Small Molecule Peak Integration | tony considine | 2022-07-18 14:48 | |||||||||||||||||||||||||||||||||||||||||||||||||||||
Hello, I am using the molecule interface of Skyline in conjunction with the ALIS system to perform RNA/protein screens looking for novel chemical matter. Since I will be screening for novel binders, I need to have a peak integration method that identifies the correct peak. I cannot go through each chromatogram to perform a manual integration as that will not be feasible due to the sizes of the screens. Instead, I have to rely on applying different filters in the exported CVS file to filter out nonspecific binders from specific binders and trust that the initial peaks identified are the correct ones. I have tried to optimize the peak integration method by spiking in known "weak" binders into pooled compound wells of 250-300 compounds per well and seeing if the correct peak is integrated. I am doing this as we don't expect to identify very strong binders in the initial screening process. However, in some cases, it identifies the wrong peak and a peak with an idotp score much lower than the correct peak with the much higher idotp score. I have tried to look under the "refine" tab and the advanced section and modify the parameters, especially the minimum idotp value. However, changing the minimum idotp value seems in mess up my method and results in the wrong compounds being identified as the peak in the respective chromatograms. I will typically know the chemical formula of all the compounds in the well and would expect an idotp score of 0.80 or greater. I have also tried to set the amount of transitions to 3 (M, M+1, M+2) and would expect to see at least main precursor and the C13 isotope. I also don't see a feature to isolate peaks based on the mass error (typically less than 10ppm) and in general, changing the parameters under the "refine" tab and the advanced section does not seem to have much of an effect. It would be super helpful if people who have experience could point me in the right direction and ways to optimize the peak integration methods so that only peaks with high idotp scores, low mass error, etc etc are identified from the chromatogram. Thank You, |
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