Cheap, Robust and Versatile Two-Dimensional Chromatography system for Proteomics of Nanogram Scale Samples

In this work we describe a low loss fractionation system comprised of a reconfigured Evosep One LC system for the first dimension and a repurposed 3D-printer as a fraction collector. The setup operates as a high-pH fractionation system capable of effectively working with nanogram scales of lysate digests. The 2D RP-RP system demonstrated superior proteome coverage over single-shot data-dependent acquisition (DDA) analysis using only 5 ng of human cell lysate digest with performance increasing with increasing amounts of material. We found that the fractionation system allowed over 70% signal recovery at the peptide level and, more importantly, we observed over 30% increase on protein level intensity which indicates the complexity reduction afforded by the system outweighs the sample losses endured. The application of data-independent acquisition (DIA) and wide window acquisition (WWA) to fractionated samples allowed more than 8,000 proteins to be identified from 50 ng of material. The utility of the 2D system was further investigated for phosphoproteomics (>21,000 phosphosites from 50 μg starting material) and pull-down type experiments and showed substantial improvements over single-shot experiments. We show that the 2D RP-RP system is highly versatile and powerful tool for many proteomics workflows.