Human HeLa proteome analysis by a nanoLC-MS/MS system using a two-meter C18 monolithic silica column under shallow gradient conditions without pre-fractionation was performed in this project. &#10;A hybrid quadrupole-orbitrap mass spectrometer (Thermo Q-Exactive) was used to analyze a tryptic digest of HeLa cells through this study. Peptide and protein assignments were filtered to a false discovery rates (FDR) of < 1% by searching against a randomized decoy database using Mascot.