Acquiring and Analyzing Data Independent Acquisition Proteomics Experiments without Spectrum Libraries

Abstract

<jats:p>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.</jats:p>

Data-independent acquisition method for ubiquitinome analysis reveals regulation of circadian biology

“…Construction of such spectral libraries requires some effort, specialized equipment for fractionation and may not always be possible for samples with low amounts such as primary cells. Alternatively, gas phase 65 or ion mobility fractionation appear to be promising strategies to simplify the workflows for project-specific spectral library construction. Furthermore, library-free approaches may also greatly simplify DIA workflows in the future.…”

Extensive and accurate benchmarking of DIA acquisition methods and software tools using a complex proteomic standard

“…Furthermore, publicly-available libraries 29 , gas-phase fractionation libraries 30 or in silico generated libraries 31 could also be used to avoid performing additional injections.…”

Recent Developments in Data Independent Acquisition (DIA) Mass Spectrometry: Application of Quantitative Analysis of the Brain Proteome

“…DIA (Ludwig et al, 2018;Zhang et al, 2020), also known as SWATH (Liu et al, 2013), enables the quantitation of thousands of proteins with low variation and high reproducibility, as demonstrated in a multi-laboratory evaluation study (Collins et al, 2017). In principle, DIA interrogates all peptides within the selected m/z windows, typically between 400 and 1,200 m/z, that contain >90% of all tryptic peptides (Koopmans et al, 2018a;Pino et al, 2020). First, the MS1 scans the entire mass range in one go.…”