The utility of porous graphitic carbon as a stationary phase in proteomics workflows: Two-dimensional chromatography of complex peptide samples

We present the first investigation into the utility of porous graphitic carbon (PGC) as a stationary phase in proteomic workflows involving complex samples. PGC offers chemical and physical robustness and is capable of withstanding extremes of pH and higher temperatures than traditional stationary phases, without the likelihood of catastrophic failure. In addition, unlike separations driven by ion exchange mechanisms, there is no requirement for high levels of non-volatile salts such as potassium chloride in the elution buffers, which must be removed prior to LC–MS analysis. Here we present data which demonstrate that PGC affords excellent peptide separation in a complex whole cell lysate digest sample, with good orthogonality to a typical low pH reversed-phase system. As strong cation exchange (SCX) is currently the most popular first dimension for 2D peptide separations, we chose to compare the performance of a PGC and SCX separation as the first dimension in a comprehensive 2D-LC–MS/MS workflow. A significant increase, in the region of 40%, in peptide identifications is reported with off-line PGC fractionation compared to SCX. Around 14,000 unique peptides were identified at an estimated false discovery rate of 1% (n = 3 replicates) from starting material constituting only 100 μg of protein extract.