment of low pH
compared to alkaline condition.
Hint 2:Different elution buffers such as ammonium bicarbonate
might elute different phosphopeptides.Desalting
XIV. Each TiO 2 tip requires one SDB-XC StageTips for desalting.
XV. Precondition the SDB-XC tips by loading 20μLsolutionB,
centrifuge at 1,000 g for 1 minute.Hint: Be careful about the height of StageTip upon the tube. Do
not let the tip touch the cover of centrifuge machine.XVI. Equilibrate the tips by loading 20μLsolutionA, 1,000×gfor
1minute.
XVII.Load the sample to the StageTip, centrifuge at 1,000×gfor1
minute. Repeat the step until all the peptide samples are loaded
into the StageTip.
XVIII.Transferthe tip to the new tube.