It is important to mention that different instruments have
differences in peptide fragmentation patterns and differences in
LC-MS runs (nano or micro flow, different LC columns pore size,
etc.). Then, if libraries created by a different mass spectrometer are
used, there will probably present some inconvenience in the posterior SWATH-MS analysis. The best option is to use IDA data
acquired using the same instrument used to perform the
SWATH-MS/DIA-MS analysis [128].
Therefore, spectral ion libraries can be generated in several
ways:
1. Analyze one single run of pooled sample created from peptides,
obtained by proteolytic digestion of all the individual samples
in DDA/IDA mode. Using this method, it is possible to obtain
a good representation of all peptides present in all samples,
which can be used in the SWATH-MS analysis.
2. Use multiple runs or technical replicates of the pooled samples.
This may improve the proteome coverage provided to the
sample library obtained from one single run.
3. In an attempt to increase the proteome coverage, even more, it
is possible to perform the ion library using previous protein/
peptide fractionation (in-gel fractionation for proteins or
HPLC fractionation for peptides) [9] and then perform the
analysis of these fractions in DDA/IDA mode. This may
increase the power of DIA-based protein identifications
because this previous fractionation increases considerably the
number of peptides used to identify the major proteins and
increase the sensitivity of MS2-based quantization for
low-abundance proteins and peptides. The methods more
commonly used for protein fractionation are (1) 1D gels
where the proteins are fractionated in accordance with their
molecular weight and (2) 2D-gels where the proteins are first
separated by their isoelectric point in the first dimension and
then in the second dimension the proteins are separated by
their molecular weight. The methods more commonly used
for peptide fractionation are [129] (1) reverse-phase HPLC
[130], (2) strong cation exchange (SCX), and (3) strong
anion exchange (SAX). In our work, the in-gel method for
protein fractionation is usually used, as other groups [131]
prior to LC and MS analysis.
In the analysis of posttranslational modifications by SWATHMS, as in the shotgun analysis, it is recommended to use enrichment strategies that can be coupled if necessary with peptide fractionation techniques.
SWATH-MS Protocols in Human Diseases
131
differences in peptide fragmentation patterns and differences in
LC-MS runs (nano or micro flow, different LC columns pore size,
etc.). Then, if libraries created by a different mass spectrometer are
used, there will probably present some inconvenience in the posterior SWATH-MS analysis. The best option is to use IDA data
acquired using the same instrument used to perform the
SWATH-MS/DIA-MS analysis [128].
Therefore, spectral ion libraries can be generated in several
ways:
1. Analyze one single run of pooled sample created from peptides,
obtained by proteolytic digestion of all the individual samples
in DDA/IDA mode. Using this method, it is possible to obtain
a good representation of all peptides present in all samples,
which can be used in the SWATH-MS analysis.
2. Use multiple runs or technical replicates of the pooled samples.
This may improve the proteome coverage provided to the
sample library obtained from one single run.
3. In an attempt to increase the proteome coverage, even more, it
is possible to perform the ion library using previous protein/
peptide fractionation (in-gel fractionation for proteins or
HPLC fractionation for peptides) [9] and then perform the
analysis of these fractions in DDA/IDA mode. This may
increase the power of DIA-based protein identifications
because this previous fractionation increases considerably the
number of peptides used to identify the major proteins and
increase the sensitivity of MS2-based quantization for
low-abundance proteins and peptides. The methods more
commonly used for protein fractionation are (1) 1D gels
where the proteins are fractionated in accordance with their
molecular weight and (2) 2D-gels where the proteins are first
separated by their isoelectric point in the first dimension and
then in the second dimension the proteins are separated by
their molecular weight. The methods more commonly used
for peptide fractionation are [129] (1) reverse-phase HPLC
[130], (2) strong cation exchange (SCX), and (3) strong
anion exchange (SAX). In our work, the in-gel method for
protein fractionation is usually used, as other groups [131]
prior to LC and MS analysis.
In the analysis of posttranslational modifications by SWATHMS, as in the shotgun analysis, it is recommended to use enrichment strategies that can be coupled if necessary with peptide fractionation techniques.
SWATH-MS Protocols in Human Diseases
131