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 proteome 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.
Gilar
Martin
M
Life Sciences Chemistry R&D, Waters Corporation, 34 Maple St., Milford, MA 01757, USA. Martin_Gilar@waters.com
Olivova
Petra
P
Daly
Amy E
AE
Gebler
John C
JC
D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D010455:Peptides / Q000737:chemistry* / Q000302:isolation & purification*; D020543:Proteome / Q000737:chemistry*
D007202:Indicators and Reagents; D010455:Peptides; D020543:Proteome