Two‐dimensional separation of peptides using RP‐RP‐HPLC system with different pH in first and second separation dimensions

Abstract

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.

Analysis of host-cell proteins in biotherapeutic proteins by comprehensive online two-dimensional liquid chromatography/mass spectrometry

“…In recent years, a different 2D-LC methodology has been developed, based on the RP separation under basic pH (pH 10) conditions in the first dimension and a low-pH RP separation in the second dimension. [26][27][28][29][30][31][32][33][34][35][36][37][38][39] In all reported work using capillary chromatography (e.g., 300 μm ID columns), [26][27][28][29][30][31][32][33][34][35][36] the first LC dimension was operated in an off-line mode using fraction collection, followed by excess organic solvent evaporation mAbs Fig. 2B) from the second dimension (lowpH) separations in four consecutive injections (experiments).…”

Comprehensive Proteomic and Metabolomic Signatures of Nontypeable Haemophilus influenzae-Induced Acute Otitis Media Reveal Bacterial Aerobic Respiration in an Immunosuppressed Environment

“…The method used two-dimensional liquid chromatography in a high-low pH reversed phase/reversed phase configuration on a nanoACQUITY UltraPerformance liquid chromatography system coupled to a Synapt G2 High Definition Mass Spectrometer (both Waters Corporation) with nanoelectrospray ionization as similarly described previously (35)(36)(37). Peptides were first trapped at 2 l/min at 97/3 v/v 20 mM ammonium formate in water, pH 10/acetonitrile on a 5 m XBridge BEH130 C18 300 m ϫ 50 mm column (Waters Corporation).…”

Capillary Electrophoresis as a Second Dimension to Isoelectric Focusing for Peptide Separation

“…2,3 But, as has been shown in a recent study, other HPLC modes could also be considered: for example, Gilar et al showed that the combination of two RP-HPLC separations, if performed at two significantly different pHs (2.6 and 10.0), can be an interesting two-dimensional separation method with rather orthogonal separation principles. 4 Although chromatographic techniques are widely used, it has already been shown that capillary electrophoresis (CE) can represent a very interesting alternative. Indeed, in comparison to HPLC, CE provides higher efficiencies and separation speeds.…”

“…The tryptic digest of a four-protein mixture has been considered as a test sample. IEF fractionation by OGE has been performed on a wide pH range (3)(4)(5)(6)(7)(8)(9)(10), and different experimental conditions to perform CE have been tested. Also, to know whether CZE can really be considered as a method complementary to OGE, some significant OGE fractions have also been analyzed by RP-HPLC.…”