Acquiring and Analyzing Data Independent Acquisition Proteomics Experiments without Spectrum Libraries.
Molecular & cellular proteomics : MCP
Data independent acquisition (DIA) is an attractive alternative to standard shotgun proteomics methods for quantitative experiments. However, most DIA methods require collecting exhaustive, sample-specific spectrum libraries with data dependent acquisition (DDA) to detect and quantify peptides. In addition to working with non-human samples, studies of splice junctions, sequence variants, or simply working with small sample yields can make developing DDA-based spectrum libraries impractical. Here we illustrate how to acquire, queue, and validate DIA data without spectrum libraries, and provide a workflow to efficiently generate DIA-only chromatogram libraries using gas-phase fractionation (GPF). We present best-practice methods for collecting DIA data using Orbitrap-based instruments, and develop an understanding for why DIA using an Orbitrap mass spectrometer should be approached differently than when using time-of-flight instruments. Finally, we discuss several methods for analyzing DIA data without libraries.
Institute for Systems Biology
Pino, Lindsay K; Just, Seth C; MacCoss, Michael J; and Searle, Brian C, "Acquiring and Analyzing Data Independent Acquisition Proteomics Experiments without Spectrum Libraries." (2020). Articles, Abstracts, and Reports. 3025.