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Mass Spectrometry Protocols

 In-Gel Digestion

The following in-gel digestion protocol is used for up to 3 - 1mm round gel pieces. The methods should be modified if starting with additional gel pieces such that all gel pieces are completely covered in solution for all steps. The amount of protease should also be adjusting accordingly.

Reagents

  • 25mM ammonium Bicarbonate (ABC)
  • 10mM dithiothreitol (DTT) in 25mM ABC
  • 100mM iodoacetamide (IA) in 25mM ABC
  • 20 μg lyophilized modified trypsin (Promega Cat# V5111)
  • 0.3% formic acid (aq)
  • Acetonitrile (ACN)

Methods

  • Add 50μl ABC to gel plugs and incubate at room temperature (RT) for 5 minutes
  • Discard solution and add 50μl ACN, incubate at RT for 5 minutes
  • Repeat both steps
  • Discard solution and add 40μl DTT, heat to 60C and incubate for 10 minutes. Let cool 15 minutes.
  • Discard solution and add 30μl IA. Incubate for 35 minutes at RT
  • Discard solution and wash two times with ABC and ACN as stated above
  • Add 15μl of trypsin solution, incubate for 10 minutes.
  • Note: Make trypsin solution immediately prior to addition. Dissolve trypsin in 0.3% formic acid to give a 0.1ug/ul solution and dilute to 5ng/ul in 25mM ABC
  • Add 10μl ABC and incubate for 4 hours at 37C
  • Add 15μl 0.3% formic acid

In-Solution Digestion

Starting with a samples of <20 μl sample buffer.

Reagents

  • 25mM ammonium Bicarbonate (ABC)
  • 10mM dithiothreitol (DTT) in 25mM ABC
  • 55mM iodoacetamide (IA) in 25mM ABC
  • 20 μg lyophilized modified trypsin (Promega Cat# V5111)
  • 0.3% formic acid (aq)
  • Acetonitrile (ACN)

Methods

  • Add 20μl ACN, incubate at RT for 20 minutes
  • Add 40μl DTT, heat to 60C and incubate for 10 minutes.
  • Let cool 15 minutes
  • Add 20μl IA. Incubate for 35 minutes at RT
  • Add 20μl of trypsin solution, incubate for 10 minutes.
  • Note: Make trypsin solution immediately prior to addition. Dissolve trypsin in 0.3% formic acid to give a 0.1ug/ul solution and dilute to 5ng/ul in 25mM ABC
  • Add 10μl ABC and incubate for 4 hours at 37C
  • Add 20μl 0.3% formic acid

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