Biology Reference
In-Depth Information
gel plug contamination and to minimize the chance of an
accidental removal of small gel plugs ( 12 ).
7.
In-gel digestion protocols vary. For example, a Sigma-Aldrich
protocol ( 10 ) recommends initially adding 20
L
trypsin solution prepared in a trypsin reaction buffer containing
40 mM ABC/9% ACN, followed by another 50
μ
L of a 20-ng/
μ
L aliquot
of the trypsin reaction buffer for a 4-h to an overnight diges-
tion at 37°C. According to this protocol, the ABC/trypsin
solution may be stored as frozen aliquots for up to 4 weeks.
A digestion protocol from Pierce ( 8 ) requires initial incubation
of a dry gel piece with a minimum volume (10-20
μ
L) of a
trypsin solution that allows the gel piece to fully swell and
hydrate at room temperature, which is then followed by the
addition of 25
μ
L of a digestion buffer. Digestion is performed
either for 4 h at 37°C or at 30°C overnight. In a similar protocol
( 11 ), dried gel pieces with or without prior cysteine alkylation
are rehydrated with 20
μ
L of sequencing-grade
modifi ed trypsin (Promega) or with 1.5 times gel volume if it
is greater than 20
μ
L of 0.02
μ
g/
μ
L, in 40 mM ABC/10% ACN for 1 h at
room temperature. An additional 50
μ
L of 40 mM ABC/10%
ACN is then added, and the digestion continues for 16-18 h
with agitation. Additional cysteine reduction and alkylation
steps have been recommended in a 1D PAGE LC-MS approach
for processing of silver-stained gel spots or for de novo sequenc-
ing (
μ
13 ). This step might be omitted to minimize the risk of
protein loss and contamination, particularly if the reduction
(dithiothreitol) and alkylation (iodoacetamide) are routinely
performed during IPG strip equilibration in an SDS buffer
prior to running the second dimension of a 2D gel. Thorough
S-S bond reduction and alkylation are recommended before
the second dimension in order to block the cysteine active thiol
groups and prevent gel procedure artifacts, e.g., acrylamide
adducts and gel streaking due to intra- and extramolecular pro-
tein covalent bonding.
8.
There are several widely used peptide extraction procedures.
In this laboratory, a robust extraction procedure utilizing C18
ZipTip purifi cation is often used. Importantly, it avoids tube-
to-tube transfers of peptide solutions and associated sample
losses. The procedure starts with an addition of 5
L of 3%
aqueous TFA to stop the digestion and to facilitate peptide
extraction from the gel plugs. The extraction is carried out at
37°C on a thermomixer (300 rpm), and the peptide-containing
solution is then loaded directly onto the primed and wet C18
ZipTips (either regular or microsize according to the estimated
peptide amount) following the ZipTip manufacturer's procedure.
The peptide-depleted solution is then returned back to the
original container of the gel piece, a microtube, for a second
μ
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