Reproducibility, Specificity and Accuracy of Relative Quantification Using Spectral Library-based Data-independent Acquisition

The use of hybrid data-dependent and -independent acquisition spectral libraries empower dual-proteome profiling

“…This relatively lower consistency is likely due to the higher amount of peptide sequences, ~35-fold, in the MS 2 PIP-predicted in silico spectral library, when compared to the extended DDA-based spectral library. The use of large database sizes already showed to increase variation in peptide and protein quantification [12]. Importantly, and as illustrated in the respective Venn diagrams (insets Figure 3, left panel), thousands of peptides not present either in the DDA results nor DDA-based spectral libraries were identified using the MS²PIP-predicted in silico spectral library, demonstrating the potential of this approach to identify peptides in DIA not found in DDA.…”

“…The sensitivity of these approaches is thus inherently limited to the stochastic limitations of DDA. Spectral libraries can be created in several ways, leading to more or less extensive libraries, with the library size influencing the accuracy and specificity of identifications and quantifications [12]. Besides the use of DDA-based spectral libraries, an alternative way of creating spectral libraries is by using algorithms that accurately predict MS2 fragmentation spectra such as MS 2 PIP [13] and Prosit [14].…”

Data-independent acquisition mass spectrometry (DIA-MS) for proteomic applications in oncology

“…Technological advances in MS instrumentation has not only led to faster scanning speeds but also increased sensitivity. These developments have resulted in the development of a next generation proteomic strategy known as DIA-MS or SWATH-MS which provides better reproducibility and sensitivity when compared to conventional DDA-MS. 13,[17][18][19] In contrast to DDA-MS, DIA-MS is based on the fragmentation of all precursor ions identified in a MS1 survey scan where fragment ions are accumulated in a fixed number of wide isolation windows that span the entire mass-to-charge ratio (m/z) range (Fig. 1B).…”

“…In DDA-MS, the stochastic nature of the automated precursor ion selection in the survey scan prior to fragmentation leads to a well-documented inability of this method to reproducibly identify the same set of proteins across technical replicate experiments. 18,19,34,35 This lack of consistency in precursor ion fragmentation results in a large number of missing values in large-scale experiments involving multiple samples which significantly impacts the level of reproducibility necessary for contemporary biological experiments. DIA-MS overcomes this challenge by the cyclic acquisition of fragment ions for all precursor ions in the survey scan thereby significantly improving reproducibility in protein identification between technical replicate experiments.…”

A mouse SWATH-MS reference spectral library enables deconvolution of species-specific proteomic alterations in human tumour xenografts

“…Conventional data-dependent acquisition (DDA) mode, where a fixed number of the most abundant precursor ions in survey scans is automatically selected for fragmentation, enables the identification of thousands of proteins in a single MS experiment. However, the stochastic nature of peptide precursor ion selection in DDA leads to low reproducibility in peptide identification between experimental runs ranging from 35% to 60% (Tabb et al, 2010;Krasny et al, 2018;Bruderer et al, 2015;Barkovits et al, 2020). Sequential window acquisition of all theoretical mass spectra (SWATH-MS) or dataindependent acquisition mass spectrometry (DIA-MS) is a nextgeneration label-free quantification method that enables highly reproducible peptide identification (ranging from 80% to 98%) and more accurate quantification in large-scale proteomic analyses across multiple experiments (Gillet et al, 2012;Muntel et al, 2019;Collins et al, 2017;Bruderer et al, 2015;Barkovits et al, 2020).…”