Analysis of intact proteins with capillary zone electrophoresis coupled to mass spectromery using uncoated and coated capillaries

• Published in: Journal of Chromatography A 2021-09, Vol.1654, p.462448, Article 462448
• Main Authors: Hamidli, N., Andrasi, M., Nagy, C., Gaspar, A.
• Format: Article
• Language: English
• Subjects: Bare fused silica; Capillary electrophoresis; Intact proteins; Linear polyacrylamide; Mass spectrometry; Polybrene; Top-down
• ISSN: 0021-9673
• DOI: 10.1016/j.chroma.2021.462448

•Intact proteins were separated by CZE-MS in different capillaries.•In bare fused silica capillaries minimal adsorption was observed at pH 1.8.•At pH=1.8 the FS capillary provided the highest N values for the studied proteins.•LPA coated capillary showed the most stable separations (0.18-0.49 RSD%).•PB coated capillary provided the highest resolution between hemoglobin variants. 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.