Protein Protocols Protein Protocols

Disulfide-Linked Peptide Detection by HPLC 581
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Detection of Disulfide-Linked Peptides by HPLC
Alastair Aitken and Michèle Learmonth
Introduction
Classical techniques for determining disulfide bond patterns usually require the fragmentation
of proteins into peptides under low pH conditions to prevent disulfide
exchange. Pepsin or cyanogen bromide are particularly useful (see Chapters 76 and 71
respectively).
Diagonal techniques to identify disulphide-linked peptides were developed by
Brown and Hartley (see Chapter 87). A modern micromethod employing reverse-phase
high-performance liquid chromatography (HPLC) is described here.
2. Materials
1. 1 M Dithiothreitol (DTT, good quality, e.g., Calbiochem).
2. 100 mM Tris-HCl, pH 8.5.
3. 4-Vinylpyridine.
4. 95% Ethanol.
5. Isopropanol.
6. 1 M triethylamine-acetic acid, pH 10.0.
7. Tri-n-butyl-phosphine (1% in isopropanol).
8 HPLC system.
9. Vydac C4, C8, or C18 reverse phase HPLC columns (see Note 1).
3. Method (see refs. 1 and 2)
1 Alkylate the protein (1–10 mg in 20–50 mL of buffer) without reduction to prevent
possible disulfide exchange by dissolving in 100 mM Tris-HCl, pH 8.5, and adding 1 µL
of 4-vinylpyridine (see Note 2).
2 Incubate for 1 h at room temperature and desalt by HPLC or precipitate with 95% ice-cold
ethanol followed by bench centrifugation.
3 The pellet obtained after the latter treatment may be difficult to redissolve and may require
addition of 10-fold concentrated acid (HCl, formic, or acetic acid) before digestion at low
pH. It may be sufficient to resuspend the pellet with acid using a sonic bath if necessary,
then commence the digestion. Vortex-mix the suspension during the initial period until
the solution clarifies.
4 Fragment the protein under conditions of low pH (see Note 3) and subject the peptides
from half the digest to reverse-phase HPLC. Vydac C4, C8, or C18 columns give particu-
From: The Protein Protocols Handbook, 2nd Edition
Edited by: J. M. Walker © Humana Press Inc., Totowa, NJ
581