Prince:FASP

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FASP Protocol

• Template File provides easy calculation of volumes and masses for Solution preparation

FASP Template

Steps

Protocols

• Preparing cells/tissue lysate
  • Lyse cells or homogenized tissue in SDT-lysis buffer using 1:10 sample to buffer ratio at 95°C for 3-5 min
  • Incubate the lysed cells for 3 min in boiling water
  • Sonicate the lysed cells using Branson SONIFIER 250 for 30 s (Amplitude 40%)
  • Incubate the mixture for 3 min in boiling water
  • Clarify the crude extract by centrifugation at 16,000 x g at 30°C for 10 min.

• Sample processing
  • Slight modifications can/should be made according to the complexity of your sample
  • Mix the lysate with UA buffer in the ratio of(1 mg protein:1 ml UA buffer) for purified proteins add 200µl of UA-DTT and vortex it
  • load samples in 30k filters.(Amicon ultra/ Vivacon 500)
    • (Sample loading and speed of centrifugation would depend on the filters used)
  • concentrate the solution for 15 min at 14000 × g
  • Add 100µl of UA and centrifuge the filters for 8 min at 14500 g(twice)
  • Add 100µl of UA-IAA, mix it well and incubated at 20° C for 20 min in dark. Spin out the UA-IAA reagent at 14500g for 8 min.
  • Add 100µl of UA centrifuge the filters for 8 min at 14500 g (twice)
  • Add 100µl of ABC and centrifuge the filters for 8 min at 14500 g (twice)
  • Add trypsin(proteomic grade)to the sample in the ratio (1:40)
    • for incomplete trypsin digestion and better coverage, microwave the sample for 1 min at 20% power in a microwave with an inverter technology.(thrice, make sure the sample cools down before, subsequent microwave)
    • for complete digestion carry out an overnight (6-18 hrs) trypsin digestion in addition to the microwave method.

Notes

Buffer Definitions

• UA
  • 8M Urea in 0.1M Tris-HCl @ pH 8.5
• UA/DTT
  • 0.1M DTT in UA buffer
• UA/IAA
  • 50mM IAA in UA buffer
• SDT Lysis Buffer
  • 2% w/v SDS, 0.1M DTT in 0.1M Tris-HCl @ pH 7.6
• ABC
  • 50mM Ammonium Bicarbonate

Measuring peptide concentration in eluant

• 1mg/ml solution has 1.1 au at 280nm

Concentration of the peptides can be estimated by UV spectrometer assuming that 0.1% solution of vertebrate proteins has at 280 nm an extinction of 1.1 absorbance units.

Reference

FASP paper (from the supplement of Nature Methods: 6(5) 2009)

Trypsin

Why you do not want to have any residual reducing agent (e.g. DTT) in your digestion buffer:

Trypsin with disulfide bonds

Porcine Trypsin [Green: Backbone, Red: Disulfide bonds]