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O
OH
H
N
H
N
H
N
R
R'
R
OH
H 2 O
O
OH
O
O
O
Pathway A
O
+
X-Asp
O
NH2
O
R'
H
N
N
H
R'
R
O
Asp-X
Pathway B
OH
O
R'
O
N
H
NH2
R'
N
H
HO
HN
H 2 O
HO
+
R
O
O
O
O
R
OH
FIGURE 1 Proposed mechanisms of acid hydrolysis. (Modi ed from reference #3.)
Microwave-supported acid hydrolysis has
been incorporated in line with chromatographic
separation. 14 Proteins in 12.5% formic acid were
exposed to microwave irradiation for 5 minutes
and 130 C via a 5
an increase
4.5%) in average sequence
coverage of proteins when compared to
microwave-supported acid hydrolysis alone. 11
In addition to digestion in solution, protein
digestion in polyacrylamide gels is an important
part of many modern proteomic work
(
รพ
L reaction loop at a
ow
m
rate of 1
L/min. The optimized method was
utilized for analysis of an E. coli lysate in which
ten proteins were successfully identi
ows.
The initial examples in the literature of
SDS-PAGE combined with in-gel microwave-
supported acid hydrolysis 3,4,9 all apply the
method to well resolved gel bands or to protein
standards. The relatively slow development of
this in-gel method may be due to reduced
recoveries of longer peptide products from the
polyacrylamide matrix. Hua and colleagues
achieved 46% sequence coverage of the stan-
dard protein, bovine serum albumin, and
observed that most of the sequence not identi-
m
ed by
LC-MS/MS. Basile and colleagues have also
reported the combination of microwave-
supported acid hydrolysis with electrochemical
oxidation. 11 The electrochemical method is
reported to cleave selectively at Trp and Tyr resi-
dues. Electrochemical oxidation has been shown
to be reproducible only on polypeptides that
weigh less than 3.4 kDa, while microwave-
supported acid hydrolysis was
thought
to
produce peptides that were too long for ef
cient
ion trap and quadrupole CID fragmentation
methods. Combining the two proteolytic
methods for E. coli lysate analysis resulted in
fied was from peptides with masses greater
than 4,500 Da. 4 Cannon has demonstrated
in-gel digestion of a whole-cell lysate from
multiple myeloma cancer cells. 25
Following
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