Résumé
Mass spectrometry–based top‐down protein analysis requires efficient separation. In the context of proteoform analysis, capillary zone electrophoresis (CZE) is very valuable. The resolution of two peaks in CZE can be increased when the absolute mobility of the counter‐directed electroosmotic flow (EOF) is close to the effective mobility of the analytes, resulting in a low apparent mobility of the analytes. The mobility of the EOF of highly efficient sulfobetaine‐modified poly(α‐L‐lysine) (α‐PLL) coatings changes depending on the number of modified side chains. Here, such coatings are used to selectively increase the peak resolution of proteoforms of model proteins and analytes in a complex protein sample (intact yeast protein extract). Whereas a high EOF system allows for the separation of proteins of a wide mobility range (complete proteome), lower EOF systems allow for a much better separation of proteins and proteoforms of low mobility, including those containing acidic post‐translation modifications (PTMs). This leads to the identification of 2.5 times more proteoforms by MS/MS experiments in the lower mobility range of the yeast proteome. The sulfobetaine‐modified α‐PLL coatings presented here exhibit a toolbox for highly resolved separation of proteins and proteoforms in targeted or untargeted top‐down protein analysis. Summary Sample complexity is one of the main challenges when analyzing a proteome on the proteoform level. In the course of this, capillary electrophoresis–mass spectrometry turned out to be an excellent tool because of its high‐performing separation, particularly for large molecules. Here, we present a method enabling the best possible separation due to efficient and EOF‐tunable coatings, allowing for flexible and dedicated selection of a range of proteins and proteoforms to be analyzed under ideal separation conditions. The high performance is demonstrated by the separation of proteoforms of common PTM‐rich model proteins as well as complex proteome samples.