31
CGE buffer from SCIEX). Figure 7 shows a separation of denaturated and native IgG, and a comparison with the conventional slab gel. This work spurred a lot of different developments of this application for recombinant IgG quality control [40]. A protocol to identify 0.5% of an impurity profiling in a recombinant antibody sample was precisely detailed by Le Potier et al. [41].
Good 2D separations were achieved by Zhu et al. The sample underwent separation in a first-dimension capillary by sieving electrophoresis (SE). Fractions were periodically transferred across an interface into a second-dimension capillary, where components were further resolved by MEKC. Five SE and five MEKC capillaries allowed separation of five samples in parallel. The FQ-labelled samples were injected into the five first-dimension capillaries, fractions were transferred across an interface to five second- dimension capillaries, and the analytes were detected in a sheath-flow cuvette containing five capillaries. A complex protein mixture of an A549 cell-line homogenate was separated in such manner [42].
d) Nucleic acids. Figure 5: CE/LIF of AA and biogenic amines.
I) Electropherogram of a plasma sample labelled by FITC where Taurine (Tau) can be identified. Separation conditions: 20 mmol/L tribasic sodium phosphate pH 11.8, 23°C, 22 kV [31]. II) Separation of CBQCA-AAs in a normal plasma sample of a child. (1 and 2): Lys; (4): Cit; (5): Gln; (6): Ans; (7): Asn; (9): Tyr; (10): Orn; (11): b-Ala; (12): Carnosine; (14): Ser; (15): Gly; (16): Ala; (18): Tau; (19): L-a-amino-butyric acid; (20): Val; (22): Met; (23): Thr; (24): N-Val; (25): Ile; (26): Phe; (27): Leu; (30): Glu; (31): Arg; (32): Asp. BGE:160 mM borate, 130 mM SDS, 7.5 mM g-CD and 20 mM NaCl at a pH of 9.5; capillary: 67 cm length (60 cm to detector) and 50 µm id. 30 kV [34].
c) Proteins.
The main problem in using CE/LIF to study proteins is that only Trp and Tyr are fluorescent, and those AA are not abundant in proteins. In consequence, very few studies can be run using CE/LIF without labelling the proteins.
As for AA analysis, different dyes can be used: 5-carboxy-tetramethylrhodamine succinimidyl ester (
5-TAMRA.SE, excitation at 488 or 532 nm), CBQCA (excitation at 488 nm), 3-(2-furoyl)-quinoline-2- carboxyaldehyde (FQ) (excitation at 488 nm) or 2, 3-naphthalenedialdehyde (NDA). NDA is a fluorogenic dye, excited at 442 nm using Helium Cadmium Laser, or a 450 nm LED can also be used. These labelling reagents react mainly on the lateral chain of Lysine.
Since this AA is quite abundantly present in proteins, many different labelled species can be seen, which will cause enlargements of the peaks in the electropherograms.
The two main applications concerning CE/ LIF applications were the applications concerning recombinant IgG purities and proteomic approaches using this technique to quantify proteins.
The impurities from pharmaceutical recombinant IgG were studied by Hunt and Nashabeh [39]. They used a purification step after the IgG labelling with 5-TAMRA. SE using a NAP-5 column (Pharmacia) which allowed them to remove excess dye. The separation was run using a non-gel sieving media called Biorad SDS buffer (no longer available but can be replaced by SDS-
Since the work of Drossman et al. [43], where the authors claimed to have rapidly separated fluorescently labelled DNA fragments generated in sequencing reactions, DNA sequencers were developed by different companies (Promega, Thermo Fisher Scientific...). In the last ten years, Berezovski et al. [44] proposed a method called ‘non-equilibrium capillary electrophoresis of equilibrium mixtures’ (NECEEM), to select aptamers from a DNA library, involving repetitive steps of partitioning without amplification between them. While the authors contributed to a real, major innovation in aptamers selection, the problem remains that for now it is impossible to detect the peak of the selected aptamer. Nevertheless, it can be collected and sequenced using the new generation of DNA sequencers as Illumina [45.
Currently, there is an ongoing effort to replace Northern-blot electrophoresis and autoradiography by CE/LIF. As an example, the separation of RNA fragments ranged from 100 to 10,000 nucleotides (nt) in polyethylene glycol (PEG) and polyethylene oxide (PEO) solutions with different molecular weight and different concentrations has been developed. The separation of small RNA fragments (<1000 nt) was improved with the increase of polymer concentration, whereas the separation performance for the large ones
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