The limitations commonly observed in data-dependent acquisition (DDA) mass spectrometric investigation of non-depleted human plasma are mainly due to the large dynamic concentration range of protein expression. Less abundant proteins are usually masked by highly abundant proteins and are therefore difficult to reliably detect. Sequential window acquisition of all theoretical fragment-ion spectra (SWATH) mass spectrometry (MS), as a representative of data-independent acquisition (DIA) approaches, provides an opportunity to improve plasma-based biomarker discovery studies because this approach does not rely on precursor intensity for fragmentation selection but rather analyzes all precursors in specified mass ranges. Here, we describe a workflow for SWATH-MS-based analysis of non-depleted plasma including sample preparation, data acquisition, and statistical analysis.
