Fast separations of intact proteins by liquid chromatography

Wang, Fei; Min, Yi; Geng, Xindu

doi:10.1002/jssc.201200339

Cited by 23 publications

(16 citation statements)

References 105 publications

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“…Since the resolution and selectivity of this MMC column were comparable to the conventional IEC and HIC columns, and it also had much higher bioactivity recovery and mass recovery for protein under both HIC and WCX modes, the single MMC column could replace two individual IEC and HIC columns to separate proteins in both WCX and HIC modes. In addition, as HIC and WCX modes are orthorhombic, this dual-retention mechanism MMC column can be called a '2D column' Wang et al, 2012), which can be used to set up 2DLC with a single '2D column' as 2DLC-1C.…”

Section: The Reproducibility Of the Wcx/hic Dual-retention Mechanism mentioning

confidence: 99%

“…Recently, our group synthesized a novel IEC/HIC (Zhao et al, 2012) dual-retention mechanism MMC column with the ligand containing the ion-exchange and hydrophobic groups. A column was reported to separate proteins with a single column in IEC and HIC modes, which was called a two-dimensional column (2D column; Geng et al, 2009;Wang et al, 2012). Later, Yang et al (2013) employed the 2D column for on-line fast separation and purification of cytochrome c (Cyt C) from bovine pancreas.…”

Section: Introductionmentioning

confidence: 99%

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Preparation and characterization of a novel dual‐retention mechanism mixed‐mode stationary phase with PEG 400 and succinic anhydride as ligand for protein separation in WCX and HIC modes

Song

Wang

Zhao

et al. 2013

Biomedical Chromatography

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A novel dual-retention mechanism mixed-mode stationary phase based on silica gel functionalized with PEG 400 and succinic anhydride as the ligand was prepared and characterized by infrared spectra and elemental analysis. Because of the ligand containing PEG 400 and carboxyl function groups, it displayed hydrophobic interaction chromatography (HIC) characteristic in a high-salt-concentration mobile phase, and weak cation exchange chromatography (WCX) characteristic in a low-salt-concentration mobile phase. As a result, it can be employed to separate proteins with both WCX and HIC modes. The resolution and selectivity of the stationary phase was evaluated under both HIC and WCX modes with protein standards, and its performance was comparable to that of conventional ion-exchange chromatography and HIC columns. The results indicated that the novel dual-retention mechanism column, in many cases, could replace two individual WCX and HIC columns as a '2D column'. In addition, the mixed retention mechanism of proteins on this '2D column' was investigated with stoichiometric displacement theory for retention of solute in liquid chromatography in detail in order to understand why the dual-retention mechanism column has high resolution and selectivity for protein separation under WCX and HIC modes, respectively. Based on this '2D column', a new 2DLC technology with a single column was developed. It is very important in proteome research and recombinant protein drug production to save column expense and simplify the processes in biotechnology.

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Section: The Reproducibility Of the Wcx/hic Dual-retention Mechanism mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Preparation and characterization of a novel dual‐retention mechanism mixed‐mode stationary phase with PEG 400 and succinic anhydride as ligand for protein separation in WCX and HIC modes

Song

Wang

Zhao

et al. 2013

Biomedical Chromatography

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“…Intact proteins have been traditionally analyzed on conventional C 18 packed silica particle columns; however, due to the complex structure of biomolecules, many issues have limited the kinetic performances such as the low diffusion coefficient, the irreversible adsorption onto column, the secondary ionic interactions and/or the high backpressure values [12,13]. Compared with silica-based stationary phases, protein recovery from monolithic columns are certainly superior.…”

Section: Introductionmentioning

confidence: 99%

Separation of intact proteins on γ‐ray‐induced polymethacrylate monolithic columns: A highly permeable stationary phase with high peak capacity for capillary high‐performance liquid chromatography with high‐resolution mass spectrometry

Simone

Pierri

Foglia

et al. 2015

J of Separation Science

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Polymethacrylate-based monolithic capillary columns, prepared by γ-radiation-induced polymerization, were used to optimize the experimental conditions (nature of the organic modifiers, the content of trifluoroacetic acid and the column temperature) in the separation of nine standard proteins with different hydrophobicities and a wide range of molecular weights. Because of the excellent permeability of the monolithic columns, an ion-pair reversed-phase capillary liquid chromatography with high-resolution mass spectrometry method has been developed by coupling the column directly to the mass spectrometer without a flow-split and using a standard electrospray interface. Additionally, the high working flow and concomitant high efficiency of these columns allowed us to employ a longer column (up to 50 cm) and achieve a peak capacity value superior to 1000. This work is motivated by the need to develop new materials for high-resolution chromatographic separation that combine chemical stability at elevated temperatures (up to 75°C) and a broad pH range, with a high peak capacity value. The advantage of the γ-ray-induced monolithic column lies in the batch-to-batch reproducibility and long-term high-temperature stability. Their proven high loading capacity, recovery, good selectivity and high permeability, moreover, compared well with that of a commercially available poly(styrene-divinylbenzene) monolithic column, which confirms that such monolithic supports might facilitate analysis in proteomics.

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“…20 However, the separation speed depends on the complexity of the sample. 2,[21][22][23][24][25][26][27][28][29] This choice is particularly important in developing proteomics in which intact proteins are fragmented directly in the mass spectrometer to achieve both protein identification and characterization, even capturing information on combinatorial post-translational modifications. 2,[21][22][23][24][25][26][27][28][29] This choice is particularly important in developing proteomics in which intact proteins are fragmented directly in the mass spectrometer to achieve both protein identification and characterization, even capturing information on combinatorial post-translational modifications.…”

Section: Introductionmentioning

confidence: 99%

Two variables dominating the retention of intact proteins under gradient elution with simultaneous ultrafast high-resolution separation by hydrophobic interaction chromatography

Geng

Liu

Wang

et al. 2015

Analyst

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The retention of intact proteins under gradient elution in hydrophobic interaction chromatography (HIC) was found to be governed by two variables, the steady region (SR) and the migration region (MR). In the SR, the proteins are immobilized by the strong interactions with the stationary phase such that the retention time is independent of the column length. In the MR, the proteins also interact with the stationary phase, but they move normally, thus the retention time depends on their partition coefficients and the column length. The SR can be used as an operation space (OP) for high-throughput protein analysis by 1D-LC using short columns at high flow rates to maintain a high resolution. The OP can also be employed for all assisted operations in online 2D-LC. Based on the steady region/migration region optimization strategy developed in this study, five successive complete separations of seven intact proteins were performed in a HIC cake in less than 5 min, and a crude extract of ribonuclease A from bovine pancreas was purified using online 2D-LC to 95.8% purity with 93.2% mass recovery in 45 min. This approach can be used to expedite the purification of drug-target proteins and should therefore be of interest to the pharmaceutical industry.

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Fast separations of intact proteins by liquid chromatography

Cited by 23 publications

References 105 publications

Preparation and characterization of a novel dual‐retention mechanism mixed‐mode stationary phase with PEG 400 and succinic anhydride as ligand for protein separation in WCX and HIC modes

Preparation and characterization of a novel dual‐retention mechanism mixed‐mode stationary phase with PEG 400 and succinic anhydride as ligand for protein separation in WCX and HIC modes

Separation of intact proteins on γ‐ray‐induced polymethacrylate monolithic columns: A highly permeable stationary phase with high peak capacity for capillary high‐performance liquid chromatography with high‐resolution mass spectrometry

Two variables dominating the retention of intact proteins under gradient elution with simultaneous ultrafast high-resolution separation by hydrophobic interaction chromatography

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