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experiments.
3
They used 2% formic acid and
a heating block to maintain the temperature
above 108
C to support reactions that proceeded
up to four hours. In the next several years, labo-
ratories reported that the temperature could be
easily achieved and maintained using micro-
wave technology, a technique that had already
been adopted by synthetic organic chemists.
Experiments utilizing microwave irradiation
were found to allow proteolysis to be achieved
in minutes, rather than hours.
4,5
Subsequently,
microwave-supported Asp-selective acid prote-
olysis has been successfully incorporated into
different proteomic work
formation of N-terminal pyroglutamate, for-
mylation, and occasionally dehydrated cyclic
intermediates. All of these are readily accommo-
dated by proteomic search engines. Comparisons
have demonstrated that the use of formic acid
resulted in formylation, a phenomenon that was
not observed with acetic acid.
20
Remily-Wood
and colleagues demonstrated that Asp-selective
hydrolysis can be catalyzed by acidic matrices
used for matrix-assisted laser desorption ioniza-
tion (MALDI) mass spectrometry.
20
Stronger
acids, such as tri
uoroacetic acid, have been
used at higher concentrations and lead to non-
speci
ows.
c cleavage, presumably with a higher pro-
portion of hydronium-catalyzed proteolysis.
20,21
Generally, longer incubation times are
required to accommodate more complex
samples. Additionally, the best results are
obtained when total protein concentration is
around 0.1 mg/mL, regardless of sample
complexity. Addition of dithiothreitol to the
reaction vessel has been shown to reduce disul-
ASPARTATE-SELECTIVE ACID
PROTEOLYSIS
Two generally accepted mechanisms are illus-
trated in
Figure 1
that allow proteolysis to occur
on the C-terminal and N-terminal sides of each
Asp residue. In these, the aspartic acid side chain
cyclizes to form 6- or 5-membered rings with
carbonyl groups adjacent on either side. Hydrol-
ysis of the
fide bonds in proteins that undergo proteol-
ysis.
9,14,16,22
O-linked glycosidic side chains and
phosphate groups are hydrolyzed in the hot
acid.
16,18
five-membered ring is favored in
model systems,
6,7
supporting observations
from Inglis and others that peptides containing
Asp residues on the carboxy terminus are
formed in higher abundance.
This reaction has been shown to proceed with
a variety of acids,
5,8,9
consistent with the hypoth-
esis that protonation of the Asp side chain is
required. According to Inglis
MICROWAVE-SUPPORTED ASP-
SE
LECTIVE ACID PROTEOLY
SIS
Microwaves are a nonionizing form of electro-
magnetic radiation, which provide insuf
cient
energy for molecular rearrangement. Under the
in
s initial character-
ization of the reaction, optimal conditions were
de
'
uence of electromagnetic radiation, dipoles
will attempt to align themselves with the rapidly
oscillating electric
ned as pH 2.0 (both HCl and formic acid
were used to achieve this condition), temperature
of 108
C, and a two-hour incubation period.
2
Hydrochloric and formic acid also act to denature
proteins, which facilitates protonation and prote-
olysis. Most proteomics laboratories use formic
acid or acetic acid at concentrations between
0.05%and 12.5%,
4,5,8,10
e
19
all resulting in peptides
cleavedat the expected cleavage sites. Several side
reactions have been observed
d
speci
field. This constant dipolar
rotation is the source of dielectric heating. Addi-
tionally, microwave energy absorption and
subsequent heating is dependent on the proper-
ties of the solvent.
23
The physics of microwave
energy absorption and mechanisms of reaction
catalysis are beyond the scope of this chapter;
a detailed review of microwave-supported
chemistry is found in Lidström and colleagues.
23
cally,
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