Protocol

Direct Enzymatic Digestion of Protein Complexes for MS Analysis

This protocol was adapted from "Identification of Novel Protein Complexes and Protein-Protein Interactions by Mass Spectrometry," Chapter 18, in Protein-Protein Interactions, 2nd ed. (eds. Golemis and Adams). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2005.

INTRODUCTION

The individual components of a purified protein complex can be determined by MS. Components can be separated by SDS-PAGE and subsequently digested, or the entire protein complex can be digested by chemical agents or by proteases. The mixture of peptides that results from digestion without prior separation is more complex than that obtained from the digestion of a single protein, and can best be resolved subsequently using HPLC in combination with a biphasic column. Protein complexes can be digested directly using several different protocols. Common methods include a double digest with endoproteinase Lys-C and trypsin, or a triple digest with elastase, subtilisin, and trypsin. Endoproteinase Lys-C cleaves carboxy-terminal to lysine residues, while trypsin cleaves carboxy-terminal to lysine and arginine residues. The triple digest uses the relatively nonspecific proteases elastase and subtilisin to create a large number of overlapping peptides for the analysis of post-translational modifications. The double and triple digest protocols begin with the reduction and alkylation of cysteine residues to disrupt disulfide bonds and, therefore, higher-order protein structure.

RELATED INFORMATION

See Overview of Affinity Purification in Combination with Mass Spectrometry.

Materials

Reagents

Acetone, ice cold

CaCl₂, 1 M

Elastase Grade II (Roche)

Endoproteinase Lys-C (Roche)

Formic acid, 90%

Iodoacetamide (IAA), 500 mM (Sigma)

Prepare fresh before each use.

Protein complex of interest, in solution

Subtilisin (Boehringer Mannheim)

Trichloroacetic acid (TCA), 20%

Tris (2-carboxyethyl)-phosphine hydrochloride (TCEP), 100 mM (Pierce)

Tris-HCl, 100 mM (pH 8.0) (Sigma)

Trypsin, sequencing grade, modified (Promega)

Urea/Tris solution

Over time, urea breaks down into ammonium cyanate, which can cause carbamylation of the amino termini of peptides as well as lysine and arginine side chains. It is therefore essential to prepare the urea/Tris solution fresh before each use.

Equipment

Incubator, 37°C

Microcentrifuge, 4°C

METHOD

Precipitation and Preparation of Purified Protein Complexes

1. Add an equal volume of 20% TCA to the protein sample (the protein concentration will vary depending on the experiment). Incubate for 30 minutes on ice.

2. Centrifuge the sample at 12,000g in a microfuge for 15 minutes at 4°C.

3. Carefully remove all the supernatant. Add 300 µl of cold acetone. Centrifuge at 12,000g for 5 minutes at 4°C.

4. Remove the supernatant. Air-dry the pellet.

5. Redissolve the protein pellet in 50-100 µl of urea/Tris solution.

6. Add 100 mM TCEP to the protein solution to a final concentration of 5 mM. Incubate for 20-30 minutes at room temperature.

7. Add 500 mM freshly prepared IAA to a final concentration of 10 mM. Incubate for 20-30 minutes in the dark at room temperature.

8. Digest the protein complex using one of the following methods:

For double digestion:

i. Add endoproteinase Lys-C to a final concentration of 0.002 µg/µl. Incubate in the dark for 4 hours at 37°C.
For complex mixtures of proteins, incubate the samples overnight.

ii. Dilute the sample to 2 M urea by adding 100 mM Tris-HCl (pH 8.0).

iii. Add 1 M CaCl₂ to a final concentration of 2 mM. Add trypsin to a final concentration of 0.01 µg/µl. Incubate overnight (12-24 hr) at 37°C.

iv. After incubation, quench the digestion by adding 90% formic acid to a final concentration of 5%.
Store at -20°C until needed for analysis by ESI MS/MS.

For triple digestion:

i. Divide the IAA-treated sample (from Step 7) into three aliquots of equal volume.
Each aliquot will be treated with a different protease and then pooled after digestion.

ii. Dilute Fractions 1 and 2 to 2 M urea with 100 mM Tris-HCl (pH 8.0). Dilute Fraction 3 to 4 M urea with 100 mM Tris-HCl (pH 8.0).

iii. To Fraction 1, add 1 M CaCl₂ to a final concentration of 2 mM. Add trypsin to a final concentration of 0.01 µg/µl. Incubate overnight (12 hr) at 37°C.

iv. To Fraction 2, add elastase to a final concentration of 0.005 µg/µl. Incubate for 4 hours at 37°C.
Store at -20°C until Fraction 1 is ready.

v. To Fraction 3, add subtilisin to a final concentration of 0.002 µg/µl. Incubate for 4 hours at 37°C.
Store at -20°C until Fraction 1 is ready.

vi. After digestion, pool the three fractions.

vii. Quench the reactions by adding 90% formic acid to a final concentration of 5%.
Store at -20°C until needed for analysis by ESI MS/MS.