Online Comprehensive RPLC x RPLC with Mass Spectrometry Detection for the Analysis of Proteome Samples

LC-MS-based shotgun proteomics relies both on the power of the separation techniques and the sensitivity of detection methods. As a viable alternative to classical approaches in this field, we developed a fully automated, comprehensive 2D LC system, in which RPLC x RPLC was coupled to MS detection, for the first time, and applied for the analysis of tryptic digests obtained from a-casein and dephosphorylated a-casein. The use of a significantly different pH in the two dimensions allowed us to attain high peak capacity, despite the employment of novel identical stationary phases. Furthermore, such a combination addresses compatibility issues, thus allowing straightforward interfacing in online 2D LC configuration, as well as direct linkage to a mass spectrometer. A theoretical peak capacity of ca. 8500 was calculated for the setup, employing four serially coupled C18 columns in the first dimension (600 x 2.1 mm, 2.7 mu m d.p.), operated under basic conditions, and 3 cm length of the same stationary phase (30 x 4.6 mm, 2.7 mu m d.p. column), under acidic conditions, for fast second dimension analysis.