Enrichment of Phosphopeptides Using PureCube Products

 

This protocol provides an example on how to enrich phosphopeptide using any kind of PureCube MagBeads that are meant for this purpose.

The protocol has been used for 250 μg HELA cell digest and 2.5 mg beads. Adjust the volumes of wash and binding buffers for higher bead/protein amounts.
0 – 5 % GA is added to the binding buffer to enhance specificity. The optimal amount depends on samples and can be optimized.

Note: Phosphopeptide Enrichment is a diverse process. Your specific assay may require modifications to this protocol.

Buffer compositions - Phosphopeptide enrichment protocol

Binding Buffer

ComponentFinal concentration
Acetonitrile (ACN) 80%
Trifluoroacetic acid (TFA) 5%
Glycolic acid (GA) 0.2%

Wash buffer

ComponentFinal concentration
Acetonitrile (ACN) 80%
Trifluoroacetic acid (TFA) 1%

Wash buffer 2

ComponentFinal concentration
Acetonitrile (ACN) 10%
Trifluoroacetic acid (TFA) 0.2%

Elution buffer

ComponentFinal concentration
Ammonium hydroxide 1%
   
A.NHS coupling protocol
 
 
  1. Use 10:1 (w/w) bead to protein ratio
  2. Calculate the required amount of beads for all samples + 1. Wash three times in 10 x volume of binding buffer and adjust the final concentration to 2.5 % in binding buffer
  3. Add 250 µl binding buffer to dried protein digest and incubate for 5 min on a thermoshaker to resuspend the peptides (250 rpm, 25°C)
  4. Add the required amount of beads and incubate for 20 min on a thermoshaker (250 rpm, 25°C)
  5. Place tube on a magnetic rack until all beads are trapped (10 - 30 sec) and remove supernatant
  6. Depending on the temperature stability of the protein, incubate at room temperature or 4°C for 2 h on an end-overend shaker or thermoshaker.
  7. Add 250 µl wash buffer 1 , incubate for 2 min on a thermoshaker (250 rpm, 25°)
  8. Place tube on a magnetic rack until all beads are trapped (10 - 30 sec) and remove supernatant
  9. Add 250 µl wash buffer 1 , incubate for 2 min on a thermoshaker (250 rpm, 25°C)
  10. Place tube on a magnetic rack until all beads are trapped (10 - 30 sec) and remove supernatant
  11. Add 250 µl wash buffer 2, incubate for 4 min on a thermoshaker (250 rpm, 25°C)
  12. Place tube on a magnetic rack until all beads are trapped (10 - 30 sec) and remove supernatant
  13. Add 80 µl elution buffer and incubate for 30 min on a thermoshaker (250 rpm, 25°C)
  14. Place tube on a magnetic rack until all beads are trapped (10 - 30 sec) and transfer supernatant to a new tube
  15. Add 20 µl 10 % FA (formic acid) to supernatant and dry in a vacuum centrifuge
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