Identification and Determination of Alanine-Proline-Glutamate Isomers by Ion Mobility Mass Spectrometry

The biological functions of tripeptides are highly dependent on their stereochemical configurations, making the development of rapid and accurate chiral analysis methods crucial for elucidating their structure-activity relationships and promoting their applications. This study developed a direct chiral analysis method based on ion-mobility-mass spectrometry (IM-MS) coupled with a supramolecular complexation strategy, enabling effective discrimination and quantitative analysis of all eight stereoisomers of the tripeptide Ala-Pro-Glu which contains three chiral centers. In this approach, β-cyclodextrin served as the chiral selector and Mg2+ as the bridging ion, forming stable ternary non-covalent complexes [β-CD+Mg + M-H]+ with the target tripeptides in the gas phase. Analysis using trapped ion mobility spectrometry-time-of-flight mass spectrometry (TIMS-TOF-MS) revealed that tripeptides of different chiral configurations led to complexes with significantly different collision cross-section (CCS, ranging from 327.5 to 349.6 Å2) and mobility (1/K0) values, achieving baseline separation for several isomer pairs (with a maximum resolution Rp-p of 2.056). The study further uncovered that complexes of certain isomers (e.g., L-Ala-D-Pro-D-Glu and L-Ala-L-Pro-D-Glu) existed in two stable conformations, displaying an intriguing mirror-symmetric distribution in terms of CCS values, highlighting the subtle control of chirality over gas-phase ion structures. Quantitative analysis demonstrated a good linear response (R2 > 0.99) for typical isomers (e.g., D-Ala-L-Pro-D-Glu and L-Ala-D-Pro-L-Glu) within the concentration range of 3.0–9.0 μM. The established IM-MS supramolecular chiral analysis strategy, requiring no chiral columns or derivatization steps, and provides a powerful and direct analytical platform for the high-throughput chiral identification and quantification of oligopeptides and other bioactive molecules.