Extending proteome coverage through an on-line 2D SCX-RP ultra-high pressure system

Data from ProteomeXchange, PXD ID: PXD000705. Experiment: 1D 600min, file: 1D 600min 1st repl.mzml. From ProteomeXchange: {{i}} ... Proteome coverage can be extended using 2-dimensional chromatography, but traditionally this comes at the expense of sample losses and much longer analysis times. Here, we asked the question whether a fast and sensitive online 2D SCX-RP UHPLC-MS/MS workflow, could compete with the current 1D long gradient analysis, making total analysis time- versus proteome coverage and sample used as benchmark parameters. Our new automated 2D-LC/MS system is robust and easy to use, consisting of a homemade SCX column, a trap column and a 50 cm analytical EASY-Spray column. We benchmark the system using small amounts of a human cell lysate digest. The 2D SCX-RP UHPLC-MS/MS workflow allowed us to identify 56600 unique peptides and 5958 proteins in a total analysis time of just over 8 hours. ... {{/i}}