Run RawTools for parsing and quantification for Windows
Now that we have our data in place, we are ready to run RawTools for parsing and quantification. This will all be done using the command line, so open Command prompt (type command prompt in the windows search bar).
To run RawTools for parsing of your file to generate 'Metrics' or 'ScanMatrix' data, follow the steps below.
First navigate in Command prompt to the directory where you stored RawTools. For us, this is the D: drive folder we created earlier.
>cd D:
>cd rawTools
First, let's load the RawTools parse help in order to see the commands available to us.
>RawTools -help
The output should appear similar to below.
As you can see there are many options available. Read through the details of each to familiarize yourself with what kinds of data analysis you can do with RawTools and the inputs that it requires.
Now we can generate a simple Metrics file that will contain a summary of our run data.
>RawTools -f D:\rawTools\ch_23Aug2018_HeLa_Std_1.raw -x
The -f flag here is directing RawTools to a single input file. If you want to process all files in a single directory, use the -d flag instead. The -x flag is telling RawTools to output a _Metrics.txt file in the same directory as the original raw file.
For a more extensive description of what is contained within the _Metrics.txt output file, please visit the page detailing the output here.
Now we can move on to more extensive data parsing. First let's make a '_Matrix.txt` output file that contains line-by-line information for all individual MS2 scans.
>RawTools -f D:\rawTools\ch_23Aug2018_HeLa_Std_1.raw -p
As above, the -f flag here is directing RawTools to the file (-d can be used instead for all files in a directory). The -p flag is telling RawTools to generate the _Matrix.txt file of scan data in the same location as the original raw file.
For a more extensive description of what is contained within the _Matrix.txt output file, please visit the page detailing the output here.
As you may have noticed, there are many other flags available with parse. These can be stacked into single commands with RawTools.
>RawTools -f D:\rawTools\ch_23Aug2018_HeLa_Std_1.raw -puxm
This command will generated _Metrics.txt (-x flag) and _Matrix.txt (-p flag) outputs in the same location as the original raw file, as well as creating an MGF output (triggered by the -m flag) that can be used in database searching. When creating an MGF, the -c flag can be used to control the masses output into the final MGF. In addition, the -R flag will trigger the recalibration of the precursor ion masses and charge states for the MGF. The additional -u flag adds precursor quantification to the _Matrix.txt output file, as described here.
Running RawTools for quantification is identical as running for parsing, only some arguments are changed. Let's run a quantification analysis on our raw file.
>RawTools -f D:\rawTools\ch_23Aug2018_HeLa_Std_1.raw -q -r TMT10
This command will generate a _Matrix.txt output file as above containing line-by-line analysis and quantification for every MS2 scan. The -q flag tells RawTools we are doing quantification, and the -r flag specifies we are using TMT 10-plex reagents. As you can see in the help output ($mono RawTools.exe -help), the available quantification reagents are TMT0, TMT2, TMT6, TMT10, TMT11, iTRAQ4, and iTRAQ8. For a more extensive description of what is contained within the _Matrix.txt output file when using quantification, please visit the page detailing the output here.
As before with general parsing, commands can be stacked to generate multiple outputs at once.
>RawTools -f D:\rawTools\ch_23Aug2018_HeLa_Std_1.raw -quxm -r TMT10
This command will generated _Metrics.txt (-x flag) and _Matrix.txt (-q flag) outputs in the same location as the original raw file, as well as creating an MGF output (triggered by the -m flag) that can be used in database searching. The additional -u flag adds precursor quantification to the _Matrix.txt output file, as described here. The _Matrix.txt file will also contain the data for the reporter ion quantification.
RawTools also has the option to generate text file outputs of chromatographic data for investigation or plotting. This can be done with the command:
>RawTools -f D:\rawTools\ch_23Aug2018_HeLa_Std_1.raw -chro 1B
As you can see in the help for RawTools ($mono RawTools.exe -help), the output of the -chro command is controlled by the type of chromatogram output you want and the MS level you are interested in (e.g. MS1, MS2, MS3). The text file for the chromatogram will be generated in the folder that contains the original raw input file.
As above, these commands can be stacked to generate multiple outputs all at once.
>RawTools -f D:\rawTools\ch_23Aug2018_HeLa_Std_1.raw -quxm -r TMT10 -chro 1B -c 132
This command will generate _Metrics.txt and _Matrix.txt files, the latter containing quantification data for TMT10 reporter ions. Chromatogram output for the MS1 base-peak will also be generated. Lastly, an MGF output is created using a mass cutoff of 132 (will remove the TMT reporter ions from the search input). All files are output into the same directory as the original raw file.
You are now ready to move on to performing a quality control analysis with RawTools. First we can setup X! Tandem.