Nanoflow IEC-HRMS to study complex proteoform mixtures

The CAST scientist Ziran Zhai published a manuscript investigating a novel method of using nanoflow strong cation exchange – native mass spectrometry to characterize non-denaturing complex proteoforms mixtures from the intact level. Zhai focuses on three critical aspects: i) extending the MW that can be observed by top-down proteomics, ii) increasing the MS sensitivity to create conditions of detecting low-abundant proteins, and iii) apply mild desolvation conditions to maintain the native structures of proteins and complexes.

Proteoforms, which are protein products arising from homologous genes due to sequence variations, alternative splicing, and post-translational modifications, play a crucial role in a wide range of critical functions. However, the standard approach to characterize proteins, known as bottom-up proteomics, faces limitations. This approach cannot directly identify proteoforms as the presence of proteins is inferred from peptides. While top-down proteomics and intact protein mass spectrometry offer solutions to these limitations, the most common top-down methods employ denaturing LC-MS approaches, which unfold proteins and lead to the loss of non-covalent protein complexes.

In this work, we directly coupled nanoflow (250 or 500 nL min¯¹) strong cation exchange chromatography (SCX) to nano-electrospray-ionization (nESI) under native MS (nMS) conditions. Proteins were separated on packed capillary SCX columns and eluted according to their pI values by a salt-mediated pH gradient method. The low flow promoted desolvation/ionization efficiency allowing for sensitive detection of low-abundant proteins and complexes. We successfully applied our method to analyze an E. coli cell lysate and observed hundreds of proteins with masses up to 150 kDa. We believe that the proposed nanoSCX-nMS is a promising approach for characterizing proteoforms and provides a universal strategy to overcome detection limitations in native top-down proteomics.

Chemometrics and Advanced Separations Team: screenshot from the publication.

The study is part of the FFF (From Form to Function) project of Zhai, Astefanei, Corthals, and Gargano and was funded by the Chinese Scholarship Council (CSC) and was recently published in Analytica Chimica Acta and can be accessed freely at the link below.