Protocol for Data Independent Acquisition – Mass spectrometry analysis – a DIA-based Organelle Proteomics

Purification of intact organelles by previously described methods (dx.doi.org/10.17504/protocols.io.bybjpskn; dx.doi.org/10.17504/protocols.io.6qpvrdjrogmk/v1) allows to profile the organelle proteome using quantitative mass spectrometry. Here we provide a detailed protocol for the Data Independent Acquisition (DIA)-based mass spectrometry (MS) data acquisition method for proteomic profiling of the Golgi. This includes a description of how to construct the nano Liquid chromatography and DIA MS methods as well as a Data Dependent Acquisition (DDA) strategy to generate deep spectral libraries to be able to use in searching the DIA data. In addition, we provide detailed search parameters for database search for both DDA and DIA and downstream MS data analysis.