|MS1 Quant with t-SIM partially solved, still questions on skyline||Tobi||2018-06-22|
Dear the whole Skyline Crew,
hope you had a good time in San Diego, I sadly could not join, so it would be nice if I can please ask you something here.
We want to do absolute peptide quantification, at first without any MS2 data or library (I read the older forum posts on this topic, hope I didnt miss anything). We measured 5 BSA Peptides of a simple solution without any background or standard with both a t-SIM only and a Full Scan t-SIM aquisition on thermo q exactive hf. Adding the peptide target list and loading the thermo raw files into skyline works fine and we get the t-SIM MS1 data.
The Problem is to get the Full Scan MS1 data with skyline from thermo raw files, which contain both t-SIM and Full Scan MS1 data (only t-SIM data gets displayed). A solution we have for now is to use MSConvert and filter the msLevel 1 into an mzXML file. When loading the mzXML file we do see the Full scan data, which is nice, but it takes a lot of time and resources.
Is there a way to selectivley display both the t-SIM and the Full Scan MS1 data with skyline from the original raw file directly? Or do you know any other workaround, like making a exact copy of the raw file just without the t-SIM data?
The second small issue, could you please help me with an opinion on if to use the high-selectivity extraction feature (halving the extraction width), especially for high resolution t-SIM data? At least for FullScans on samples with complex background, the area to background ratio gets improved, but the detected m/z changes slightly. For now, I am just not sure if the accuracy of the picked peak remains as high as without high-selectivity extraction.
For the end, I would just mention the observation, that displayed peak area replicate comparisons are sometimes shown as a line graph even if bar graph type is selected, just for your information.
Skyline seems pretty nice and I wish you continued succes with it in the future.