Automated Two-Dimensional Separation of Peptides by Ion-Pair Reversed-Phase High-Performance Liquid Chromatography-Electrospray Ionization-Mass Spectrometry at High and Low pH

IntroductIon Protein samples of biological origin can be very complex, and require powerful separation tools like multidimensional liquid chromatography (MDLC) to resolve components of interest. MDLC can be performed using on-line and off-line approaches. In the off-line MDLC approach, due to a fractionation step, a high flexibility in terms of column dimensions, optimization of flow rate, buffer composition, and pH can be realized for each separation dimension independently. The optimization of both separation dimensions can increase peak capacity, although to fully exploit the peak capacity, the separation mechanism used in the first-dimension should be orthogonal to that of the second-dimension separation. The combination of strong-cation-exchange and reversedphase chromatography (SCX/×/RP) is known for its high degree of orthogonality, but can have difficulties resolving higher valence tryptic peptides. However, reversedphase HPLC using low and high pH (RP/x/RP) offers an alternative approach. In this technical note, the off-line RP/×/RP 2-D LC approach is examined in terms of selectivity, separation efficiency, orthogonality, repeatability, and assessment as to how complementary it is to SCX/×/RP. EquIpmEnt Automated off-line two-dimensional chromatographic experiments were performed using an UltiMate® 3000 Proteomics MDLC system (Dionex Corporation) consisting of a dual ternary gradient pump (DGP-3600) with membrane degasser (SRD-3600), flow manager module (FLM-3100) equipped with a 1:100 flow splitter, autosampler with integrated micro-fraction collector (WPS-3000), and two UV detectors (VWD-3400). The second-dimension separation was performed using the reduced-solvent-consumption option.1 With this option, solvent consumption is reduced by a factor of 10, allowing continuous operation for up to one month.