Protocol

Digestion of Insoluble Protein Fractions for Multidimensional Protein Identification Technology (MuDPIT) Analysis

This protocol was adapted from "The Use of Mass Spectrometry in Proteomics," Chapter 8 in Proteins and Proteomics (ed. Simpson). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2003.

INTRODUCTION

Proteins associated with insoluble particulate matter from a whole-cell lysate, or carefully prepared membrane samples, can be analyzed using MuDPIT analysis. However, the preliminary steps provided in this protocol are required to prepare a usable complex protein mixture from these types of samples.

RELATED INFORMATION

For information on MuDPIT analysis, please see Analysis of Complex Protein Mixtures Using Multidimensional Protein Identification Technology (MuDPIT).

MATERIALS

Reagents

Ammonium bicarbonate, saturated

Cyanogen bromide

Formic acid (90%)

Equipment

Chemical fume hood

pH paper

Vacuum concentrator

METHOD

WARNING: These steps MUST be carried out in a chemical fume hood, exercising proper safety precautions.

1. Lyophilize the insoluble fraction.

2. Add 100 µl of 90% formic acid to the lyophilized pellet and incubate the tube for 5 minutes at room temperature.

3. Add 100 µg of cyanogen bromide to the protein, mix the solution well, and incubate it overnight at room temperature in the dark (wrap the tube in aluminum foil).

4. Add saturated ammonium bicarbonate to the tube until the pH reaches 8.5. Add the ammonium bicarbonate solution very slowly, because a large amount of bubbling occurs during this step. Use pH paper to check the pH of the sample.

5. Concentrate the fraction to ~200 µl by lyophilization in a vacuum concentrator.

6. Proceed with Step 2 in Analysis of Complex Protein Mixtures Using Multidimensional Protein Identification Technology (MuDPIT).