If users already have a list of peptide sequences, users can run quantitative analysis without running protein identification. The advantage of the Targeted Quantitative Analysis (TQA) tool is that it looks for peptides in the entire ms1 spectra. Sometimes low abundant peptides may not be identified by the search engine, but they can be detected by the TQA tool. TQA tool uses precursor mass and isotope peaks to extract chromatograms. It applies smoothing and Gaussian curve fitting to measure the area under the curve (AUC). Users can input multiple samples, and group them for comparison.
Users can combine replicates into a group. Later, users can compare two different groups quantitatively like control vs treated group. If users want to quantify only one experiment, they can create only one group containing only one experiment. In this example, we set the group name to ‘group 1’, and selected three experiments. Then, click ‘Add to group’ button’
When you finish adding groups, click the next button.
Type analysis name in the “Analysis name to save” box. On the result page, users will recognize analyses by this name.
Mass tolerance is the precursor mass tolerance in ppm.
Charge state range is the range to search precursors. If values are 2 and 5, IP2 will search the precursor with charge state 2,3,4 and 5.
Then, input peptide list. Each sequence should be on a separate line. If users input with prefix and suffix in the sequences, IP2 will take only the sequence between prefix and suffix. For example, If users input
R.AVFVDLEPTVIDEVR.T, IP2 will use AVFVDLEPTVIDEVR to quantify. If the sequence does not contain a prefix and suffix, IP2 will use the whole sequence as it is.
If you have differential modifications in the sequence, IP2 can handle them. For example, if your sequence is like R.DPEPSTMEK.E, IP2 can handle PTM mass 79.9663 for precursor mass calculation.
In the atomic table, carbamidomethylation (+57 Da) of cysteine was already considered.
When ready, click the ‘submit’ button to run.
Users can download the result text file by clicking ‘download result file’. To view the result, click the name link. In this example, click ‘my first run’ link.
When users input more than one group to compare, IP2 will calculate p-value and q-value. AUC column is the area under the curve. Each value is for each experiment. When there are replicates, each value is separated by a comma. Zero value means the peptide was not found. When users click the peptide sequence, IP2 displays extracted chromatogram graphs underneath.