Hi, and thanks for taking a look at this.

I performed ID-MRM for four target peptides (3 transitions/peptide), with triplicate analyses at each of the 12 light peptide concentrations (2x-serial dilution series), at two matrix concentrations. This involved the use of 4 L-peptide sequences that were matched with the corresponding H-peptide sequences with AAA verification. The heavy peptide concentrations for different peptide target sequences were all slightly different from each other, although the spike level for individual H-peptides was the same in prepared samples for analysis. Double-blanks were used. Equal amounts of BSA were included in samples, prior to trypsin digest, as a digestion control. It is of interest to use QuaSAR to analyze the data.

The GenePattern QuaSAR Documentation indicates (p. 6) "...QuaSAR is unable to handle multiple concentrations for IS targets at this time." Does this mean I can't simply enter the different H-peptide spike levels, for the different peptides in the QuaSAR concentration map, and proceed?

What makes me worry is that:

• I've not been able to edit the document grid IS spike level for the different H-peptide sequences. When I change it for one peptide sequence, it changes for all the others.

• In the QuaSARInput report, I can't edit the H-peptide concentration/spike level to reflect the actual H-peptide concentrations for the different peptide sequences. If the edit .csv could then be processed by QuaSAR, maybe it would be a work around?

In the event QuaSAR can't handle multiple concentrations for IS targets, could I just perform the analysis using four different .sky documents, where I edit each of them to contain the correct H-spike concentrations? Maybe this is what the GenePattern QuaSAR Documentation was indicating on p. 4, and I just didn’t understand?

We're running Win7 Enterprise SP1, Skyline 4.2.0.19009 , and QuaSAR 1.32.

Thank you.