the sample will reach the trap column and several volumes of
the trap column will pass through to achieve efficient desalting.
If guanidine or phosphates are used in sample preparation,
make the desalting slightly longer. This step usually lasts from
1 to 4 min, depending on the flow and volumes mentioned
above.
17. Duration of the gradient and its slope depend on the column
and the LC system used. In general, the gradient should be
fast, lasting just a few minutes. However, during this short
period, the most efficient elution should be achieved. Therefore, gradient running from 5 or 10% acetonitrile (solvent B)
and ending at 25–35% acetonitrile should be used. In specific
cases, the upper acetonitrile percentage can be raised to 40 or
50% or even higher.
18. For example, here the data are processed in DataAnalysis using
Find>Compounds AutoMS(n) feature and exported into a
MASCOT generic file (*.mgf). Two different algorithms are
used for peak picking (FTMS and SNAP) and thus two *.mgf
files are created for each analysis. The whole procedure can be
automated using Visual Basic macros in Data Analysis. These
macros can be obtained from the authors.
19. If the coverage map shows many gaps and the covered parts are
represented by short peptides, the protein was probably overdigested. Faster flow (even in hundreds of microliters per
minute) should be tested. One should, however, keep in
mind the trapping efficiency of the trap column at a given
flow rate. Alternatively, another protease may be used. These
steps are also advisable if higher redundancy is desired. In cases
where the peptides are too long, temperature should be
increased or addition of a denaturing agent considered. Use
of alternative aspartic proteases beside pepsin is advantageous
for DNA binding proteins since they tend to be rich in basic
residues. While pepsin exerts nearly no cleavage after Lys, His,
and Arg residues, the other proteases like nepenthesins, aspergillopepsin, and rhizopuspepsin cleave at these sites with high
frequency [17, 24, 26]. These proteases may thus yield good
spatial resolution of basic proteins or basic protein regions.
20. It is often stated that the use of robotics leads to higher
reproducibility; however, trained user and careful pipetting
can lead to very similar outcomes. The main advantage in
using automation is the ability to perform the analysis in an
unattended manner and thus using the instrument time more
efficiently. Most importantly, the manual way utilizes freezing
of samples and their subsequent thawing. These steps require
additional 0.5–2 min, during which the sample undergoes
back-exchange or (in the case of low D 2 O dilution by the
214
Ruzena Filandrova et al.
the trap column will pass through to achieve efficient desalting.
If guanidine or phosphates are used in sample preparation,
make the desalting slightly longer. This step usually lasts from
1 to 4 min, depending on the flow and volumes mentioned
above.
17. Duration of the gradient and its slope depend on the column
and the LC system used. In general, the gradient should be
fast, lasting just a few minutes. However, during this short
period, the most efficient elution should be achieved. Therefore, gradient running from 5 or 10% acetonitrile (solvent B)
and ending at 25–35% acetonitrile should be used. In specific
cases, the upper acetonitrile percentage can be raised to 40 or
50% or even higher.
18. For example, here the data are processed in DataAnalysis using
Find>Compounds AutoMS(n) feature and exported into a
MASCOT generic file (*.mgf). Two different algorithms are
used for peak picking (FTMS and SNAP) and thus two *.mgf
files are created for each analysis. The whole procedure can be
automated using Visual Basic macros in Data Analysis. These
macros can be obtained from the authors.
19. If the coverage map shows many gaps and the covered parts are
represented by short peptides, the protein was probably overdigested. Faster flow (even in hundreds of microliters per
minute) should be tested. One should, however, keep in
mind the trapping efficiency of the trap column at a given
flow rate. Alternatively, another protease may be used. These
steps are also advisable if higher redundancy is desired. In cases
where the peptides are too long, temperature should be
increased or addition of a denaturing agent considered. Use
of alternative aspartic proteases beside pepsin is advantageous
for DNA binding proteins since they tend to be rich in basic
residues. While pepsin exerts nearly no cleavage after Lys, His,
and Arg residues, the other proteases like nepenthesins, aspergillopepsin, and rhizopuspepsin cleave at these sites with high
frequency [17, 24, 26]. These proteases may thus yield good
spatial resolution of basic proteins or basic protein regions.
20. It is often stated that the use of robotics leads to higher
reproducibility; however, trained user and careful pipetting
can lead to very similar outcomes. The main advantage in
using automation is the ability to perform the analysis in an
unattended manner and thus using the instrument time more
efficiently. Most importantly, the manual way utilizes freezing
of samples and their subsequent thawing. These steps require
additional 0.5–2 min, during which the sample undergoes
back-exchange or (in the case of low D 2 O dilution by the
214
Ruzena Filandrova et al.