Protein Protocols Protein Protocols

582 Aitken
larly good resolution depending on the size range of fragments produced. Typical separation
conditions are;- column equilibrated with 0.1% (v/v) aqueous trifluoro acetic acid
(TFA) , elution with an acetonitrile–0.1% TFA gradient. A combination of different
cleavages, both chemical and enzymatic, may be required if peptide fragments of interest
remain large after one digestion method.
5 To the other half of the digest (dried and resuspended in 10 µL of isopropanol) add 5 µL
of 1 M triethylamine-acetic acid pH 10.0; 5 µL of tri-n-butyl-phosphine (1% in
isopropanol) and 5 µL of 4-vinylpyridine. Incubate for 30 min at 37°C, and dry in vacuo,
resuspending in 30 µL of isopropanol twice. This procedure cleaves the disulfides and
modifies the resultant –SH groups.
6 Run the reduced and alkylated sample on the same column, under identical conditions on
reverse-phase HPLC. Cysteine-linked peptides are identified by the differences between
elution of peaks from reduced and unreduced samples.
7 Collection of the alkylated peptides (which can be identified by rechromatography on
reverse-phase HPLC with detection at 254 nm) and a combination of sequence analysis
and mass spectrometry (see Note 1 and Chapter 86) will allow disulfide assignments
to be made.
4. Notes
1. If the HPLC separation is combined with mass spectrometric characterization, the level of
TFA required to produce sharp peaks and good resolution of peptide (approx 0.1% v/v)
results in almost or complete suppression of signal. This does not permit true on-line
HPLC-MS as the concentration of TFA in the eluted peptide must first be drastically
reduced. However, the new “low TFA,” 218MS54, reverse-phase HPLC columns from
Vydac (300 Å pore size) are available in C4 and two forms of C18 chemistries. They are
also supplied in 1 mm diameter columns that are ideal for low levels of sample eluted in
minimal volume. We have used as little as 0.005% TFA without major loss of resolution
and have observed minimal signal loss. There may be a difference in selectivity compared
to “classical” reverse-phase columns; for example, we have observed phosphopeptides
eluting approx. 1% acetonitrile later than their unphosphorylated counterparts (the opposite
to that conventionally seen). This is not a problem, but it is something of which one
should be aware, and could be turned to advantage.
2. The iodoacetic acid used must be colorless. A yellow color indicates the presence of
iodine; this will rapidly oxidize thiol groups, preventing alkylation and may also modify
tyrosine residues. It is possible to recrystallize from hexane. Reductive alkylation may
also be carried out using iodo-[ 14C]-acetic acid or iodoacetamide (see Chapter 59). The
radiolabelled material should be diluted to the desired specific activity before use with
carrier iodoacetic acid or iodoacetamide to ensure an excess of this reagent over total
thiol groups.
3. Fragmentation of proteins into peptides under low pH conditions to prevent disulfide
exchange is important. Pepsin, Glu-C, or cyanogen bromide are particularly useful (see
Chapters 76 and 71 respectively). Typical conditions for pepsin are 25°C for 1–2 h at
pH 2.0–3.0 (10 mM HCl, 5% acetic or formic acid) with an enzyme:substrate ratio of
about 1:50. Endoproteinase Glu-C has a pH optimum at 4 as well as an optimum at pH 8.0.
Digestion at the acid pH (typical conditions are 37°C overnight in ammonium acetate at
pH 4.0 with an enzyme/substrate ratio of about 1:50) will also help minimize disulfide
exchange. CNBr digestion in guanidinium 6 M HCl/ 0.1–0.2 M HCl may be more suitable
acid medium due to the inherent redox potential of formic acid which is the most
commonly used protein solvent. When analyzing proteins that contain multiple disulfide