Phosphopeptide Enrichment

Phosphopeptide Enrichment

 

Currently we use TiO2 columns to enrich phosphopeptides before mass spectrometry runs. This is done with a commercial TopTip, using either lactate or glycolic acid.

TiO2  Columns using commercial TopTip ----- Lactate method

Buffers

Lactate Loading buffer: 240mg/mL Lactate in 80% (v/v) Acetonitrile, 1% (v/v) TFA.

  • 200µl of lactate solution 1.2g/ml (Fluka 69785)
  • 800µl ACN
  • 10µl TFA
  • Washing buffer: 80% (v/v) Acetonitrile, 5% (v/v) TFA
  • 150µl  water
  • 800µl ACN
  • 50µl TFA
  • Elution buffer: Ammonium water (20ul NH3 in 980ul H2O), pH 10.5.

    Method

    • Resuspend iTraq labeled peptide mixture in 150µl of lactate loading buffer
    • Equilibrate tip with 2 x 75µl of lactate loading buffer
    • Load sample as 2 x 75µl aliquots Keep flow-through and apply to a new tip (sample B).
    • Wash tip with 2 x 60ul lactate loading buffer.
    • Wash 4 x 60µl with Wash buffer.
    • Elute phosphopeptides in 60uL elution buffer (sample A)
    • Repeat procedure with initial flow through (sample B)
    • Combine samples A and B
    • Dry down sample in the Speed-vac

    TiO2  Columns using commercial TopTip ---- Glycolic acid method

    Buffers

    Loading buffer.

    • 1M Glycolic acid
    • 80%l ACN
    • 5% TFA

    Washing buffer: 80% (v/v) Acetonitrile, 5% (v/v) TFA

    • 150µl  water
    • 800µl ACN
    • 50µl TFA

    Elution buffer: Ammonium water (20ul NH3 in 980ul H2O), pH 10.5.

    Method

    • Resuspend iTraq labeled  peptide mixture in 6 µl 4M Urea, 9 µl 1%SDS, then dilute in 1M Glycolic acid, 80%ACN, 5%TFA.
    • Equilibrate tip with 2 x 75µl of Loading buffer
    • Load sample as 2 x 75µl aliquots Keep flow-through and apply to a new tip (sample B).
    • Wash tip with 2 x 60ul loading buffer.
    • Wash 4 x 60µl with Wash buffer.
    • Elute phosphopeptides in 60uL elution buffer.