Hydroxyproline (hPro) is a non-essential amino acid commonly found in collagen, where it plays a major role in stabilizing the triple helix structure. The amount of free hPro in plasma and urine may indicate collagen catabolism and be a potential biomarker for certain diseases. There are several isomeric forms of hPro. Differentiation between them by mass spectrometry (MS) is a challenge, given that they have the same molecular mass and, when protonated, undergo similar dissociation pathways. Specifically, collision-induced dissociation of protonated hPro isomers proceeds via loss of H2O and CO. Therefore, other MS-based approaches, including alternative charge carriers, ion mobility spectrometry, and infrared multiple photon dissociation (IRMPD) spectrometry could be useful in achieving hPro isomer differentiation on MS platforms. In this talk, the literature on hPro isomer differentiation by mass spectrometry will first be reviewed, then two recent advances from our research group will be discussed in context. The first advance, reported in a recently published study, demonstrated that IRMPD spectroscopy can be used to differentiate three lithiated hPro isomers, namely cis-3-hPro, cis-4-hPro, and trans-4-hPro. While this approach allows for the differentiation of all three isomers, the IRMPD spectra of the stereoisomers were much more distinct than that of the positional isomers, which can be explained based on the fact that both cis- isomers adopt a charge-solvated configuration and the trans- isomer adopts a salt-bridge configuration. In this previous work, we found that the stereoisomers investigated (cis-4-hPro and trans-4-hPro) produce similar laser-induced dissociation (LID) mass spectra (despite the more distinct IRMPD spectra), whereas the positional isomers (cis-4-hPro and cis-3-hPro) produced distinct LID mass spectra. This implies that by looking at both the IRMPD spectra and dissociation pathways all three isomers can be readily differentiated with high confidence. This brings us to the second advance to be presented in this talk, aimed at understanding why specific dissociation pathways (e.g., loss of H2O and CO) were absent in the dissociation spectrum of lithiated cis-3-hPro, yet present in the other investigated isomers’ MS/MS spectra. To this end, we are applying computational chemistry approaches to investigate the thermodynamics and mechanisms associated with these various dissociation pathways.

Differentiation of lithiated hydroxyproline isomers on mass spectrometry platforms

ACS Spring Meeting 2021

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