Herein we demonstrate the first application of ozone-induced cleavage of endocyclic CC double bonds for improved steroid isomer separation using ion mobility-mass spectrometry. Steroids represent a challenging biomolecular class for ion mobility (IM) separations due to their structural rigidity and subtle stereochemical differences. In this work, we compare the effects of ozonolysis on the relative mobilities of a model stereoisomer pair, testosterone and epitestosterone. A solution-phase ozonolysis approach is used due to its simplicity, relatively low cost, and potential for rapid, online analysis. Despite the presence of solvent-based addition products, we observe that these steroids undergo an ozone-based cleavage resulting in unique, stable gas-phase conformations. The resulting resolution between testosterone and epitestosterone, with collision cross section values of 176.6 and 193.3 Å2, respectively, demonstrates a significant improvement in comparison with previous IM-based approaches. The significantly smaller conformation observed for epitestosterone is stabilized by a three-point interaction between the oxygen-containing functional groups and a sodium ion; this same conformation cannot be sterically achieved by testosterone. Identification of this specific structural difference is strengthened by experimental results showing the disappearance of this conformation following in-source water loss, which eliminates the potential for that three-point interaction. Computational modeling of the lowest energy gas-phase structures for these ozone products corroborates the experimental results. In conclusion, this approach provides tremendous potential as a rapid IM separation method for steroid isomers and other endocyclic CC double bond containing molecules.
The Paterno-Buchi (PB) reaction is a common organic reaction in which a carbonyl radical formed by exposure to UV radiation reacts with an alkene to form an oxetane ring. Recent analytical applications of this reaction have included the determination of CC bond position in lipid fatty acyl tails using tandem mass spectrometry. Our group has recently investigated methods for structurally modifying steroid isomers to improve their identification and resolution using ion mobility spectrometry. Herein, we report the first application of the Paterno-Buchi reaction to form steroid oxetanes using a simple, low-cost, and high efficiency method with a low pressure mercury lamp. This methodology is performed on several endogenous steroid isomers, resulting in unique ion mobility spectra that provide a unique fingerprint for each. These fingerprint spectra can add confidence in identification of those compounds, especially in complex biological matrixes. Testosterone and epitestosterone, an epimer pair commonly interrogated in a number of applications such as for their use as performance enhancing drugs, displayed one and three unique ion mobility peaks, respectively. These spectra and their measured collision cross sections (CCS) allow for unambiguous differentiation of these and several other steroid isomer groups analyzed in this work. Finally, multiple anabolic androgenic steroids prohibited by the World Anti-Doping Agency were tested with this method and resulted in unique CCS for their PB reaction products. This approach can offer improved confidence in their identification as well as for many other banned substances.
β-methylamino-L-alanine (BMAA) has been linked to the development of neurodegenerative (ND) symptoms following chronic environmental exposure through water and dietary sources. The brains of those affected by this condition, often referred to as amyotrophic lateral sclerosis-parkinsonism-dementia complex (ALS-PDC), have exhibited the presence of plaques and neurofibrillary tangles (NFTs) from protein aggregation. Although numerous studies have sought to better understand the correlation between BMAA exposure and onset of ND symptoms, no definitive link has been identified. One prevailing hypothesis is that BMAA acts a small molecule ligand, complexing with critical proteins in the brain and reducing their function. The objective of this research was to investigate the effects of BMAA exposure on the native structure of ubiquitin. We hypothesized that formation of a Ubiquitin+BMAA noncovalent complex would alter the protein's structure and folding and ultimately affect the ubiquitinproteasome system (UPS) and the unfolded protein response (UPR).Ion mobility-mass spectrometry revealed that at sufficiently high concentrations BMAA did in fact form a noncovalent complex with ubiquitin, however similar complexes were identified for a range of additional amino acids. Collision induced unfolding (CIU) was used to interrogate the unfolding dynamics of native ubiquitin and these Ubq-amino acid complexes and it was determined that complexation with BMAA led to a significant alteration in native protein size and conformation, and this complex required considerably more energy to unfold. This indicates that the complex remains more stable under native conditions and this may indicate that BMAA has attached to a critical binding location. File list (4) download file view on ChemRxiv Wilson -Native Protein Ubq-BMAA -2020.pdf (1.28 MiB) download file view on ChemRxiv Wilson -Native Protein Ubq-BMAA -2020 Supporting In... (533.53 KiB) download file view on ChemRxiv Wilson -Native Protein Ubq-BMAA -2020 Supporting In... (353.75 KiB) download file view on ChemRxiv Wilson -Native Protein Ubq-BMAA -2020.docx (900.50 KiB)
β-methylamino-L-alanine (BMAA) has been linked to the development of neurodegenerative (ND) symptoms following chronic environmental exposure through water and dietary sources. The brains of those affected by this condition, often referred to as amyotrophic lateral sclerosis-parkinsonism-dementia complex (ALS-PDC), have exhibited the presence of plaques and neurofibrillary tangles (NFTs) from protein aggregation. Although numerous studies have sought to better understand the correlation between BMAA exposure and onset of ND symptoms, no definitive link has been identified. One prevailing hypothesis is that BMAA acts a small molecule ligand, complexing with critical proteins in the brain and reducing their function. The objective of this research was to investigate the effects of BMAA exposure on the native structure of ubiquitin. We hypothesized that formation of a Ubiquitin+BMAA noncovalent complex would alter the protein’s structure and folding and ultimately affect the ubiquitinproteasome system (UPS) and the unfolded protein response (UPR). Ion mobility-mass spectrometry revealed that at sufficiently high concentrations BMAA did in fact form a noncovalent complex with ubiquitin, however similar complexes were identified for a range of additional amino acids. Collision induced unfolding (CIU) was used to interrogate the unfolding dynamics of native ubiquitin and these Ubq-amino acid complexes and it was determined that complexation with BMAA led to a significant alteration in native protein size and conformation, and this complex required considerably more energy to unfold. This indicates that the complex remains more stable under native conditions and this may indicate that BMAA has attached to a critical binding location.
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