Table 1. Advantages and disadvantages of data-dependent acquisition, parallel reaction monitoring/multiple reaction monitoring, and data-independent acquisition methods.

Method	Instrument setup	Ease of data analysis	Precision of peptide quantification	Reproducibility of peptide identification	Breadth of peptide identification
DDA	Easiest Requires user selection of precursor m/z range and frequency of precursor scans. Is the default mode of use on most commercial instruments.	Easiest Many convenient and comprehensive pipelines for the analysis of DDA spectra have been developed over more than 20 years 48, 51, 52.	Low/Moderate/High Spectral counts (low), isobaric-tag labels (moderate), or SILAC (high) can be used for relative quantification of protein abundance across samples. Hard to use for absolute quantification.	Lowest Run-to-run peptide identification overlap for a given sample is around 60% 53.	Highest Samples and identifies a single time as many peptides as can be individually isolated.
PRM/MRM	Hardest Requires prior identification of peptides and, in MRM, selection of reproducible fragments that do not exhibit interference 54.	Moderate A few pipelines have been developed over the past few years but require some manual curation to identify and quantify fragment chromatograms 54.	Highest Provides good relative peptide quantification and can be coupled with heavy labeled reference peptide for absolute quantification. Most sensitive method because of high signal-to-noise ratio 54.	Highest Run-to-run peptide identification overlap for a given sample is more than 85% 54.	Low Repeatedly samples and identifies a small set of pre-specified peptides 54.
DIA	Easy Requires user selection of precursor m/z windows for MS1 and MS2 scans.	Hardest Requires multiple steps from multiple experiments to compile spectral libraries, with more parameters to choose in recently developed, not-yet- established pipelines.	Moderate/High Similar to PRM/MRM but more vulnerable to variation caused by interference from other peptides 32– 37, 39– 45, 47, 50– 54.	High Similar to PRM/MRM but more vulnerable to variation caused by interference from other peptides 32– 37, 39– 45, 47, 50– 54.	High Repeatedly samples every peptide within pre-specified m/z windows and identifies those whose signals can be successfully deconvolved 54.

DDA, data-dependent acquisition; DIA, data-independent acquisition; 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 (or some) 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.