The website introduces the
procedures of preparing input data for our tools. The user guide is also
avaliable in pdf format. Download
User
Guide for Preparing Input Data
The quantitation tools developed by the Systems Proteomics Lab, Institute of
Information Science, Academia Sinica require spectral
data files (.mzXML) and search results as the input data files. In
the following, we introduce conversion of raw spectral data to mzXML files and
preparation of search results. The workflows for Mascot users and
PeptideProphet/ProteinProphet users are shown in the following figures.
Figure. Data preparation for Mascot
users
Figure. Data
preparation for PeptideProphet/ProteinProphet users (Also for users using
Mascot, SEQUEST, and X!tandem coupled with
PeptideProphet/ProteinProphet)
I.
Convert Spectral data files to mzXML Files
All the spectral data files generated from instrument
have to be converted into mzXML format. Here we list the mzXML converter for
RAW, Wiff, and BAF files. For the detail information of mzXML data format, you
can check the following website of Seattle Proteome Center: ttp://tools.proteomecenter.org/wiki/index.php?title=Formats:mzXML
a. For Wiff data format
To convert Wiff files to mzXML format, we recommend
use mzWiff, which is available at http://tools.proteomecenter.org/wiki/index.php?title=Software:mzWiff.
- Note that Analyst® QS has to be pre-installed.
b. For RAW (ThermoFinnigan) data format
To convert RAW files to mzXML format for ThermoFinnigan users
, we recommend use ReAdW, which is available at http://tools.proteomecenter.org/wiki/index.php?title=Software:ReAdW.
- Note that Thermo's XCalibur data acquisition software
has to be pre-installed.
c. For RAW (Waters) data format
To convert RAW files to mzXML format for Waters users,
we recommend use massWolf, which is available at http://tools.proteomecenter.org/wiki/index.php?title=Software:massWolf.
- Note that Waters' MassLynx data acquisition software
has to be pre-installed.
d. For BAF data format
The detailed information of conversion BAF files
to mzXML format by CompassXport can be found at http://tools.proteomecenter.org/wiki/index.php?title=Software:CompassXport.
II.
Prepare Search Results
The tools accept search results from Mascot, SEQUEST, X!Tandem,
and PeptideProPhet /ProteinProphet.
a. For Mascot Users
The tools accept XML format from Mascot output. When exporting search results from Mascot, please make sure that
the option “Query title" is checked in “Peptide Match Information
Section”, as shown below.
Troubleshooting: If you get any error message when executing our tools,
please first make sure your search result files belong to the following four
types that contain required information for subsequent quantitation.
For Bruker instruments:
- The current supported title contains Cmpd, which indicates the
scan number of an identified peptide, e.g.,
Cmpd 1167, +MSn(404.25), 17.4 min
Note:
Since the peptide query title provides insufficient information for
quantitation, after completing Mascot search, please make sure that the name of
search result file is consistent with that of mzXML file.
For
Waters instruments:
- If you use Mascot Distiller
to convert raw files to MGF files for
Mascot search, the peptide query title needs to contain Scan and FilePath,
e.g.,
527: Scan 2784 (rt=3304.43, f=2, i=233) [FilePath.FileExtension]
- If you use Mascot Distiller
to convert raw files to mzData files
for Mascot search, the peptide query title needs to contain acqNumber,
which indicates the scan number of an identified peptide, e.g.,
spectrumId=2043 Polarity=positive ScanMode=MassScan Time In Seconds=7275.01 acqNumber=6242
Note: Since the peptide query title provides insufficient information for quantitation, after completing Mascot search, please make sure that the name of search result file is consistent with that of mzXML file.
For ThermoFinnigan instruments:
- If you use extract_msn
to convert *.raw files to DTA files for
Mascot search, the peptide query title needs to contain FileName, followed by a number
(marked in red color in the following example) to indicate the raw data of
an identified peptide, e.g.,
FileName.1024.1027.2
- If you use DTASuperCharge
to convert *.raw files to DTA files or MGF files for Mascot search, the peptide query title
needs to contain FileName and scan number (marked in red color in the
following example) to indicate the raw data of an identified peptide, e.g.,
Elution from: 34.373 to 34.373 period: 0 experiment: 1 cycles: 1 precIntensity: 413158.0 FinneganScanNumber: 4871 MStype: enumIsNormalMS rawFile: SILAC-A1.raw
- If you use raw2msm
to convert *.raw files to MGF files for
Mascot search, the peptide query title needs to contain FileName and scan
number (marked in red color in the following example) to indicate the raw
data of an identified peptide, e.g.,
Elution from: 27.18 to 30.09 period: Lysate_1.raw experiment: 1 cycles: 1 precIntensity: 13989851.3 FinneganScanNumber: 4871
For SCIEX/ABI Q-STAR instrument:
- If you use Analyst QS 1.1
or 1.2 with Mascot dll script to convert .wiff files to MGF files for Mascot search, the peptide
query title needs to contain FileName and four essential numbers:
sample number, Period, Cycle, and Experiment (marked in red color in the
following example) to indicate the raw data of an identified peptide,
e.g.,
File: FileName.FileExtension, Sample: wei (sample number 1), Elution: 22.387 min, Period: 1, Cycle(s): 1061 (Experiment 4)
Note: .table file is required, which can be generated by our developed tool, wiff2scan.
- We provide a program, called Wiff2Scan, to
generate .table files. The procedure to use Wiff2Scan is given below.
- Click “Open Dir” button and select the directory where
.WIFF files locate. Then all WIFF files in the folder are listed (as shown
below).
- Check the WIFF files and click “GO” button to generate
.table files. If a checked WIFF file contains spectral data from more than
one LC-MS runs, please check the option “Convert to individual files”.
- When the process is completed, the “status” field will
show “Done!” and .table files are shown in the folder.
b. For SEQUEST, X!Tandem
and PeptideProphet-ProteinProphet Users
SEQUEST and X!Tandem users need to go through PeptideProphet and ProteinProphet
to generate PepXML and ProtXML files for peptide identification and protein
identification results, respectively . Other PeptideProphet and ProteinProphet
users also use the generated PepXML and ProtXML files as search result input in
our quantitation tools.
Systems
Proteomics Laboratory
Institute of Information Science
Academia Sinica, Taipei 115, TAIWAN
Last updated 10/24/2008
Website: http://ms.iis.sinica.edu.tw/
E-mail: bioinfo@iis.sinica.edu.tw