Proteolytic Digestion

Introduction

Proteins are specifically cleaved into small fragments to facilitate their identification. Specific cleavage can be achieved by chemical or enzymatic methods. For example, cyanogen bromide (CNBr) cleaves polypeptides only on the carboxyl side of methionine residues and trypsin specifically hydrolyzes peptide bonds at the carboxyl side of lysine (Lys, K) and arginine (Arg, R) residues. CNBr and trypsin are examples of chemical and enzymatic digestion, respectively.

In general, mass spectrometry (MS) of mixtures is challenging. However, matrix-assisted laser desorption ionization (MALDI) mass spectrometry is a very useful method for the analysis of peptide mixtures resulting from proteolysis [1-3]. The objective of this application note is to describe enzymatic digestion of proteins by trypsin for MALDI-MS analysis.

Trypsin is a pancreatic serine (Ser,S) protease (Mr ~24 kDa). The aspartate residue in the binding site enables trypsin to form a salt bridge with positively charged residues (e.g., Lys and Arg side-chains) of the substrate, thus enablind the cleavage reaction to occur. Trypsin can undergo autolysis and lose activity. The presence of autolysis peptides can interfere in the MS analysis. Autolysis of trypsin can be prevented by addition of calcium; however, excess calcium in the reaction is normally not desired. A chemically modified trypsin is commercially available from Promega [4]. The modified trypsin does not undergo autolysis and retains high enymatic activity.

There are two common methods for enzymatic digestion of proteins: solution based and in-gel based. The following sections describe the procedures used to perform each experiment.

Solution Base Digestion

Solution based digest methods typically work better than in-gel methods and produce better yields. Protein and enzyme are dissolved in solution. Denatured proteins typically produce enhance peptide fragment yields.

Materials and methods:

  1. Prepare a buffer 50 mM solution of ammonium bicarbonate (NH4HCO3, pH = 8.0) dissolved in deionized water.
  2. Prepare a 1-5 uM solution of protein dissolved in the NH4HCO3 buffer prepared in step 1.
  3. Prepare 0.1 ug/uL solution of trypsin (Promega sequence grade) in storage buffer (pH = 2-3, provided by Promega) . Best if prepared just prior to use.
  4. Mix protein and trypsin solutions together in 20:1 to 50:1 (w:w) ratio.
  5. Incubate at 37 oC for 30 min to 24 hours. The solution can be incubated at room temperature but the activity of trypsin is much lower and the digestion requires significantly longer time to produce high yields of peptide fragments.

MALDI analysis:

  1. Mix 5 uL of digest mixture with 5 uL of matrix solution (e.g., 0.02-0.03 M alpha-cyano-4-hydroxy-cinnamic acid in 0.1% TFA/acetonitrile 1:2). The acidity of the matrix solution disables the activity of the trypsin.
  2. Transfer 0.5 - 1 uL of mixture from step 1 to a MALDI sample plate and allow to dry.
  3. Analyze samples with mass spectrometer.

Results and Discussion

MALDI time-of-flight MS analysis of tryptic digests of 10 and 30 minutes as well as 4 and 24 hours are shown in Figure 1. Each spectrum is the average of 30 laser shots. There are no signals that correspond to autolysis peaks of trypsin in any of the spectra. As the digestion time increases, a shift in signal intensity is observed. For example, in the 10 and 30 min digest spectra, a significant amount of lysozyme is present; whereas, in the 4 and 24 hour spectra the intensity of lysozyme is minimal if not insignificant and the spectra contain very intense peptide fragment ion signals. The presence of numerous peptide fragments in high abundance allows for the amino acid sequence of lysozyme to be mapped completely.

10 minutes

30 minutes

4 hours

24 hours

Figure 1. MALDI TOF MS of tryptic digestion of chicken-egg white lysozyme. 2.5 pmol of lysozyme was digested and applied in alpha-cyano-4-hydroxy-cinnamic acid matrix.

This experiment can be used to follow the kinetics of the enzymatic digestion to determine the optimum time to terminate the digestion and analyze the fragments. Furthermore, kinetic studies can be used to learn about the three dimensional structure of proteins if one knows where in the sequence of a protein specific peptide fragments originate.

On to In-Gel Digest Method.