A significant advance in SWATH-MS technology is that this
method can improve proteome coverage, reduce quantitative variability, and minimize missing data because it can enhance the number of identified peptides by protein, considerably. To perform this
kind of analysis, first, the proteins are digested and usually separated
by reverse-phase liquid chromatography (LC) before the mass
spectrometer analysis. In a Triple TOF mass spectrometer, precursor selection is made filtering precursors through mass-to-charge
windows (usually 4–10 m/z wide), sequentially across the entire
m/z range of interest. As the width of the isolation windows is so
great, there are possibilities that two or more co-eluting precursors
are fragmented at the same moment to give an MS2 spectrum,
which, in this case, can contain fragment ions from these multiple
precursor ions. To improve the ability to find and accurately identify these peptides, it is possible to associate specific peptides with
defined regions within the chromatographic elution profile. It is
made by using the retention time (RT) determination samples
alignment across all the chromatographic time. To perform this
alignment, it can use both exogenous commercial peptides [23] or
endogenous RT standards [24] that are composed of some peptides
that can be observed across all the RT elution profiles. On the other
side, it is necessary for the optimization of m/z window number,
and the dwell time/ion accumulation time per window. Both
things can be performed, making that the mass spectrometer cycles
through the entire desired precursor m/z range (e.g.,
400–1250 m/z). Therefore, this depends on the mass spectrometer
and the sample type. In the case of the Triple TOF, it is possible to
go until 2250 m/z, but normally, for tryptic digests, the mass
analyzed is performed between 400 and 1250 m/z [25].
Once it is performed, and the mass data are obtained, it is
searched against a peptide/protein library specific for a specific
experiment. Then, the transition ion chromatograms are extracted
for all the peptides. Using several factors, the peaks groups are
scored in order to establish if this peptide is “true” or “false”; it
means that the peptide is yes (false) or not (true) from spectral
noise. All these values are used to determine a specific cutoff score
that can ascertain an acceptable false discovery rate. The relative
peptide abundance is then joined for the extracted ion chromatograms (XICs) of each transition, and after that, statistical tools are
used to transform these values into peptide intensity estimations
and, consequentially, the protein intensity [25], given as a protein
quantification between groups.
1.1 SWATH-MS
Application in
Proteomics
Although SWATH-MS technology is a very recent technique [16],
there is a high number of references reporting the use of this
technology; see Anjo et al. [1]. Therefore, SWATH-MS is a translational and powerful technique that can be applied to the biomarker
search in translational investigations. This technology is able to see
SWATH-MS Protocols in Human Diseases
109
method can improve proteome coverage, reduce quantitative variability, and minimize missing data because it can enhance the number of identified peptides by protein, considerably. To perform this
kind of analysis, first, the proteins are digested and usually separated
by reverse-phase liquid chromatography (LC) before the mass
spectrometer analysis. In a Triple TOF mass spectrometer, precursor selection is made filtering precursors through mass-to-charge
windows (usually 4–10 m/z wide), sequentially across the entire
m/z range of interest. As the width of the isolation windows is so
great, there are possibilities that two or more co-eluting precursors
are fragmented at the same moment to give an MS2 spectrum,
which, in this case, can contain fragment ions from these multiple
precursor ions. To improve the ability to find and accurately identify these peptides, it is possible to associate specific peptides with
defined regions within the chromatographic elution profile. It is
made by using the retention time (RT) determination samples
alignment across all the chromatographic time. To perform this
alignment, it can use both exogenous commercial peptides [23] or
endogenous RT standards [24] that are composed of some peptides
that can be observed across all the RT elution profiles. On the other
side, it is necessary for the optimization of m/z window number,
and the dwell time/ion accumulation time per window. Both
things can be performed, making that the mass spectrometer cycles
through the entire desired precursor m/z range (e.g.,
400–1250 m/z). Therefore, this depends on the mass spectrometer
and the sample type. In the case of the Triple TOF, it is possible to
go until 2250 m/z, but normally, for tryptic digests, the mass
analyzed is performed between 400 and 1250 m/z [25].
Once it is performed, and the mass data are obtained, it is
searched against a peptide/protein library specific for a specific
experiment. Then, the transition ion chromatograms are extracted
for all the peptides. Using several factors, the peaks groups are
scored in order to establish if this peptide is “true” or “false”; it
means that the peptide is yes (false) or not (true) from spectral
noise. All these values are used to determine a specific cutoff score
that can ascertain an acceptable false discovery rate. The relative
peptide abundance is then joined for the extracted ion chromatograms (XICs) of each transition, and after that, statistical tools are
used to transform these values into peptide intensity estimations
and, consequentially, the protein intensity [25], given as a protein
quantification between groups.
1.1 SWATH-MS
Application in
Proteomics
Although SWATH-MS technology is a very recent technique [16],
there is a high number of references reporting the use of this
technology; see Anjo et al. [1]. Therefore, SWATH-MS is a translational and powerful technique that can be applied to the biomarker
search in translational investigations. This technology is able to see
SWATH-MS Protocols in Human Diseases
109