Influence\nof Software Settings on the Identification\nRate, Quantification Results, and Reproducibility in Profiling Post-Translational\nModifications by Microflow Liquid Chromatography–Ion Mobility-Quadrupole\nTime-Of-Flight Analysis Using PEAKS So

The influence of data evaluation parameters on qualitative\nand\nquantitative results of untargeted shotgun profiling of enzymatic\nand nonenzymatic post-translational modifications (PTMs) was investigated\nin a model of bovine whey protein α-lactalbumin heated with\nlactose. Based on the same raw data, individual adjustments to the\nprotein database and enzyme settings of PEAKS studio software increased\nthe identification rate from 27 unmodified peptides to 48 and from\n322 peptides in total to 535. The qualitative and quantitative reproducibility\nwas also assessed based on 18 measurements of one sample across three\nbatches. A total of 570 peptides were detected. While 89 peptides\nwere identified in all measurements, the majority of peptides (161)\nwere detected only once and mostly based on nonindicative spectra.\nThe reproducibility of label-free quantification (LFQ) in six measurements\nof the same sample was similar after processing the data by either\nthe PTM algorithm or the LFQ algorithm. In both cases, about one-third\nof the peptides showed a coefficient of variation of above 20%. However,\nthe LFQ algorithm increased the number of quantified peptides from\n75 to 179. Data are available at the PRIDE Archive with the data set\nidentifier PXD050363.