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.
- It is not unusual to get MS/MS for just one component of a pair. In such cases,
the identity of the other precursor can be inferred from its predicted mass and retention time.
When checked, Allow mass time match allows a matching
component to be selected using mass and (optionally) time.
(allow_mass_time_match, required)
- Check Allow elution shift to allow a matching component
to have a systematic shift in elution time. The algorithm is: 1. For all pairs
of peptides where both partners can be confidently identified,
determine an elution time delta. 2. Make a least squares fit of this
delta against elution time 3. Use this function to interpolate a correction
for pairs where only one partner can be identified.
(allow_elution_shift, optional, default false)
- When checked, All charge states extends the mass and time matching to cover contiguous charge states.
In other words, if there is a positive match on 2+, the software will look for 1+ and 3+.
It will not look for 4+ unless 3+ is found, etc.
(all_charge_states, optional, default false)
An example of Mascot search results for metabolic labelling with 15N
can be found here.
We are grateful to Joost Gouw of the
Department of Biomolecular Mass Spectrometry
at the University of Utrecht for this data set, which was acquired on
a Waters QTof.
To see the quantitation method details, follow the method details link in the report
header or click here.
Queries 71 and 72 are a good example of a pair where both light and heavy components give strong matches.
More detailed information about quantitation reports for the precursor protocol will be available as soon as the
Mascot Distiller Quantitation Toolbox is released.
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