Biology Reference
In-Depth Information
Microwaves are electromagnetic radiation
with a wavelength between one millimeter and
one meter. The speci
Ribosomal and other basic proteins contain
high numbers of Arg and Lys residues, which
makes them poor candidates for proteomic anal-
ysis based on tryptic digestion, because many of
the peptide products are too small for routine
proteomic analysis.
Figure 2
illustrates small
(red) and large (teal) peptides predicted for
each ribosomal protein and the protein coverage
each peptide provides. The bottom graph
predicts peptides produced by tryptic digestion
and the top graph shows peptides produced by
Asp-selective acid digestion.
The chemical properties of peptides resulting
from Asp-selective acid hydrolysis are distinct
from those that result from tryptic digestion.
Tryptic speci
c frequency of 2.5 GHz
has been most commonly used to support chem-
ical reactions. Initial work from this laboratory
utilized a domestic microwave oven operated
at 745 W
5
; however, later work from this labora-
tory and others
8,10
e
15
advanced to research-
grade microwave devices with tunable power
settings. Although Asp-selective acid digestion
has been achieved in domestic microwave
ovens, and with thermal heating,
3,4,9,20
micro-
wave instruments designed speci
cally for
chemical
ts.
Domestic microwaves irradiate in a nonuniform
manner. Although this isn
reactions have signi
cant bene
t an issue for thawing
food, which usually occupies a signi
'
city on the C terminus side of
Lys and Arg results in a basic site at the C
terminus of every peptide. Protonation of the
C-terminal residue and also the basic amino
terminus typically results in product ion spectra
well suited to high-throughput database search-
ing.
24
Swatkoski and colleagues demonstrated
that product ion spectra of peptides lacking
a C-terminal basic residue can still be interpreted
and searched successfully against databases.
Interestingly, in the
S. cerevisiae
ribosomal
sample, the majority of peptides produced con-
tained two or more internal basic sites, which
bolstered sensitivity for MS nanospray analysis.
Swatkoski and colleagues
18
appraised the reac-
tion by performing bioinformatic searches on
peptides without designating any enzymatic
speci
cant
amount of space in a domestic microwave, one
can imagine that the microscale sample volumes
typically used in proteomics would require
more focused microwave exposure. Commercial
microwave devices have been designed for
focused irradiation within a con
ned area and
uniform energy distribution. Additionally,
research-grade microwave ovens allow the
temperature to be regulated to
5
C.
METHODS DEVELOPMENT WITH
MICROWAVE-SUPPORTED ACID
HYDROLYSIS
Following effective demonstrations of residue
selective microwave-supported acid hydrolysis
on standard proteins and on bacteria, Swatkoski
and colleagues evaluated its application in both
MALDI and electrospray-based proteomic anal-
yses of a complex mixture.
8
city, which allowed evaluation of cleav-
age speci
city based on fragmentation data,
a stronger basis for identi
cation compared to
methods applied previously. In general agree-
ment with the results obtained by Li and
colleagues,
3
it was observed that 70% of the total
peptides were formed with the expected
cleavage C or N terminal to Asp. Mascot and
some other search programs now offer the
option of designating acid cleavage (
In this experiment,
ribosomes were puri
ed from
S. cerevisiae
and
digested for twenty minutes in 12.5% acetic
acid at 140
5
C. In an automated high-
throughput LC-MS/MS analysis of the same
sample, 247 peptides were identi
“
formic
ed repre-
senting 58 of the 78 expected yeast ribosomal
proteins.
18
acid
in Mascot) as a parameter, and accommo-
date the possibilities of cleavage on either side
of Asp residues.
”
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