Quantitation: Average protocol
This is label-free, absolute quantitation for the proteins in a mixture based on the
application of a rule to the intensities of extracted ion chromatograms (XICs) for the
peptide matches in a database search result. The method is described in
Silva, J. C.,
et al., Absolute quantification of proteins by LCMSE - A virtue of parallel MS
acquisition, Molecular & Cellular Proteomics 5 144-156 (2006). The observation
was that the average MS signal response for
the three most intense tryptic peptides per mole of protein
was constant within a coefficient of variation of less than
±10%.
This 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.
- An updated copy of
quantitation.xml
is available for Mascot Server version 2.2 (only). Right click the link and choose Save As.
- Quantitation is based on the intensities of a fixed number of
peptides per protein, (num_peptides, optional, default 3).
- The Selection type control determines whether the n peptides
must have different sequences (unique_sequence, default), or whether to accept
different modification states of same sequence (unique_mr), or
even to accept peptides with same sequence and modifications in
different charge states (unique_mz).
- If a protein accession is entered into Reference Accession,
and this protein appears in the quantitation table, it will be
selected as the reference protein (reference_accession, optional). If no accession is specified,
or if the accession is not present, the first protein hit is
selected as reference. The selection can be changed using the
checkboxes in the Ref column of the table.
- Reference Amount allows you to enter an absolute amount for
the protein selected in the quantitation table as the reference
protein (reference_amount, optional, default 1.0). For example, if the first protein has a total intensity
of 1000 and the second 500 and you enter an amount of 6, the
second protein will be listed as having an amount of 3. The
default setting for Reference Amount is 1
To see how this method works in practice, try Distiller on
30 day evaluation.
Pretty much any MS/MS dataset with a reasonable number of matches to more than one protein can be used.
Follow through the tutorial in the Distiller help (F1) to become familiar with the way Distiller works,
then process your own data. If you don't have
an in-house Mascot 2.2 Server, you can use the
free, public Mascot server, as long as you stay within the limit of 1200 spectra.
If you have an in-house server, no such limits apply, but make sure it is
patched to 2.2.06 or later.
|