Optimised data-independent acquisition strategy recaptures the classification of early-stage hepatocellular carcinoma based on data-dependent acquisition.

Proteomics is increasingly used for exploring disease biomarkers and therapeutic targets. The data-independent acquisition (DIA) method collects all peptide signals in a sample, and provides a convenient way to archive disease-related molecular features for further exploration. In this study, we first established a high-coverage human hepatocellular carcinoma (HCC) spectral library containing 9393 protein groups, 119,903 peptides. Furthermore, we optimised the DIA method with respect to four key parameters: settings for mass spectrometry acquisition, gradient length, amount of sample loading, and length of analytical column. More than 6000 proteins from HepG2 cells could be stably quantified using the optimised one-shot DIA approach with a 2 h gradient time. One-shot DIA identified a similar number of proteins as did multi-fraction data-dependent acquisition (DDA) from the same group of HCC samples, but at a quarter of the total acquisition time. DIA data could recapture the classification results obtained from DDA data, thus paving the way for large-scale, multi-centre proteomics analysis of clinical samples. SIGNIFICANCE: The organ-specific spectral library for HCC and the optimised 2 h DIA approach met the urgent demands for large-scale quantitative proteomics analysis of HCC clinical samples. Compared with multi-fraction-DDA, the optimised one-shot DIA could reach a similar identification while consuming shorter acquisition time, thus making it possible to analyse thousands of clinical samples.