Two‐dimensional reverse phase‐reverse phase chromatography: A simple and robust platform for sensitive quantitative analysis of peptides by LC/MS. Hardware design

Abstract

We have revised current two-dimensional RP-RP approaches and developed a new robust 2-D RP-RP platform. This platform was implemented on an Agilent 1100 2-D liquid chromatography system and is based on high pressure switching between two high-resolution RP columns. An independent binary gradient was implemented for each dimension. The powerful combination of dual analytical columns with independent gradient elution achieves high analyte purity, effectively eliminates matrix effects, and maximizes MS sensitivity in Q1 SIM comparable to the sensitivity enhancements of MS/MS-based methods. Implementation of dual simultaneous gradient profiles (overlapped gradients) reduces 2-D method run-time to the scale of 1-D method run-times. This robust and sensitive approach is particularly suitable for hydrophobic peptides and small proteins and can be used as a routine standard technique for enhanced on-line peptide purification coupled with mass spectrometric detection.

The human embryonic stem cell proteome revealed by multidimensional fractionation followed by tandem mass spectrometry

“…The discrepancy between the two datasets not only relates to the biological variations between cell lines and differences in the analyzing techniques but also reflects upon the complexity of the hES cell proteome. It is noteworthy that the two RP LC separations in our experiments were both operated under acidic condition, which could be improved to provide better orthogonality by employing two RP‐LC systems under different pHs or pressures .…”

Synthesis and chromatographic evaluation of phenyl/tetrazole bonded stationary phase based on thiol‐epoxy ring opening reaction

“…RP/RP 2‐D LC is the ideal mode for 2‐D chromatography due to the separation efficiency and mobile phase compatibility. The widely used RP/RP 2‐D LC method was constructed with reversed‐phase separation under different pH in first and second dimensions for the separation of peptides . But for non‐ionic compounds, this kind of pH‐based 2‐D LC system was not suitable.…”

“…In summary, the PTAS stationary phase showed good selectivity and peak shape for several kinds of polar compounds, demonstrating that it was a good complement to the C18 column. reversed-phase separation under different pH in first and second dimensions for the separation of peptides [31][32][33][34][35][36]. But for non-ionic compounds, this kind of pH-based 2-D LC system was not suitable.…”

Application of Fused-Core Particle Column in Two Dimensional Reversed Phase - Reversed Phase LC/MS Analysis of Biological Samples. Impact of Extra-Column Volume

“…We have previously reported a 2D RP-RP LC/MS method designed for measurement of human C-peptide and endogenous peptide hormones in biological samples [10,11]. In the first dimension, we used a 2×100 mm, 5 µm Jupiter C5 column.…”