Abstract
Although, in general, the application of coated capillaries is recommended for the separation of intact proteins, bare silica capillary is still the most often used capillary due to its simplicity and cheapness. In this work, the performance of bare fused silica capillary for intact protein analysis was compared to that of different (dynamically coated polybrene (PB) and permanently coated linear polyacrylamide (LPA)) coated capillaries using capillary zone electrophoresis - mass spectrometry (CZE-MS). In cases where low pH (pH=1.8) was used in bare silica capillaries, good precision (0.56-0.78 RSD% and 1.7-6.5 RSD% for migration times and peak areas, respectively), minimal adsorption and separation efficiency (N= 27 000/m - 322 000/m) similar to or even better than those obtained with the coated capillaries (created by an intricate multi-step process) was achieved. The PB and the LPA capillaries demonstrated their slightly better resolving power in terms of separating the different forms/variants of the same protein (e.g., hemoglobin subunits). Among the studied capillaries the one with LPA coating showed the most stable separations in the long term (n=25: 0.18-0.49 RSD% and 3.1-4.9 RSD% for migration times and peak areas, respectively). For the separation of a few proteins or even a larger number of proteins in biological samples (e.g., snake venom) the application of the simple and cheap bare fused silica capillary can be considered as an efficient choice.
Highlights
Proteins and protein complexes perform vital roles in the cells, from transportation of metabolites and assisting biochemical reactions to regulating gene expression and ensuring structural stability
In order to find suitable conditions for the separation of intact proteins in a bare silica capillary, five different proteins were analysed using background electrolytes (BGE) with „extreme” pH values and pH more commonly used for CE separations
The narrowest peak was obtained in the case of BGE of pH=1.8 for insulin and HSA (Fig. 1), while in ”moderate” pH range, wider and shorter and tailed peaks were attained as a result of electrostatic interactions
Summary
Proteins and protein complexes perform vital roles in the cells, from transportation of metabolites and assisting biochemical reactions to regulating gene expression and ensuring structural stability. Many reviews [6,9,17,19,30,31] discussed and evaluated these results and applications These works clearly prefer the use of coated capillaries for intact protein separation to prevent the adsorption effects and to obtain constant migration times. In several papers the comparison of the different capillaries was made in an “unfair” way These studies typically employed the same separation conditions (which was ideal only for the coated capillary) for both coated and uncoated capillaries − neglecting the fact that the parameters (mainly the pH) may not be ideal for the bare silica capillary [22,25,26,31]. The bare silica capillary is well applicable for intact protein separation only in very low or very high pH conditions [34]
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