ABSTRACTIn silico spectral library prediction of all possible peptides from whole organisms has a great potential for improving proteome profiling by data-independent acquisition (DIA) and extending its scope of application. In combination with other recent improvements in the field of mass spectrometry (MS)-based proteomics, including sample preparation, peptide separation and data analysis, we aimed to uncover the full potential of such an advanced DIA strategy by optimization of the data

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... isition. The results demonstrate that the combination of high-quality in silico libraries, reproducible and high-resolution peptide separation using micro-pillar array columns as well as neural network supported data analysis enables the use of long MS scan cycles without impairing the quantification performance. The study demonstrates that mean coefficient of variations of 4 % were obtained even at only 1.5 data points per peak (full width at half maximum) across different gradient lengths, which in turn improved proteome coverage up to more than 8000 proteins from HeLa cells using empirically-corrected libraries and more than 7000 proteins using a whole human in silico predicted library. These data were obtained using a Q Exactive orbitrap mass spectrometer with moderate scanning speed (12 Hz) and perform very well in comparison to recent studies using more advanced MS instruments, which underline the high potential of this optimization strategy for various applications in clinical proteomics, microbiology and molecular biology.