Exercise: Looking at an LC-MS file from different perspectives
In this exercise we will look at an LC-MS/MS file from an Orbitrap instrument.
We will convert between formats, analyse the raw file do a MS/MS search.
The sample is a total cell extract of the yeast Saccharomyces cerevisiae. The file
is named YeastGlu1.mzML.gz, and has been converted from Thermo RAW format
to open mzML format. The file contains extracted spectra from timepoints 720 s
to 1800 s.
The file can be downloaded from this link:
https://www.immun.lth.se/uploads/media/Yeast_Glu1.mzML_01.gz
Background info - File formats:
Closed raw data formats:
Data generated by the MS instruments is stored in the proprietary format which
can be only read through software with vendor license.
Waters .RAW directories
Thermo .RAW files
Open XML formats for raw data:
The proprietary raw files can be converted to open format which are XML based:
mzXML, developed at Institute of Systems Biology (ISB), Seattle
mzData developed by the Proteomics Standards Initiative (PSI)
(http://www.psidev.info)
mzML to replace the above formats (http://www.psidev.info/)
Peak list formats for database searching:
(http://www.matrixscience.com/help/data_file_help.html)
DTA (DaTA, Orginally for Sequest, text format)
PKL (PeaK List, Waters file format, text format)
MGF (Mascot Generic File format, text format)
mzData , mzML (XML-based standard formats)
1 File conversion from closed formats to open formats:
ProteoWizard is an open-source cross-platform tool that can be used to convert
the files from “.raw” format to mzML format. It can also be used to convert files
to the MGF format.
Note: Conversion from “.raw” format is only possible on Windows OS as vendor
licenses only work on Windows OS. While conversion from mzML to MGF is
platform-independent.
- Download and install Proteowizard http://proteowizard.sourceforge.net
- Convert the file to MGF using msconvert. This can be done using the command
line:
msconvert --mgf YeastGlu1.mzML.gz
or through the graphical user interface MSConvertGUI
- Also convert it to uncompressed mzML:
msconvert YeastGlu1.mzML.gz
- Look at the MGF and mzML files as text. Tip: Use the text editor Notepad ++
Can you find the first MS/MS spectrum?
2 Looking at the file in a file viewer.
- Select one of the following:
SeeMS (Proteowizard – Windows only). http://proteowizard.sourceforge.net
or
ToppView (OpenMS – all platforms) http://openms.de
Using SeeMS or OpenMS, open the mzML file and look at the structure.
a) Can you find peaks in MS1 spectra that have been subjected to MS/MS?
b) Are peaks in the MS1 spectra centroided or in profile?
c) What is the charge and exact mass of the MS1 peak eluting at 1365 s, closest
to m/z 649.3?
d) What is the charge and exact mass of the MS1 peptide eluting at 1297 s,
closest to m/z 622.8? Are there any MS/MS spectra of this peak? Comments?
Alternative - If you have trouble using ToppView in MAC OS / Linux OS try
with msInspect (all platforms) http://proteomics.fhcrc.org/CPL/msinspect/
Download from this link: http://raspberry.fhcrc.org/latest_msInspect.html
Using msInspect perform the following steps:
a) Can you find peaks in MS1 spectra that have been subjected to MS/MS?
b) Are peaks in the MS1 spectra centroided or in profile?
c) What is the charge, mass and retention time of the MS1 peak with the m/z
649.3382?
d) How many features are found with the charge state 3?
3 – MS/MS searching
Identify sequences for peptides subjected to MS/MS in the file. First the file
needs to be converted to a format acceptable for the search engines. mzML and
MGF will work in X!Tandem
Search in the yeast database using X!Tandem at
http://h003.thegpm.org/tandem/thegpm_tandem.html
Search in the Saccharomyces cerevisiae database, mixed with reverse sequences.
a) Are any reverse proteins identified
b) Can you find the peptide from 2d in the result?
c) Which MS2 ions were matched in the MS/MS spectrum for this peptide?
d) What is the fragment mass error and how large was it for this match?
4 2D view exploration and Feature detection
You will need java for this step
Open the file in msInspect (http://raspberry.fhcrc.org/latest_msInspect.html):
Note that msInspect doesn't read compressed files. To decompress (mzML.gz>.mzML) use msconvert with no parameters on the .mzML.gz file.
java –Xmx1G –jar viewerApp.jar
YeastGlu1.mzML
a) Save the identified features to a text file.
b) How many features are found with charge +2 or +3