Online 2D High-pH and Low-pH Reversed-Phase Nano-LC-MS/MS System for Deep Proteome Analysis

In bottom-up proteomic profiling, the complexity of proteome composition and wide dynamic range has created challenges on the limited number of protein identification and proteome coverage, especially in sample input-limited nanoflow (nano) LC-MS/MS analysis. Herein, we developed a fully automatic online 2D nano-LC-MS/MS system using both high-pH and low-pH reverse phase (RP) LCs on a single LC instrument toward comprehensive proteomics analysis. Compared to conventional microflow 2D-LC, the high-pH RP trapping column demonstrated a low sample requirement of cellular protein digest at the μg level with good fractionation resolution of >90% peptides in a single fraction. Compared to the offline 2D RP-RP nano-LC-QTOF using C18-HPLC column and C18-Stage Tip, and 1D nano-LC-QTOF system, superior coverage was observed on the higher number of identified protein groups/unique peptides by 1.35-/1.68-, 1.46-/1.75-, and 3.21-/4.35-fold, respectively, using an online 2D RP-RP nano-LC-QTOF mass spectrometer. On the evolution of quantitation performance, the online 2D high-/low-pH RP data-independent acquisition (DIA) showed a higher reproducibility in protein groups intensity (R² > 0.977) and more quantified proteins than that obtained using the offline 2D high-/low-pH RP DIA approach. Using an advanced Orbitrap Exploris 480 mass spectrometer, ∼1.9-fold higher proteome coverage was also observed in our 2D online RP-RP system (6039 protein groups) compared to the 1D nano-LC system (3133 protein groups). In summary, the online 2D nano-LC-MS/MS platform can be a sensitive and robust approach compatible with conventional nano-LC instruments for deep proteome coverage of trace amounts of samples.