Data-Independent acquisition (SWATH) mass spectrometry analysis of protein content in primary neuronal cultures

Sequential Window Acquisition of all THeoretical fragment-ion (SWATH) is a recently developed discovery proteomics technique based on Data-Independent Acquisition (DIA) mass spectrometry. In this approach, MS/MS is performed simultaneously on all peptides contained in a predefined wide-open mass window of up to 25 Da. The mass window is sequentially stepped through over the entire mass range, usually between 400 and 1200 Da that covers most peptides. As quantitative MS/MS information is generated for all observable peptides in the sample, the missing data and variability between replicates are substantially reduced when compared to a Data-Dependent Acquisition approach. To identify each peptide from the high complexity of the MS/MS spectra generated from multiple peptides, a comprehensive reference spectral library derived prior from a similar sample by Data-Dependent Acquisition, rather than a conventional genome-wide database, should be used. In this chapter, we describe a general protocol that benefits from the advances of SWATH-MS for the quantification of the primary neuronal culture proteome.