We often get questions about doing PTM analysis with Scaffold DIA. We spoke with Brian Searle, OSU Assistant Professor, and want to share this excerpt on the topic:
EncyclopeDIA/Scaffold DIA is a peptide-centric search tool, but there are other search strategies. For example, DirectDIA in Spectronaut is a spectrum-centric tool. Peptide-centric searching implicitly FDR corrects based on the number of peptides searched. It takes a significant penalty when searching really large peptide spaces, where unexpected PTMs can explode that search space. Spectrum-centric searching implicitly FDR corrects based on the number of peaks/RAW files. It takes a significant penalty when searching lots of raw files. So EncyclopeDIA/Scaffold DIA is better for getting consistent quantification across lots of files, while DirectDIA is better at considering unexpected PTMs.
If you’ve got fewer samples, DirectDIA is probably a better tool. If you’ve got a lot of data, EncyclopeDIA/Scaffold DIA is probably a better tool.