Automated Nanoflow Two-Dimensional Reversed-Phase Liquid Chromatography System Enables In-Depth Proteome and Phosphoproteome Profiling of Nanoscale Samples

Two-dimensional reversed-phase capillary liquid chromatography (2D RPLC) separations have enabled comprehensive proteome profiling of biological systems. However, milligram sample quantities of proteins are typically required due to significant losses during offline fractionation. Such a large sample requirement generally precludes the application samples in the nanogram to low-microgram range. To achieve in-depth proteomic analysis of such small-sized samples, we have developed the nanoFAC (nanoflow Fractionation and Automated Concatenation) 2D RPLC platform, in which the first dimension high-pH fractionation was performed on a 75-mu m i.d. capillary column at a 300 nL/min flow rate with automated fraction concatenation, instead of on a typically used 2.1 mm column at a 200 mu L/min flow rate with manual concatenation. Each fraction was then fully transferred to the second-dimension low-pH nanoLC separation using an autosampler equipped with a custom-machined syringe. We have found that using a polypropylene 96-well plate as collection device as well as the addition of n-Dodecyl beta-D-maltoside (0.01%) in the collection buffer can significantly improve sample recovery. We have demonstrated the nanoFAC 2D RPLC platform can achieve confident identifications of similar to 49,000-94,000 unique peptides, corresponding to similar to 6,700-8,300 protein groups using only 100-1000 ng of HeLa tryptic digest (equivalent to similar to 500-5,000 cells). Furthermore, by integrating with phosphopeptide enrichment, the nanoFAC 2D RPLC platform can identify similar to 20,000 phosphopeptides from 100 mu g of MCF-7 cell lysate.