3x More Proteoforms With Online 2D nanoLC-CE-MS

Summary. Researchers at Aalen University and Kiel University in Germany reported in Analytical Chemistry, published online on 14 October, a comprehensive online coupling of nanoflow liquid chromatography with capillary zone electrophoresis-mass spectrometry (2D nanoLCxCZE-MS) for deep top-down proteomics. Top-down proteomics analyses intact proteins to characterize proteoforms, the different molecular forms in which a single protein can exist after processing or modification. Capillary zone electrophoresis separates proteoforms well but accepts only small sample loads, which has limited its use; placing a reversed-phase nanoLC separation in front of it in a single automated online system addresses that limitation.

Research context (RUO). The authors describe a highly automated platform with robust performance that also supports proteoform quantitation, which they demonstrated with isobaric labelling experiments. Because reversed-phase liquid chromatography and capillary zone electrophoresis separate proteins by largely independent properties, the combined system reached a peak capacity of about 2,200 and roughly tripled the number of identified proteoforms in a human cell-line lysate compared with nanoLC-MS alone. Electrophoretic mobilities measured by CZE also allowed many more proteoforms to be assigned to a proteoform family at the MS1 level, before fragmentation. The authors present the platform as flexible and suitable for both untargeted and targeted analysis of complex biological samples. The paper is an analytical-methods report with no clinical component. For mass-spectrometry and protein-characterization laboratories, it offers a route to deeper separation of intact proteins and their modified forms than one-dimensional workflows provide.