Evaluation of Comprehensive Multidimensional Separations Using Reversed-Phase, Reversed-Phase Liquid Chromatography/Mass Spectrometry for Shotgun Proteomics

Abstract

Two-dimensional liquid-chromatographic (LC) separation followed by mass spectrometric (MS) analysis was examined for the identification of peptides in complex mixtures as an alternative to widely used two-dimensional gel electrophoresis followed by MS analysis for use in proteomics. The present method involves the off-line coupling of a narrow-bore, polymer-based, reversed-phase column using an acetonitrile gradient in an alkaline mobile phase in the first dimension with octadecylsilanized silica (ODS)-based nano-LC/MS in the second dimension. After the first separation, successive fractions were acidified and dried off-line, then loaded on the second dimension column. Both columns separate peptides according to hydrophobicity under different pH conditions, but more peptides were identified than with the conventional technique for shotgun proteomics, that is, the combination of a strong cation exchange column with an ODS column, and the system was robust because no salts were included in the mobile phases. The suitability of the method for proteomics measurements was evaluated.

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).…”

“…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.…”

A Fast Workflow for Identification and Quantification of Proteomes

“…Previous studies have revealed the outstanding capability of 2-D RP-RP system in peptide separation (13,24,37). RP-RP approaches using two different pH values were further demonstrated to have good potential in fractionating complex protein mixtures (14,38,39). Based on previous knowledge, we chose an opposite way from current proteomics strategies by using a dual-short RP/RPLC-MS/MS approach (Fast-seq strategy) in proteome deep-coverage (Fig.…”

Hydrophobic Interaction Chromatography: Fundamentals and Applications in Biomedical Engineering

“…Then, total or partial depletion of these proteins allows detecting low abundance proteins. Different methods can be used to deplete these proteins, being liquid chromatography the most popular one (Nakamura et al, 2008). Different chromatographic strategies are available for this purpose, including affinity dye-based chromatography for albumin depletion, affinity to protein A and G for immunoglobulin depletion, specific antibodyaffinity columns (Linke et al, 2007), and affinity columns containing lectins, peptides or inorganic ligands (Salih, 2005).…”