Original protocol from Cold Spring Harbor protocols. Enjoy.

Cleavage at Tryptophan by ο-Iodosobenzoic Acid

Richard J. Simpson

This protocol was adapted from “Peptide Mapping and Sequence Analysis of Gel-Resolved Proteins,” Chapter 7, in Proteins and Proteomics (ed. Simpson). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2003.

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INTRODUCTION

Chemical reagents that selectively cleave at tryptophan residues have contributed greatly to the elucidation of protein structure and function. This protocol uses ο-iodosobenzoic acid for specific cleavage at tryptophan residues.

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MATERIALS

Reagents

p-Cresol

Glacial acetic acid (Puriss grade, Merck)

Guanidine hydrochloride

ο-Iodosobenzoic acid

Lyophilized protein sample

Equipment

Column, size-exclusion (Sephadex G-25, Amersham Biosciences or equivalent) (optional; see Step 4)

Concentrator (SpeedVac, Thermo Savant)

Nitrogen supply

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METHOD

1. Dissolve the ο-iodosobenzoic acid (10 mg) in 1.0 ml of 80% (v/v) acetic acid containing 4 M guanidine-HCl and 20 μl of p-cresol.

2. Incubate the mixture for 2 hours at room temperature.

3. Dissolve protein sample in the above solution to a final concentration of 5-10 mg/ml. Flush the tube with a stream of nitrogen to displace oxygen, cap the tube, and incubate for 24 hours at room temperature in the dark.

4. Terminate the reaction by adding H2O (~10 volumes) and dry using a SpeedVac concentrator (or equivalent). Alternatively, obtain the peptides by applying the digest directly to a size-exclusion column or run on the gel (adjust the PAA % and running buffer accordingly to the expected sizes of the cleaved peptides).

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DISCUSSION

Selective cleavage of a protein at tryptophan, a relatively rare amino acid, generates a distinctive set of large peptide fragments. The yield of cleavage is moderate to high (up to 80%) and, besides some cleavage at tyrosine residues, there are very few side reactions. This protocol has been adapted from the method of Mahoney et al. (1981). For a detailed review of other, albeit less specific, procedures for cleaving proteins at tryptophan, see Fontana and Gross (1986).

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REFERENCES

↵ Fontana A., Gross E. (1986) in Practical protein chemistry, Fragmentation of polypeptides by chemical methods, ed Darbyre A (Wiley, Chichester, UK), pp 67–120. Search Google Scholar

↵ Mahoney W.C., Smith P.K., Hermodson M.A. (1981) Fragmentation of proteins with o-iodosobenzoic acid: Chemical mechanisms and identification of o-iodosobenzoic acid as a reactive contaminant that modifies tyrosyl residues. Biochemistry 20:443–448. eLinkMedline

Hi OLeg,

thanks a lot for the clear protocol

does cleavage leaves any residues at the cleaved fragments ???