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

Ultra high pressure liquid chromatography (UHPLC) systems combined with state of the art mass spectrometers have pushed the limit of deep proteome sequencing to new heights making it possible to identify thousands of proteins in a single LC/MS experiment within a few hours. However, the relationship between gradient length and the number of proteins identified is not linear and as gradient time increases to several hours, performance gain diminishes. 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. On the same system a 1D RP UHPLC-MS/MS workflow gave only 22000 peptides and 4200 unique proteins in 6 hours analysis time, increases of 157% and 42% at the peptide and protein level, respectively. These and other data reported here reveal that with this fast online SCX-RP UHPLC-MS/MS workflow proteome coverage can be substantially extended without compromising too much analysis time and sample usage