Quantification of Proteins by Label-Free LC-MS/MS - LC-MS/MS in Proteomics: Methods and Applications - page 227

Fig. 13.5. Intensity measurements of technical replicates are averaged.

calculating the mean intensity value including all three replicates
such that for a given peptide there is only one intensity value for
each sample. An optional step following combining of technical
replicates is combine peptide intensities to the protein level by
summing the peptide intensities per protein. This should only
include peptides that were not modified by a posttranslational
modification. This final step, however, depends on the experimen-
tal design and purpose.

4. Notes

This protocol should be run prior to every quantitative study as
it ensures the operational performance of the nanoLC-MS/MS
system.
1. Inject five consecutive runs of 50 fmol digested yeast enolase
using the gradient in Table 13.3 .
2. Process the five raw data files using the data processing and
searching software.
3. Create a table as shown in Table 13.1 and fill it out based
on the results from the processing software.
4. If the specifications in Table 13.1 are met then proceed to
inject mix 1 and mix 2 in triplicates using the gradient in
Table 13.2 .
5. Process the six injections using PLGS.
6. Run the 'Expression Analysis' (time alignment) for these two
samples. Set the alcohol dehydrogenase (accession P00330)
as the internal standard for normalization. The fold changes
of the remaining three proteins should be within the specifi-
cation listed in Table 13.4 .

4.1.System
Verification