Selective Comprehensive Online NanoLCxCZE-MS Platform for Top-Down Proteoform Analysis

Abstract

Efficient separation is crucial for an in-depth analysis of proteoforms in complex samples. Two-dimensional (2D) separation platforms exhibit increased selectivity and peak capacity compared to their one-dimensional components. Here, we present for the first time a platform for online selective comprehensive coupling of reversed-phase nanoLC and capillary zone electrophoresis-mass spectrometry (CZE-MS) and its application for intact top-down (TD) proteoform analysis. The coupling of these techniques is promising since reversed-phase nanoLC allows for large-volume injection and separation of different proteins by hydrophobicity, whereas CZE-MS is powerful for the separation of proteoforms varying in charge and/or size. Combining reversed-phase nanoLC in the first dimension and highly efficient successive multiple ionic-polymer layers CZE-MS in the second dimension, we evaluated our setup by analyzing a human cell lysate. Using our 2D platform for intact TD proteoform characterization, we were able to detect two to four times more proteoforms compared to one-dimensional (1D) nanoLC-MS measurements in the sampled nanoLC time window. A targeted data evaluation approach for Histone H4 revealed additional proteoforms varying in acetylation and methylation, previously not detected using 1D nanoLC-MS. Combining the power of nanoLC and CZE, the here presented platform will advance the targeted proteoform analysis on the intact protein level in complex biological samples.