Tryptic digest analysis by comprehensive reversed phase×two reversed phase liquid chromatography (RP‐LC×2RP‐LC) at different pH's

Abstract

As an alternative to the classical approach of combining strong cation exchange SCX-LC and RP-LC for the separation of complex proteomic samples, this essay describes the online comprehensive RP-LCxRP-LC separation of BSA and human blood serum. High orthogonality and peak capacity are achieved through the application of a significantly different pH in the two dimensions. The coupling of fused-core columns in series ensures high efficiency in the first dimension, while a previously designed interface with parallel second dimension columns further enhances the separation capability of the comprehensive system.

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).…”

“…The Hi3 quantification methodology is likely to be responsible for the discrepancy between these methods because the accuracy of this method is reported to be in the range of 20-40%. 38,39 In almost all measurements, the Hi3 method slightly overestimated the HCP concentrations. One explanation for this observation is that that the Hi3 method combines all potential HCP isoforms into one measurement, whereas the MRM method is isoform-specific.…”

“…26,27,30,31 This observation prompted much interest in coupling two RP columns, operated at two pH extremes (pH 10 and pH 2.5), as a 2D chromatographic system for peptide separation, using online or off-line configurations. [26][27][28][29][30][31][32][33][34][35][36][37][38][39] Although the coupling of high-pH RP/low-pH reversed-phase separations was shown to be less orthogonal than the classical SCX/RP multidimensional system for the separation of complex peptide mixtures in proteomic experiments, 30 the separation resolution offered by the high-pH RP in the first chromatographic dimension is far superior to the SCX separation. RP separation elutes peptides almost equally over the entire retention window (trapezoidal distribution of peptides) allowing for a greater spread of peptides across the same number of fractions.…”

Theory and Practice of UHPLC and UHPLC–MS

“…Desmet et al described a useful approach illustrating the influence of maximal backpressure (DP max ) on achievable N and t 0 , based on the Van Deemter data (u, H), column permeability (K v,0 ) and mobile phase viscosity (h) with Eqs. (1.4) and (1.5) (Desmet et al, 2005a(Desmet et al, ,b, 2006aBillen et al, 2007;Desmet and Cabooter, 2009;.…”

“…1.5 always correspond to a pressure drop equal to DP max with variable column lengths and mobile phase flow rates. For a better understanding of the employed procedure to construct this 2D map, readers can refer to a few didactic papers on kinetic plot methodology (Desmet et al, 2005a(Desmet et al, ,b, 2006aBillen et al, 2007;Desmet and Cabooter, 2009;.…”