Prince:FASP

From OpenWetWare

Jump to: navigation, search

Image:Prince lab logo.png

Home        Lab Members         Research         Publications         Internal         Mass Spec         Contact        


Contents

FASP Protocol

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

Steps

Cell/Tissue Samples:

  • Preparing cells/tissue lysate
    • Prepare SDT lysis buffer making sure you have a 1:10 sample:lysis-buffer ratio
    • Bring this amount of lysis buffer to a boil and drop in frozen sample pellet, this is to reduce the activity of phosphotases and kinases as much as possible
    • Incubate the lysed cells for 10 min at 95°C
    • Sonicate the lysed cells (Branson SONIFIER 250 for 30 s) (Amplitude 40%) until no longer viscous (DNA broken)
    • Incubate the mixture for 10 min at 95°C
    • Clarify the crude extract by centrifugation at 16,000 x g at room temperature for 10 min.
  • Sample processing (for complex sample) times are for 30K filters, when using large filters multiply volume amounts by 50
    • load up to 40 µl (30μL is best) of supernatant with 200µl of UA buffer in 30k filters (Amicon ultra/ Vivacon 500) and vortex it
    • concentrate the solution for 15 min at 14000 × g
    • Add 100µl of UA and centrifuge the filters for 15 min at 14000 × g (twice)
    • Add 100µl of UA-IAA, mix it well and incubate at room temperature for 20 min in dark. Spin out the UA-IAA reagent at 14000 × g for 15 min.
    • Add 100µl of UA centrifuge the filters for 15 min at 14000 × g (twice)
    • Add 100µl of ABC and centrifuge the filters for 20 min at 14000 × g (twice)
    • Add trypsin (proteomic grade) to the sample in the ratio (1:40)
      • mix the required amount of trypsin in 50µl of ABC and add to the filter, vortex it well and pulse the filters to 2000 × g
      • Incubate the samples for 4 h-18 h at 37 °C
    • Transfer the filter units to new collection tubes and centrifuge at 14000 × g
    • wash the filter with 40µl of ABC and centrifuge at 14000 × g
    • Acidify the solution to 1% formic acid

Purified Protein Samples:

  • Sample processing (for purified proteins) times are for 30K filters
    • load ~20 µg (have done up to 100) of purified protein in a 1:4 sample:UA buffer ratio in 30k filters and centrifuge for 15 min at 14000 × g
    • Add 100µl of UA-DTT and mix the solution thoroughly centrifuge for 10 min at 14000 × g
    • Add 100µl of UA and centrifuge for 10 min at 14000 × g
    • Add 100µl of UA-IAA, mix it well and incubate at room temperature for 20 min in dark. Spin out the UA-IAA reagent at 14000 × g for 15 min.
    • Add 100µl of UA centrifuge the filters for 15 min at 14000 × g (twice)
    • Add 100µl of ABC and centrifuge the filters for 15 min at 14000 × g (twice)
    • Add trypsin, (0.8 µg proteomic grade) to the sample in the ratio (1:40)
      • mix the required amount of trypsin in 40 µl of ABC and add to the filter, vortex it well and pulse the filters to 2000 × g
      • microwave (inverter based) the sample at 20% power for 1 min and let it cool to room temperature repeat the microwave process again.
    • Transfer the filter units to new collection tubes and centrifuge for 15 min at 14000 × g
    • wash the filter with 40µl of ABC and centrifuge for 15 min at 14000 × g
    • Acidify the solution to 1% formic acid

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
    • 4% w/v SDS, 0.1M DTT in 0.1M Tris-HCl @ pH 7.6
  • ABC
    • 50mM Ammonium Bicarbonate (in MS grade water)

Ordering Information

  • Trypsin
    • Sequencing Grade Trypsin from Promega (C/N #V5113) or Trypsin Gold, Mass Spectrometry Grade from Promega (C/N #V5280)
  • Filters
    • Sartorius stedim Vivacon 500 Line Vivacon Product Information
      • 100K
      • 50K
      • 30K
      • 10K requires additional 30 minutes/spin
      • 2K we haven't tried yet

Notes

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]

In Gel FASP Protocol

Also, try our new and improved In-gel FASP method!

Personal tools