Proteomics aims to deep proteome profiling in tissues, cells, or body fluids to understand the biological processes and signaling pathways. The bottom-up proteomics analyzes complex peptides mixture after enzymatic digestion of protein. These peptides in single sample may contain millions of peptides with dynamic range of more than 10 orders. In order to overcome high complexity and wide dynamic range of sample, many proteomics studies employ a fractionation method, such as non-contiguous fractionating and concatenating (NCFC) fractionation utilizing high-pH RPLC, before each fraction is subjected to LC-MS/MS experiments. These NCFC fractionation method was usually performed offline with many manual steps, which result in sample loss and less reproducibility.

In this study, we present a dual online reverse-phase/reverse-phase liquid chromatography system that employs an online non-contiguous fractionating and concatenating device (NCFC fractionator). The DO-2D-NCFC-RP/RPLC shows significant improvements in separation spaces by improved orthogonality of two RPLCs (mid-pH and low-pH RPLC) and “hidden” isocratic elution implemented for the last dimension RPLC by the mutually independent operation of the dual online RPLC. The DO-2D-NCFC-RP/RPLC system produces up to 24 NCFC online fractions and each fraction was subsequently analyzed by the dual online RPLC-MS/MS experiments. The DO-2D-NCFC-RP/RP-MS/MS experiments were fully automated by a method script that time-controls the valve positions. As a result, this system provides minimal sample loss and high reproducibility. From 25 mg of HeLa peptides, this novel system identified more than 10,000 proteins in one day of MS data acquisition time. Also, by combining DO-2D-NCFC-RP/RPLC with online high-field asymmetric waveform ion mobility spectrometry (FAIMS), the resultant online 3D separation resulted in identification of more than 100,000 phosphopeptides (>60,000 phosphosites) from 3 mg of HeLa peptides, the largest phosphoproteome reported to data from HeLa cells.