Quantitation: Precursor protocol
Quantitation based on
the relative intensities of extracted ion chromatograms (XICs)
for precursors within a single data set. This is by far the most widely used protocol, which can be used
with any chemistry that creates a precursor mass shift. For example,
- 18O: Miyagi, M.
and Rao, K. C. S., Proteolytic O-18-labeling strategies for quantitative proteomics,
Mass Spectrometry Reviews 26 121-136 (2007)
- AQUA: Gerber, S. A.,
et al., Absolute quantification of proteins and phosphoproteins from cell lysates by
tandem MS, Proceedings of the National Academy of Sciences of the United States of
America 100 6940-6945 (2003)
- ICAT: Gygi, S. P.,
et al., Quantitative analysis of complex protein mixtures using isotope-coded
affinity tags, Nature Biotechnology 17 994-999 (1999)
- ICPL: Schmidt, A.,
et al., A novel strategy for quantitative proteornics using isotope-coded protein labels,
Proteomics 5 4-15 (2005)
- Metabolic: Beynon, R. J.
and Pratt, J. M., Metabolic labeling of proteins for proteomics, Molecular & Cellular
Proteomics 4 857-872 (2005)
- SILAC: Ong, S. E.,
et al., Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and
accurate approach to expression proteomics, Molecular & Cellular Proteomics 1
376-386 (2002)
The precursor protocol requires information from the raw data file that is not
present in the peak list, so the quantitation report is
generated in Mascot Distiller, which has access to both the Mascot
search results and the raw data.
Ideally, the raw data should be true profile data. Many users of Thermo instruments save
centroided data to the raw file, because this makes the files much smaller. For high resolution
instruments, such as Orbitrap and FTMS, this is not a problem. For standard trap data, it can
make it difficult to get good quantitation results unless the original peak detection was reliable.
Where to begin
You need Mascot Distiller, including the Search Toolbox and Quantitation Toolbox,
and access to a Mascot 2.2 Server. You can automate the peak picking and Mascot search submission using
Mascot Daemon (except for multi-file projects), but you have to open the resulting Distiller project
file to generate the final quantitation report. Automation of this final step will be possible once
Mascot 2.3 is released.
- An updated copy of
quantitation.xml
is available for your in-house Mascot Server. (Right click the link and choose Save As.)
- It is not unusual to get MS/MS for just one component of a pair.
Check Allow mass time match to enable the partner to be inferred from
its predicted mass.
- Do not check Allow elution shift unless your tag includes Deuterium, which causes
a systematic shift in elution time. With other tags, setting this option to true slows
down processing and may degrade the results for weak signals that give noisy XIC's.
To see screen shots of examples of Precursor quantitation, refer to
this presentation
from our 2008 ASMS User Meeting
A better way to see how it works is to try Distiller on
30 day evaluation
and follow through the
tutorial in the help file. This uses data from a three component SILAC experiment acquired on an
AB SCIEX QStar instrument. You can follow the tutorial and experiment with the processing
settings even if you don't have an in-house Mascot 2.2 Server.
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