J. Sep. Sci. 2005, 28, 1694 – 1703 www.jss-journal.de i 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Martin Gilar Petra Olivova Amy E. Daly John C. Gebler Waters Corporation, 34 Maple St., Milford, MA 01757, USA Two-dimensional separation of peptides using RP- RP-HPLC system with different pH in first and second separation dimensions Two-dimensional high performance liquid chromatography is a useful tool for pro- teome analysis, providing a greater peak capacity than single-dimensional LC. The most popular 2D-HPLC approach used today for proteomic research combines strong cation exchange and reversed-phase HPLC. We have evaluated an alternative mode for 2D-HPLC of peptides, employing reversed-phase columns in both separation dimensions. The orthogonality of 2D separation was investigated for selected types of RP stationary phases, ion-pairing agents and mobile phase pH. The pH appears to have the most significant impact on the RP-LC separation selectivity; the greatest orthogonality was achieved for the system with C18 columns using pH 10 in the first and pH 2.6 in the second LC dimension. Separation was performed in off-line mode with partial fraction evaporation. The achievable peak capacity in RP-RP-HPLC and overall performance compares favorably to SCX-RP-HPLC and holds promise for proteomic analysis. Key Words: Two-dimensional; Liquid chromatography; Peptides; Proteomic; Orthogonal; Received: March 15, 2005; revised: April 20, 2005; accepted: April 22, 2005 DOI 10.1002/jssc.200500116 1694 Gilar, Olivova, Daly, Gebler 1 Introduction Despite the recent progress in ultrahigh-performance liquid chromatography [1, 2] and mass spectrometry (MS), the liquid chromatography (LC)-MS analysis of highly complex proteomic samples remains a challenging task [3 – 5]. Since the top-down LC-MS analysis of intact proteins is difficult [6 – 8], the proteomic analysis is usually performed on a peptide level after sample proteolysis with trypsin (or alternative enzymes). However, the tryptic cleavage generates multiple peptides per protein, proteo- mic samples typically consist of hundreds of thousands of peptides. To date, no separation method is capable of resolving so many components in a single analytical dimension prior to the MS analysis. Consequently, multi- ple peptides enter the mass spectrometer at any given time and overwhelm the MS/MS capability of the instru- ment. This results in a reduced number of peptide identifi- cations, and greatly adds to the LC-MS/MS analysis (and database search) variability [3, 9, 10]. Off-line and on-line two-dimensional high-performance liquid chromatography (2D-HPLC) separations have been applied for analysis of proteomic samples, both having unique advantages and disadvantages [3, 11–13]. While the on-line approach minimizes the manipulation with the sample and its potential losses, it requires a sophisticated instrumental setup with additional pumps, switching valves, and trapping columns [14]. The requirements for the mobile phase compatibility for direct fraction transfer to the second separation dimension narrows the choice of useful LC separation modes. An off-line approach is sim- pler to implement, for example, via an appropriate fraction collector. The collected fractions can be further pro- cessed, concentrated and/or chemically modified prior to analysis in the second LC-MS dimension. Therefore, the off-line 2D-HPLC has less stringent requirements on mobile phase compatibility, and offers more freedom for timing of analysis. The implementation of both 2D-HPLC approaches is further complicated by the fact that analysis is often performed in capillary- or nano-LC format. Dedi- cated and well maintained instrumentation is absolutely essential to maintain data quality. Current 2D-HPLC approaches for proteomic analysis usually combine a strong cation exchange (SCX) chroma- tography with reversed-phase (RP) HPLC [3, 9, 11]. Besides the differences in selectivity, the rationale for the choice is also a good compatibility of SCX mobile phases with the second separation dimension (RP). The peptides/ salt fractions can be directly introduced on a RP-HPLC; while peptides are retained on sorbent, salts (required to elute peptides from SCX) are washed out unretained. High concentrations of salts are typically diverted to waste via an additional switching valve in order to avoid the contam- ination of the MS ionization source. The choice of mode in the second LC dimension is generally RP, due to its high Correspondence: Dr. Martin Gilar, Life Sciences Chemistry R & D, Waters Corporation, 34 Maple St., Milford, MA 01757, USA. Tel: +1 508 482 2000. E-mail: Martin_Gilar@waters.com