Data Independent Acquisition (DIA)-MS data is traditionally analysed using spectral reference libraries (SRLs) created from Data Dependent Acquisition (DDA) experiments. DDA inherent missingness, low reproducibility and biased to high abundant peptides, have allowed the surge of alternative strategies such as SRL-free methods and gas phase fractionation (GPF). In this study, we compare the performance of several SRL strategies in the context of complex clinical samples using DIA-NN.
1,261 fresh frozen cancer samples encompassing 73 cancer types were grouped to produce 39 sample sets. Each set was fractionated using high-pH RP-HPLC, fractions were analysed in Triple TOF 6600 instruments in technical duplicate, DDA and DIA (fractionated DDA and fractionated DIA), respectively. Additionally, every set went through GPF and resulting fractions were acquired in DIA (GPF DIA). Lastly, in silico SRLs were produced per set from unfractionated sample DIA files (SRL free DIA). The four approaches were evaluated in terms of library size, quantitated peptides, q-value scoring, missingness and peptide intensity.
In a prostate adenocarcinoma set, fractionated DDA, fractionated DIA, SRL-free DIA, and GPF DIA, approaches identified 5,087, 7,876, 6,974, and 6,927, proteins, respectively with a peptide overlap of 32%. Whilst fractionated DIA had the highest number of identifications, the SRL-free DIA method had the lowest number of missing values at 38%. Analysis of the proteins uniquely identified in each show that the SRL-free method produced the most intense values with the highest confidence score peptides and the lowest missingness.
Conventional DDA-SRLs performed poorly relative to the other methods in terms of uniquely quantified peptides and proteins and peptide confidence score. Fractionated SRLs also have a cost of a high proportion of missing values and low intensity peptides. Our results show that the SRL-free approach offers a higher quality alternative for large scale clinical proteomic analysis where sample availability may be limited.