Preparation of a capillary isoelectric focusing column with monolithic immobilized pH gradient and its application on protein separation based on an online capillary isoelectric focusing platform

Liu, Rangdong; Pan, Hong; Zheng, Yiting; Hu, Jing; Cheddah, Soumia; Wang, Weiwei; Wang, Yan; Yan, Chao

doi:10.1002/elps.201900125

Cited by 9 publications

(4 citation statements)

References 34 publications

(35 reference statements)

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“…In a similar approach, a CIEF column with monolithic immobilized pH gradient was prepared [116]. First, in the presence of MeOH and decan‐1‐ol as porogens, a partially filled capillary monolithic column was prepared by in situ reaction of glycidyl methacrylate and poly(ethylene glycol) diacrylate.…”

Section: Separations By the Particular Ce And Cec Methodsmentioning

confidence: 99%

Recent developments in capillary and microchip electroseparations of peptides (2019–mid 2021)

Kašička

2021

Electrophoresis

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The review provides a comprehensive overview of developments and applications of high performance capillary and microchip electroseparation methods (zone electrophoresis, isotachophoresis, isoelectric focusing, affinity electrophoresis, electrokinetic chromatography, and electrochromatography) for analysis, microscale isolation, and physicochemical characterization of peptides from 2019 up to approximately the middle of 2021. Advances in the investigation of electromigration properties of peptides and in the methodology of their analysis, such as sample preparation, sorption suppression, EOF control, and detection, are presented. New developments in the individual CE and CEC methods are demonstrated and several types of their applications are shown. They include qualitative and quantitative analysis, determination in complex biomatrices, monitoring of chemical and enzymatic reactions and physicochemical changes, amino acid, sequence, and chiral analyses, and peptide mapping of proteins. In addition, micropreparative separations and determination of significant physicochemical parameters of peptides by CE and CEC methods are described.

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Section: Separations By the Particular Ce And Cec Methodsmentioning

confidence: 99%

Recent developments in capillary and microchip electroseparations of peptides (2019–mid 2021)

Kašička

2021

Electrophoresis

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“…It is also possible in CIEF to use immobilized pH gradients as applied in slab gels. This technique is still under development, but can bring many advantages [6, 8–10]. In this case, the imaged CIEF with carrier ampholytes was used, i.e., an on‐line imaging detection system that does not require the mobilization of the analytes after focusing [11].…”

Section: Rbd/fc Rbd/his S1/his S1/s2/his Hace2/hismentioning

confidence: 99%

Isoelectric point determination by imaged CIEF of commercially available SARS‐CoV‐2 proteins and the hACE2 receptor

et al. 2021

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In order to contribute to the scientific research on the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), we have investigated the isoelectric points (pI) of several related proteins, which are commercially available: the receptor‐binding domain (RBD) with His‐ and Fc‐tag, the S1 subunit with His‐tag, the S1/S2 subunits with His‐tag and the human angiotensin‐converting enzyme 2 (hACE2) with His‐tag. First, the theoretical pI values, based on the amino acid (AA) sequences of the proteins, were calculated using the ProtParam tool from the Bioinformatics Resource Portal ExPASy. The proteins were then measured with the Maurice imaged CIEF system (native fluorescence detection), testing various measurement conditions, such as different ampholytes or ampholyte mixtures. Due to isoforms, we get sections with several peaks and not just one peak for each protein. The determined pI range for the RBD/Fc is 8.24–9.32 (theoretical pI: 8.55), for the RBD/His it is 7.36–9.88 (8.91) and for the S1/His it is 7.30–8.37 (7.80). The pI range of the S1/S2/His is 4.41–5.87 (no theoretical pI, AA sequence unknown) and for hACE2/His, the determined global range is 5.19–6.11 (5.60) for all experimental conditions chosen. All theoretically derived values were found within these ranges, usually close to the center. Therefore, we consider theoretical values as useful to make predictions about the isoelectric points of SARS‐CoV‐2 proteins. The experimental conditions had only a minor influence on the pI ranges obtained and mainly influenced the peak shapes.

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“…iCIEF separates proteins with different p I s in the pH range of the ampholyte [28–30]. As long as the p I values of the proteins are different, it is possible to separate them, and the separation sensitivity is high [31–33]. The PEG‐UHC was obtained by modifying the ε‐amino group of lysine residues in the UHC protein to near‐saturation with mPEG‐SPA‐5k.…”

Section: Discussionmentioning

confidence: 99%

Determination of PEGylation homogeneity of polyethylene glycol‐modified canine uricase

Huo

Shen

et al. 2020

Electrophoresis

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Polyethylene glycol‐modified canine uricase (PEG‐UHC) was prepared by modifying the ε‐amino group of lysine residues on the canine uricase (UHC) protein to near‐saturation with 5 kDa monomethoxyl‐polyethylene glycol succinimide (mPEG‐SPA‐5k). In order to accurately determine the PEGylation uniformity of PEG‐UHC, CZE, 3–8% gradient gel SDS‐PAGE, and imaging CIEF (iCIEF) analyses were compared. CZE could not effectively separate PEG‐UHC proteins with different degrees of modification, 3–8% gradient gel SDS‐PAGE could separate PEG‐UHC into seven gel bands; however, most of the gel bands were smeared or blurred, and the separation of PEG‐UHC samples by iCIEF was significantly better than that by 3–8% gradient gel SDS‐PAGE. Under denatured conditions, iCIEF separated 12 pI peaks, and could also accurately quantify the relative monomer PEG‐UHC content. More than 85% of the total monomeric PEG‐UHC was conjugated with 7–12 PEG molecules; of this 85%, approximately 40% was conjugated with 9–10 PEG molecules. These results demonstrated that iCIEF exhibits good potential for determining the PEGylation homogeneity of PEGylated protein drugs.

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Preparation of a capillary isoelectric focusing column with monolithic immobilized pH gradient and its application on protein separation based on an online capillary isoelectric focusing platform

Cited by 9 publications

References 34 publications

Recent developments in capillary and microchip electroseparations of peptides (2019–mid 2021)

Recent developments in capillary and microchip electroseparations of peptides (2019–mid 2021)

Isoelectric point determination by imaged CIEF of commercially available SARS‐CoV‐2 proteins and the hACE2 receptor

Determination of PEGylation homogeneity of polyethylene glycol‐modified canine uricase

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