UCDHSC Anesthesiology Research - Clinical Nutrition Research Unit

The CNRU Mass Spectrometry Core Facility is your source for Metabolomics research.

IN-SOLUTION DIGESTION PROTOCOL

MATERIALS: Ammonium Bicarbonate 25 mM ammonium bicarbonate (1.9 mg/ml of water)
8M Urea
Dilute 480 milligrams of urea in 1.0 mL of 25 mM ammonium bicarbonate solution
Reducing Reagent
Dissolve 30 mg of DTT in 0.20 mL of 25 mM ammonium bicarbonate solution to make 1 M DTT
Alkylating Reagent
Dissolve 36 mg (0.036 g) in 1 mL of ammonium bicarbonate solution to make 200 mM iodoacetamide.
Trypsin solution Trypsin
is made up by dissolving 20 ug of trypsin in 200 uL of bicarbonate buffer. This 0.10 ug/uL solution can be used according the protein content in the sample in a 1:30 ratio (enzyme:substrate by weight). This is an approximate value for the trypsin catalyst. Alternatively, you can make up a 1 mg/ml solution of trypsin in water or ammonium bicarbonate and add 1 - 2 µl to each sample.

DIGESTION PROCEDURE

Reconstitute sample in approximately 20 uL of 8.0 M urea in a 0.5 mL microfuge tube.
Add 1 uL of Reducing Reagent and mix the sample by gentle vortex.
Reduce the mixture for 1 hour at room temperature or in an oven at 37°C. Do not go over 37°C or the urea will react with the sample and generate carbamylated artifacts. Allow the sample to cool to room temperature.
Add 20 uL of Alkylating Reagent and alkylate for 1 hour at room temperature in the dark (you can use aluminum foil to cover up the sample).
Add 4 uL of Reducing Reagent to consume any leftover alkylating agent (so the trypsin is not alkylated).
Add 60 uL of ammmonium bicarbonate solution to dilute the urea before digesting it with trypsin.
Add trypsin in appropriate ratio (1:30) to approximate amount of protein by weight. Digest overnight at 37°C.
In the morning, add 1 ul of 100% formic acid to the sample. Vortex and centrifuge. Freeze unused sample at -20 - -80° C.

A portion of this sample is generally analyzed by LC-MS and another aliquot can be removed, concentrated and cleaned up using a C₁₈ coated tip, then analyzed using MALDI.

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