Advancing Peptide and Protein Stereoisomer Analysis with Conformation‐Based Chiral Separation via Liquid Chromatography and Ion Mobility‐Mass Spectrometry

Peptides and proteins with D‐amino acids and other stereoisomers in their sequences are now considered to be widespread in different organisms. Their significance is attributed to the altered functions of these molecules, such as having some pathological significance or enhancing biological activity. Only slight shifts in structural and other biophysical parameters make their full characterization and complete distinction technically challenging for traditional tools like mass spectrometry (MS). Ion mobility (IM) spectrometry in conjunction with liquid chromatography (LC) adds an extra dimension to the separation in space as it depends on the mobility of ions, being determined by their overall shape and the orientation of the molecules. Thus, peptide isomers having measurable mobility and collisional cross‐section values can thus be resolved by IM‐MS. In addition, by combining with tandem MS techniques, IM‐MS with adequate conformation‐resolving capability is likely to localize the isomerization sites. This review briefly introduces the basic principles of conformation‐based chiral separation through LC and capillary electrophoresis, in conjunction with IM‐MS for enhanced peptide/protein stereoisomer differentiation, and then comprehensively summarizes the recent advancements in IM‐MS‐based peptide/protein stereoisomer analysis, focusing on the development of conformation‐based chiral separation strategies, including instrumental modifications, chemical modifications, and computational tools.