Abstract
The ultimate aim of proteomics is to fully identify and quantify the entire complement of proteins and post-translational modifications in biological samples of interest. For the last 15 years, liquid chromatography-tandem mass spectrometry (LC-MS/MS) in data-dependent acquisition (DDA) mode has been the standard for proteomics when sampling breadth and discovery were the main objectives; multiple reaction monitoring (MRM) LC-MS/MS has been the standard for targeted proteomics when precise quantification, reproducibility, and validation were the main objectives. Recently, improvements in mass spectrometer design and bioinformatics algorithms have resulted in the rediscovery and development of another sampling method: data-independent acquisition (DIA). DIA comprehensively and repeatedly samples every peptide in a protein digest, producing a complex set of mass spectra that is difficult to interpret without external spectral libraries. Currently, DIA approaches the identification breadth of DDA while achieving the reproducible quantification characteristic of MRM or its newest version, parallel reaction monitoring (PRM). In comparative de novo identification and quantification studies in human cell lysates, DIA identified up to 89% of the proteins detected in a comparable DDA experiment while providing reproducible quantification of over 85% of them. DIA analysis aided by spectral libraries derived from prior DIA experiments or auxiliary DDA data produces identification and quantification as reproducible and precise as that achieved by MRM/PRM, except on low‑abundance peptides that are obscured by stronger signals. DIA is still a work in progress toward the goal of sensitive, reproducible, and precise quantification without external spectral libraries. New software tools applied to DIA analysis have to deal with deconvolution of complex spectra as well as proper filtering of false positives and false negatives. However, the future outlook is positive, and various researchers are working on novel bioinformatics techniques to address these issues and increase the reproducibility, fidelity, and identification breadth of DIA.
Highlights
For the last 15 years, liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomics has provided broad detection and relative quantification—through chemical or metabolic labeling—of thousands of proteins across a variety of biological samples using a data-dependent acquisition (DDA) strategy[1,2,3,4]
All LC-MS/MS methods discussed in this article are bottom-up proteomics: Proteins are enzymatically digested into peptides which are separated using high-performance liquid chromatography (HPLC), ionized, isolated, fragmented, and detected in the mass spectrometer as they elute from the HPLC
DDA, data-dependent acquisition; DIA, data-independent acquisition; multiple-reaction monitoring (MRM), multiple reaction monitoring; MS1, scan in which the peptide ions entering the mass spectrometer at a given time are identified; MS2, scan in which the fragments of all of the peptides that are in the mass spectrometer at a given time are identified; m/z, mass-to-charge ratio; PRM, parallel reaction monitoring; SILAC, stable isotope labeling by amino acids in cell culture
Summary
For the last 15 years, liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomics has provided broad detection and relative quantification—through chemical or metabolic labeling—of thousands of proteins across a variety of biological samples using a data-dependent acquisition (DDA) strategy[1,2,3,4]. The ultimate goal of proteomics is the development of acquisition strategies that have both the breadth of DDA and the precision of MRM/PRM to provide reproducible identification and quantification of every protein in any biological sample. Irreproducibility and imprecision are fundamental to DDA’s design; if too many peptide species co-elute and appear in a single MS1 scan, DDA stochastically samples only the most abundant peptides and misses the rest This approach diminishes reproducibility and prevents the measurement of low-abundance peptides[9]. DIA is like MRM/PRM in that it repeatedly samples the same peptides for more precise quantification, but it differs from them and DDA by dispensing with the isolation of individual peptide species and instead repeatedly selecting mixtures of peptide species within large, pre-specified mass ranges (Figure 1 and Figure 2B) for MS2 scans.
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