The QconCATquantSTAR.py script allows the quantification of endogenous proteins for which a QconCAT internal standard was spiked-in. Protein abundances from a DDA method analysed with ProteomeDiscoverer (Thermo Fisher Scientitific, Waltham, MA, USA) are used for protein quantification in combination with concentration values of spiked-in QconCAT standard proteins.
The repository is supporting material for the STAR protocol "Design, expression and purification of labeled peptides for absolute quantification of Gram-negative bacterial proteins".
The DDA PeptideGroups results table (.CSV file) should be exported from ProteomeDiscoverer. This results table should at least include the following headers: Annotated Sequence, Modifications, Master Protein Accessions, and Abundances (Normalized): F{X}: Sample for each sample X. An example DDA PeptideGroups results table is located in the Examples folder as DDA_PeptideGroups_example.csv.
The QconCAT input table (.CSV file) should have the following structure including headers. The FullPeptideName column should have the sequence of each peptide included in the QconCAT internal standards, the ProteinName column should have the UniProt identifier of the endogenous protein of the corresponding peptide sequence, and the Concentration column should have the spiked-in concentration of each QconCAT peptide. An appropriate concentration unit would be fmol/µg total protein, since the spiked-in QconCAT protein and the corresponding QconCAT peptides have equimolar concentration. The unit of concentration used in the QconCAT input table will be the unit of concentration in the output table with the quantified endogenous proteins.
| FullPeptideName | ProteinName | Concentration |
|---|---|---|
| sequence 1 | UniProt ID 1 | concentration 1 |
| sequence 2 | UniProt ID 2 | concentration 2 |
| ... | ... | ... |
| sequence N | UniProt ID N | concentration N |
An example QconCAT input table is located in the Examples folder as QconCATinfo_example.csv.
The QconCATquantSTAR.py script can be used as follows:
python QconCATquantSTAR.py -i ./Examples/DDA_PeptideGroups_example.csv -Q ./Examples/QconCATinfo_example.csv
The DDA PeptideGroups results table and the QconCAT input table should be given as the i and the Q argument, respectively.
The output will be generated at QconCATproteins.csv. The output table is formatted with the following column headers: ProteinName (UniProt identifier of the quantified endogenous protein) and conc_sample{X} (concentration of the quantified endogenous protein). There will be as many columns with concentrations as there are samples. An example output table is located in the Examples folder as QconCATproteins_example.csv.