Protein Protocols Protein Protocols

Enzymatic Digestion of Membrane-Bound Protein 525
acts as both a reagent for peptide extraction and a blocking reagent for preventing
enzyme adsorption to the membrane during digestion. Remaining cystine bonds are
reduced with dithiotreitol (DTT) and carboxyamidomethylated with iodoacetamide.
After incubation with the enzyme of choice, the peptides are recovered in the digestion
buffer. Further washes of the membrane remove the remaining peptides, which can be
analyzed by microbore HPLC. Purified peptides can then be subjected to automated
sequence analysis.
Additional studies have been reported that have enhanced this procedure. Best et al.
(8) have reported that a second aliquot of enzyme several hours later improves the
yield of peptides. Reduction and alkylation of cysteine is possible directly in the digestion
buffer, allowing identification of cysteine during sequence analysis (9). Finally,
octyl- or decylglucopyranoside can be substituted for RTX-100 in order to obtain
cleaner mass spectrometric analysis of the digestion mixture (10).
2. Materials
The key to success with this procedure is cleanliness. Use of clean buffers, tubes,
and staining/destaining solutions, as well as using only hydrogenated Triton X-100 as
opposed to the nonhydrogenated form, greatly reduces contaminant peaks obscured
during reverse-phase HPLC. A corresponding blank piece of membrane must always
be analyzed at the same time as a sample is digested, as a negative control. Contaminants
can occur from may sources and particularly protein contaminants are a cocnern.
Human Kertain may contaminate samples if gloves are not worn. Proteins from Western
blotting can contaminate the PVDF membrane if previously used dishes are used
for staining. UV absorbing contaminants can arise from dirty tubes and sub quality
detergents. All solutions should be prepared with either HPLC-grade water or doubleglass-distilled
water that has been filtered through an activated charcoal filter, and passed
through a 0.22-µm filter (11).
2.1. Preparation of the Membrane-Bound Sample
Protein should be analyzed by SDS-PAGE or 2D-IEF using standard laboratory techniques.
Electrophoretic transfer of proteins to the membrane should be performed in a
full immersion tank rather than a semidry transfer system to avoid sample loss and
obtain efficient transfer (12). PVDF membranes with higher protein binding capacity
such as Immobilon Psq (Millipore, Bedford, MA), Problott (Applied Biosystems, Foster
City, CA), and Westran (Bio-Rad, Hercules, CA), are preferred owing to greater
protein recovery on the blot, although all types of PVDF and nitrocellulose can be used
with this procedure. The following stains are compatible with the technique: Ponceau
S, Amido black, india ink, and chromatographically pure Coomassie brilliant blue with
a dye content >90%. A blank region of the membrane should be excised to serve as
a negative control.
2.2. Enzymatic Digestion Buffers
Digestion buffer should be made as described in Table 1. Make up 1 mL of buffer at
a time and store at –20°C for up to 1 wk. Hydrogenated Triton X-100 (RTX-100) (protein
grade, cat. # 648464, Calbiochem, LaJolla, CA) is purchased as a 10% solution,
which should be stored at –20°C. Note: Only hydrogenated Triton X-100 should be used