Acquiring and Analyzing Data Independent Acquisition Proteomics Experiments without Spectrum Libraries

Mol Cell Proteomics. 2020 Jul;19(7):1088-1103. doi: 10.1074/mcp.P119.001913. Epub 2020 Apr 20.


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.

Keywords: DIA; Data evaluation; data independent acquisition; label-free quantification; mass spectrometry; protein identification; quantification.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Chromatography, Gas / methods*
  • Chromatography, Liquid / methods*
  • Databases, Protein
  • HeLa Cells
  • Humans
  • Mass Spectrometry / methods*
  • Peptide Library*
  • Peptides / chemistry*
  • Proteome / analysis
  • Proteomics / methods*
  • Software


  • Peptide Library
  • Peptides
  • Proteome