that can be fine-tuned by the final user are mass ranges of the
analysis in both MS and MS/MS experiments together with all
specific source settings, which may be specific for every instrument
and chromatographic conditions. All methods were created using
the Acquisition software provided by Sciex.
3.6.4 SWATH-MS Data
Analysis Using PeakView
2.2 and SWATH-MS
Microapp 2.0 (See Note
10)
Introduction to SWATH-MS
Data Analysis Procedure
There are several software options for data processing and visualization, although, for practical reasons, we only describe the workflows provided by the manufacturer that in other publications have
also been described as one of the best choices [132]. Briefly, data
are imported into PeakView 2.2 software and aligned to available
libraries using a “SWATH quantitation” plug-in. During data
alignment, the algorithm integrated into the software, first do a
Data Extraction and second Data Scoring. For Data Extraction, the
software uses the scoring information present in the library, aiming
to avoid using low score or no proteotypic peptides. Once this filter
gets passed, peptides are taken according to its intensity. Regarding
fragments, three different tiers are applied for fragment selection:
first, fragments above Q1 window starting with the most intense;
second, fragments below Q1 window above y3/b3, and lastly,
fragments in Q1 window. At the moment, only “b” or “y” ions
are used for processing.
For data validation, a Scoring is also applied at both chromatographic and MS levels. At chromatographic level, all the
extracted ion chromatograms for every fragment and peptide are
grouped in the so-called peptide groups. Within these groups,
consistency of Peak areas, Peak apex, and Peak width at half maximum is measured. At MS level, mass accuracy and internal ion
ratios are used after background subtraction. All the information
is integrated by the algorithm to calculate a score that is used to
determine the rate of false positives. The whole process largely
facilitates the identification of inconsistencies that could fake the
final results.
Other alternatives to this software are Skyline [133], Open
SWATH [122, 134], and Spectronaut.
Here, we want to show how we process the data using Sciex
software tools. We recommend using the Sciex software user manuals for additional guidance.
Creation of Spectral Ion
Library Using Protein Pilot
Paragon Method
1. Download the protein reference database necessary for your
samples from UniProt (http://www.uniprot.org/proteomes)
as a FASTA document. Typically, we use the revised database
for our organism of interest. FASTA proteome databases
should be saved in the database folder in the ProteinPilot
software files on your computer.
2. In Protein Pilot, select the option for an LC-MS search and
include all of the raw data files from the library runs you would
like to include to build the ion library. Select the database and
SWATH-MS Protocols in Human Diseases
133
analysis in both MS and MS/MS experiments together with all
specific source settings, which may be specific for every instrument
and chromatographic conditions. All methods were created using
the Acquisition software provided by Sciex.
3.6.4 SWATH-MS Data
Analysis Using PeakView
2.2 and SWATH-MS
Microapp 2.0 (See Note
10)
Introduction to SWATH-MS
Data Analysis Procedure
There are several software options for data processing and visualization, although, for practical reasons, we only describe the workflows provided by the manufacturer that in other publications have
also been described as one of the best choices [132]. Briefly, data
are imported into PeakView 2.2 software and aligned to available
libraries using a “SWATH quantitation” plug-in. During data
alignment, the algorithm integrated into the software, first do a
Data Extraction and second Data Scoring. For Data Extraction, the
software uses the scoring information present in the library, aiming
to avoid using low score or no proteotypic peptides. Once this filter
gets passed, peptides are taken according to its intensity. Regarding
fragments, three different tiers are applied for fragment selection:
first, fragments above Q1 window starting with the most intense;
second, fragments below Q1 window above y3/b3, and lastly,
fragments in Q1 window. At the moment, only “b” or “y” ions
are used for processing.
For data validation, a Scoring is also applied at both chromatographic and MS levels. At chromatographic level, all the
extracted ion chromatograms for every fragment and peptide are
grouped in the so-called peptide groups. Within these groups,
consistency of Peak areas, Peak apex, and Peak width at half maximum is measured. At MS level, mass accuracy and internal ion
ratios are used after background subtraction. All the information
is integrated by the algorithm to calculate a score that is used to
determine the rate of false positives. The whole process largely
facilitates the identification of inconsistencies that could fake the
final results.
Other alternatives to this software are Skyline [133], Open
SWATH [122, 134], and Spectronaut.
Here, we want to show how we process the data using Sciex
software tools. We recommend using the Sciex software user manuals for additional guidance.
Creation of Spectral Ion
Library Using Protein Pilot
Paragon Method
1. Download the protein reference database necessary for your
samples from UniProt (http://www.uniprot.org/proteomes)
as a FASTA document. Typically, we use the revised database
for our organism of interest. FASTA proteome databases
should be saved in the database folder in the ProteinPilot
software files on your computer.
2. In Protein Pilot, select the option for an LC-MS search and
include all of the raw data files from the library runs you would
like to include to build the ion library. Select the database and
SWATH-MS Protocols in Human Diseases
133