Different sample preparation methods were tested for HeLa proteome analysis. A sample obtained using sodium deoxycholate-based lysis allowed identification of the highest number of proteins. For this sample, a dilution series was acquired in triplicates ranging from 0.2ng to 200ng. All measurements were performed on Bruker timsTOF Pro 2 operated in dia-PASEF mode and analysed library-free using DIA-NN 1.8.