Chromatographic Considerations for Native and Denaturing ESI for Large Molecule Characterization

Intact mass is a common workflow for the analysis of large molecules by high-resolution MS. It can be useful for the identification of primary sequence, as well as the characterization of different isoforms. Intact mass can be divided into two different approaches. The first uses reversed phased LC, which is denaturing, as high temperature, low pH, and an organic solvent is required. Although this method can be more easily implemented, structural information is lost and identification of lower level variants can be challenging. As such, other so-called “native” separation modalities may also be implemented, especially useful for further characterization outside of simple primary sequence confirmation. In this discussion, we explore native and denaturing ESI for large molecule analysis, discussing how chromatography can impact both.