Investigating cationic and zwitterionic successive multiple ionic-polymer layer coatings for protein separation by capillary electrophoresis.

Successive multiple ionic-polymer layers (SMILs) have long since proved their worth in capillary electrophoresis as they ensure stable electroosmotic flow (EOF) and relatively high separation efficiency. Recently, we demonstrated that plotting the plate height (H) against the solute migration velocity (u) enabled a reliable quantitative evaluation of the coating performances in terms of separation efficiency. In this work, various physicochemical and chemical parameters of the SMIL coating were studied and optimized in order to decrease the slope of the ascending part of the H vs u curve, which is known to be controlled by the homogeneity in charge of the coating surface and by the possible residual solute adsorption onto the coating surface. SMILs based on poly(diallyldimethylammonium chloride) (PDADMAC) and poly(sodium styrene sulfonate) (PSS) were formed and the effect of each polyelectrolyte molar mass and of the number of polyelectrolyte layers (up to 21 layers) was studied. The use of polyethylene imine as an anchoring first layer was considered. More polyelectrolyte couples based on PDADMAC, polybrene, PSS, poly(vinyl sulfate), and poly(acrylic acid) were tested. Finally, zwitterionic polymers based on the poly(α-l-lysine) scaffold were synthesized and used as the last layer of SMILs, illustrating their ability to finetune the EOF, while maintaining good separation efficiency.

Augmented region of interest for untargeted metabolomics mass spectrometry (AriumMS) of multi-platform-based CE-MS and LC-MS data.

In mass spectrometry (MS)-based metabolomics, there is a great need to combine different analytical separation techniques to cover metabolites of different polarities and apply appropriate multi-platform data processing. Here, we introduce AriumMS (augmented region of interest for untargeted metabolomics mass spectrometry) as a reliable toolbox for multi-platform metabolomics. AriumMS offers augmented data analysis of several separation techniques utilizing a region-of-interest algorithm. To demonstrate the capabilities of AriumMS, five datasets were combined. This includes three newly developed capillary electrophoresis (CE)-Orbitrap MS methods using the recently introduced nanoCEasy CE-MS interface and two hydrophilic interaction liquid chromatography (HILIC)-Orbitrap MS methods. AriumMS provides a novel mid-level data fusion approach for multi-platform data analysis to simplify and speed up multi-platform data processing and evaluation. The key feature of AriumMS lies in the optimized data processing strategy, including parallel processing of datasets and flexible parameterization for processing of individual separation methods with different peak characteristics. As a case study, Saccharomyces cerevisiae (yeast) was treated with a growth inhibitor, and AriumMS successfully differentiated the metabolome based on the augmented multi-platform CE-MS and HILIC-MS investigation. As a result, AriumMS is proposed as a powerful tool to improve the accuracy and selectivity of metabolome analysis through the integration of several HILIC-MS/CE-MS techniques.

Critical parameters for highly efficient and reproducible polyelectrolyte multilayer coatings for protein separation by capillary electrophoresis.

Since the introduction of polyelectrolyte multilayers to protein separation in capillary electrophoresis (CE), some progress has been made to improve separation efficiency by varying different parameters, such as buffer ionic strength and pH, polyelectrolyte nature and number of deposited layers. However, CE is often overlooked as it lacks robustness compared to other separation techniques. In this work, critical parameters for the construction of efficient and reproducible Successive multiple ionic-polymer layers (SMIL) coatings were investigated, focusing on experimental conditions, such as vial preparation and sample conservation which were shown to have a significant impact on separation performances. In addition to repeatability, intra- and inter-capillary precision were assessed, demonstrating the improved capability of poly(diallyldimethylammonium chloride) / poly(sodium styrene sulfonate) (PDADMAC / PSS) coated capillaries to separate model proteins in a 2 M acetic acid background electrolyte when all the correct precautions are put in place (with run to run%RSD(tm) < 1.8%, day to day%RSD(tm) < 3.2% and cap to cap%RSD(tm) < 4.6%). The approach recently introduced to calculate retention factors was used to quantify residual protein adsorption onto the capillary wall and to assess capillary coating performances. 5-layer PDADAMAC / PSS coatings led to average retention factors for the five model proteins of ∼4×10-2. These values suggest a relatively low residual protein adsorption leading to reasonably flat plate height vs linear velocity curves, obtained by performing electrophoretic separations at different electrical voltages (-10 to -25 kV).

Successive Multiple Ionic-Polymer Layer Coatings for Intact Protein Analysis by Capillary Zone Electrophoresis-Mass Spectrometry: Application to Hemoglobin Analysis.

Adsorption of analytes, e.g., proteins, often interfere with separation in CE, due to the relatively large surface of the narrow capillary. Coatings often are applied to prevent adsorption and to determine the electroosmotic flow (EOF), which is of major importance for the separation in CE. Successive multiple ionic-polymer layer (SMIL) coatings are frequently used for protein analysis in capillary electrophoresis resulting in high separation efficiency and repeatability. Here, the coating procedure of a five-layer SMIL coating is described using quaternized diethylaminoethyl dextran (DEAEDq) as polycation and poly(methacrylic acid) (PMA) as polyanion. Depending on the analyte, different polyions may be used to increase separation efficiency. However, the coating procedure remains the same.To demonstrate the applicability of SMIL coatings in CE-MS, human hemoglobin was measured in a BGE containing 2 M acetic acid. DEAEDq-PMA coating was found to be the most suitable for hemoglobin analysis due to relatively low reversed electroosmotic mobility leading to increased electrophoretic resolution of closely related proteoforms. Thereby, not only alpha and beta subunit of the hemoglobin could be separated, but also positional isoforms of glycated and carbamylated species were separated within 24 min.