Native Cation and Anion Exchange Chromatography-Mass Spectrometry for the Parallel Analysis of Acidic and Basic Proteoforms in Cell Lysates

ABSTRACT: The characterization of proteoforms in complex biological samples (e.g., cell lysates) is a significant analytical challenge due to their complexity and heterogeneity. While native ion-exchange chromatography (IEC) hyphenated with native mass spectrometry (nMS) is a powerful method for resolving protein charge variants under non-denaturing conditions, its application to complex proteome samples is still limited. Two key limitations constrain the analysis: (i) the restricted availability of volatile buffer systems, which frequently leads to non-linear pH gradients and subsequent co-elution of proteoforms; and ii) reliance on a single ion-exchange mode (either anion or cation exchange), which provides insufficient resolving power for complex protein mixtures spanning a broad range of isoelectric points. To address these challenges, we developed a novel online nanoflow dual IEC-nMS platform. Mobile phases for strong anion-exchange (SAX) and strong cation-exchange (SCX) were first optimized with various volatile salts through analytical-scale chromatographic separations. This enabled methods with wide linear pH ranges spanning from 2.6 to 5.0 for SAX and 5.0 to 8.5 for SCX. Subsequently, nanoflow SAX- and SCX-nMS were developed using self-packed capillary columns operated at a flow rate of 500 nL/min. To facilitate the highly efficient and simultaneous analysis of both acidic and basic proteoforms, we created a double-barrel setup that integrates the nanoSAX and nanoSCX in a non-interfering manner. The enhanced sensitivity and selectivity of the developed methods allowed for the detection of more than 1,000 proteoforms in a complex E. coli cell lysate, with molecular weights ranging from 10 to 150 kDa.

INSTRUMENT(S): Q Exactive Plus

ORGANISM(S): Proteins

SUBMITTER: Ziran Zhai  

PROVIDER: MSV000099515 | MassIVE | Thu Oct 16 08:19:00 BST 2025

REPOSITORIES: MassIVE