on 06-18-202003:22 PM - edited on 10-15-202111:30 AM by AnalyteGuru
Tabiwang N. Arrey1, Runsheng Zheng2, Alexander Boychenko2, Alexander Harder1 ASMS 2020 Although high proteome coverages have been obtained with single shot liquid chromatography−tandem mass spectrometry (LC-MS/MS) analysis, multiple dimensional separation are necessary for deeper proteome profiling. Offline high-pH reversed-phase LC (high-pH RPLC) followed by low-pH reversed-phase LC (low-pH RPLC) has gradually replaced the strong cation exchange (SCX) coupled with low-pH reversed-phase LC-MS/MS. This is due to better efficiency of peptide separation under RP conditions, better nanoLC-MS compatibility, and lack of need for an extra desalting step after the first-dimension sample fractionation. However, an offline high-pH RP fractionation method still has potential for improvement. For example, it is time-consuming, affording low throughput, and requires large amounts of sample. It is also prone to sample loss during fraction collection and transfer between fractionation devices and analytical instruments. Here we present a simple-to-use online low-flow high-pH RP x low-pH RP separation platform (termed online 2D-nanoLC) for deep proteome profiling using a Thermo Scientific™ UltiMate™ 3000 RSLCnanosystem coupled to a Thermo Scientific™ Orbitrap Exploris™ 480 mass spectrometer.