New Framework Expands Isomer Identification Possibilities with Mass Spectrometry


New statistical framework developed by researchers at the University of California in Riverside allows confident identification of isomeric molecules by mass spectrometry, even when no unique mass-to-charge peaks are generated between the isomeric pairs.

A new statistical framework has been developed for confidently identifying isomeric molecules using mass spectrometry (MS). Isomeric molecules, which have the same molecular formula but differ in their structural arrangement, can be difficult to distinguish using MS due to their similar fragmentation patterns. The new framework, developed by Ryan R. Julian from the University of California in Riverside and colleagues and published in Analytical Chemistry, focuses on differences in peak intensity to calculate a statistical probability that the spectra derive from different analytes (1). The framework allows for confident identification of peptide isomers by comparing mass spectra that differ only in terms of peak intensity.

Sample preparation for Mass spectrometry in a scientific laboratory. Filling test tubes with samples with a pipette and measuring instruments on the background | Image Credit: © luchschenF -

Sample preparation for Mass spectrometry in a scientific laboratory. Filling test tubes with samples with a pipette and measuring instruments on the background | Image Credit: © luchschenF -

The framework can identify a range of isomer types, including D/L amino acid substitutions, Leu/Ile, and Asp/IsoAsp, and can be used with data collected by either direct infusion or liquid chromatography MS. The method can also accommodate changes in instrumental settings such as source voltages, isolation widths, and resolution without influencing the analysis.

Leu/Ile and Asp/IsoAsp are pairs of amino acid isomers that differ only in the position of a single atom. Leucine (Leu) and isoleucine (Ile) differ in the position of a methyl group, while aspartic acid (Asp) and isoaspartic acid (IsoAsp) differ in the position of a carboxyl group. These isomers can have different biological activities and can be important in the study of protein structure and function.

D/L amino acid substitutions refer to the change of a naturally occurring L-amino acid to its D-isomer counterpart. This alteration can have important effects on the biological activity and stability of peptides and proteins. Leu and Ile are both hydrophobic amino acids with very similar chemical properties and structures, differing only by a single methylene group on the side chain. Asp and IsoAsp are also similar in structure, with IsoAsp being an isomer of Asp that results from the spontaneous rearrangement of the side chain carboxyl group. These substitutions can lead to isomeric forms of peptides and proteins that can be challenging to distinguish and characterize, making them a target of interest for analytical techniques such as mass spectrometry.

The researchers demonstrated that the framework enables quantification of the composition of isomeric mixtures with calibration curves that are highly linear and reproducible. This development expands the possibilities for identifying isomeric molecules in many biological and chemical arenas, where such molecules are important analytes.

The framework can be implemented with multiple MS fragmentation techniques, including collision-induced dissociation, higher-energy collisional dissociation, electron-transfer dissociation, and radical-directed dissociation. Although the framework was presented in the context of isomer characterization, it has potential for many other contexts where similar mass spectra are generated.

This new framework for identifying differences in similar mass spectra has the potential to significantly improve the identification of isomeric molecules, making it a valuable tool in the field of analytical chemistry and beyond.


(1) Wu, H-T.; Riggs, D. L.; Lyon, Y. A.; Julian, R. R. Statistical Framework for Identifying Differences in Similar Mass Spectra: Expanding Possibilities for Isomer Identification. Anal. Chem. 2023, 95, 17, 6996–7005. DOI:

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