Parallel Analysis of Acidic and Basic Proteoforms in Cell Lysates via Native Cation and Anion Exchange Chromatography—Native Mass Spectrometry

Comprehensive characterization of proteoforms in complex biological systems remains a significant challenge. While native ion-exchange chromatography (IEC) hyphenated with native mass spectrometry (nMS) is a powerful tool for resolving proteoforms, three critical limitations persist: restricted volatile buffers causing nonlinear pH gradients; insufficient coverage of the proteome's broad isoelectric point (pI) range; and inadequate sensitivity for low-abundance proteins. To bridge this gap, we developed an online nanoflow dual IEC-nMS platform. Volatile salt systems for strong anion (SAX) and cation exchange (SCX) were optimized to achieve exceptionally wide and linear pH ranges (2.6-5.0 for SAX and 5.0-8.5 for SCX). These methods were implemented on self-packed 100 µm ID capillary columns at 500 nL/min to enhance sensitivity. To enable simultaneous analysis of acidic and basic species, we introduced a double-barrel column configuration that integrates nanoSAX and nanoSCX in a noninterfering, parallel manner. This platform was applied to an E. coli cell lysate, identifying 301 unique proteoform masses (D-score > 40) ranging from 10 to 150 kDa (with about 120> 50 kDa). Notably, the two modes were highly complementary, with only 10 overlapping species. The double-barrel nanoflow IEC-nMS platform provides a robust, sensitive, and high-resolution strategy for native top-down proteomics, enabling in-depth study of complex proteomes.