improvement of the analysis software to see and characterize peptide fragmentations [14, 15]. This method can be divided into two
groups (Fig. 1): (1) acquire simultaneously the fragmentation spectra of the all range mass (shotgun) and (2) make an m/z scan in
sequential isolation windows [14, 16] (SRM/MRM). Both methods are coupled with the same data analysis processing [17].
More recently, the combination between SWATH-MS
(sequential window acquisition of all theoretical mass spectra) and
DIA-MS allows not only a direct searching of individual sample
proteins but also gives a protein quantification between different
sample groups [1, 18]. Therefore, this method combines the targeted data extraction from DIA-MS files from previously identified
proteins, which seems an adaptation from methodologies such as
MRM (multiple reaction monitoring) or PRM (parallel reaction
monitoring) but using higher-resolution mass analysis [1] to perform a more accurate protein quantification. This technology has
been proposed as an emergent and alternative proteomics technology that has a good reproducible and accurate quantification of
more than a 100 simultaneous proteins [19] even in complex
samples (Figs. 1 and 2). This methodology is the most effective
for protein identification. It is extremely valuable for translational
research by allowing the identification of biomarkers that can be
used to distinguish healthy from diseased conditions [1].
SWATH-MS analysis needs to perform two processes (Fig. 2)
[1]. Generate a good quality MS-spectral library. This process is
generally made by making one pooled sample for each group of
experimental samples and run these samples in DDA-MS acquisition mode. These libraries contain all the peptide information such
as retention time (t R ), precursor m/z, and MS/MS spectra. This
information gives the chromatographic elution traces from a group
of specific fragment ions for each confidence peptide. Moreover, it
contains other information such as charge state and relative intensity that can be used to evaluate the peptide identified confidence
[1] to improve the identified protein FDR (false discovery
rate) [20].
The good quality of this library is the most important factor in
the correct quantification process. Once this high-quality library is
created, samples are individually run in a DDA-MS (IDA-MS)
acquisition mode to quantify the proteins identified in the library
(Fig. 3).
This methodology involves a process with a repeat acquisition
of fragment ion spectra from the ion precursors. The windows of
isolation will be 25 kDa [16], and quantification will be performed
by the area under the curve (AUC) obtained from ten peptides by
protein and seven transitions by peptide [21]. All these characteristics make this new technology a right tool for disease biomarker
discovery [1, 22].
SWATH-MS Protocols in Human Diseases
107
groups (Fig. 1): (1) acquire simultaneously the fragmentation spectra of the all range mass (shotgun) and (2) make an m/z scan in
sequential isolation windows [14, 16] (SRM/MRM). Both methods are coupled with the same data analysis processing [17].
More recently, the combination between SWATH-MS
(sequential window acquisition of all theoretical mass spectra) and
DIA-MS allows not only a direct searching of individual sample
proteins but also gives a protein quantification between different
sample groups [1, 18]. Therefore, this method combines the targeted data extraction from DIA-MS files from previously identified
proteins, which seems an adaptation from methodologies such as
MRM (multiple reaction monitoring) or PRM (parallel reaction
monitoring) but using higher-resolution mass analysis [1] to perform a more accurate protein quantification. This technology has
been proposed as an emergent and alternative proteomics technology that has a good reproducible and accurate quantification of
more than a 100 simultaneous proteins [19] even in complex
samples (Figs. 1 and 2). This methodology is the most effective
for protein identification. It is extremely valuable for translational
research by allowing the identification of biomarkers that can be
used to distinguish healthy from diseased conditions [1].
SWATH-MS analysis needs to perform two processes (Fig. 2)
[1]. Generate a good quality MS-spectral library. This process is
generally made by making one pooled sample for each group of
experimental samples and run these samples in DDA-MS acquisition mode. These libraries contain all the peptide information such
as retention time (t R ), precursor m/z, and MS/MS spectra. This
information gives the chromatographic elution traces from a group
of specific fragment ions for each confidence peptide. Moreover, it
contains other information such as charge state and relative intensity that can be used to evaluate the peptide identified confidence
[1] to improve the identified protein FDR (false discovery
rate) [20].
The good quality of this library is the most important factor in
the correct quantification process. Once this high-quality library is
created, samples are individually run in a DDA-MS (IDA-MS)
acquisition mode to quantify the proteins identified in the library
(Fig. 3).
This methodology involves a process with a repeat acquisition
of fragment ion spectra from the ion precursors. The windows of
isolation will be 25 kDa [16], and quantification will be performed
by the area under the curve (AUC) obtained from ten peptides by
protein and seven transitions by peptide [21]. All these characteristics make this new technology a right tool for disease biomarker
discovery [1, 22].
SWATH-MS Protocols in Human Diseases
107