Two IMS-MS Modalities Assist Nonenzymatic Posttranslational Modification Studies Without Mass Loss

In the pursuit of unraveling the complex world of posttranslational modifications (PTMs), a combined team of scientists at the University of Tennessee and the University of Illinois at Urbana-Champaign has made what they consider to be significant strides in a new study. Their work, recently published in the Journal of the American Society for Mass Spectrometry, delves into the intricate realm of nonenzymatic PTMs, offering fresh insights into their mechanisms and implications (1).

A wild California Sea Hare, Aplysia californica, a hermaphroditic species commonly used in laboratory research | Image Credit: © elharo - stock.adobe.com

PTMs play a critical role in cellular homeostasis and have been implicated in various pathological conditions. However, understanding these modifications has been a complex challenge. In their research, authors Connor C. Long, Aleksandra Antevska, David H. Mast, Samuel Okyem, Jonathan V. Sweedler, and Thanh D. Do employed two ion mobility spectrometry–mass spectrometry (IMS-MS) modalities, drift-tube IMS (DT-IMS) and trapped IMS (TIMS), to study three essential nonenzymatic PTMs that do not induce mass loss. These PTMs include L/D isomerization, aspartate/isoaspartate isomerization, and cis/trans proline isomerization.

Their focus was on a single peptide system, specifically the pleurin peptides, Plrn2, derived from Aplysia californica. The results of their investigation have opened new avenues for understanding the intricacies of these modifications and their implications.

One of the key findings of this study was that DT-IMS-MS/MS can effectively capture and pinpoint asparagine deamidation, transforming it into aspartate; the subsequent isomerization becomes isoaspartate. This is a significant discovery, because isoaspartate serves as a critical biomarker for age-related diseases. The ability to detect and locate this modification is a crucial step forward in understanding the underlying mechanisms of age-related conditions.

Moreover, the study also explored nonenzymatic peptide cleavage via in-source fragmentation, assessing differences in the intensities and patterns of fragment peaks between the various PTMs. The researchers discovered that peptide fragments resulting from in-source fragmentation, after preceding peptide denaturation by liquid chromatography (LC) mobile phase, exhibited cis/trans proline isomerization. This insight into the effects of in-source fragmentation profiles reveals the structural changes in Plrn2 and their fragment ions.

The choice of fragmentation voltage at the source and solution-based denaturation conditions was another aspect under scrutiny. It was confirmed that LC denaturation and in-source fragmentation have a profound impact on N-terminal peptide bond cleavages of Plrn2 and the structures of their fragment ions. This underscores the significance of carefully controlling these conditions in mass spectrometry experiments.

In conclusion, the research conducted by these authors showcases the power of the combined mass spectrometry techniques coupled with in-source fragmentation in the identification of three vital posttranslational modifications, bringing separation scientists one step closer to understanding the intricacies of nonenzymatic PTMs and their role in various biological processes. This work demonstrates the subtle yet crucial modifications that play a role in cellular processes and disease mechanisms, and as researchers continue to explore these nonenzymatic PTMs, their findings may have implications in our understanding of age-related diseases and other health conditions.

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Reference

(1) Long, C. C.; Antevska, A.; Mast, D. H.; et al. Nonenzymatic Posttranslational Modifications and Peptide Cleavages Observed in Peptide Epimers. J. Am. Soc. Mass Spectrom. 2023, 34 (9), 1898–1907. DOI: https://doi.org/10.1021/jasms.3c00092