Lipid Peroxidation and Carbonyl Stress

Oxidative stress has been implicated in age-associated decline in function, but reactive oxygen species (ROS) refers to a diverse class of molecules whose downstream effects are vast, including those that are essential. Lipid hydroperoxides are a class of ROS molecules that has been implicated in cell damage, particularly to induce ferroptosis.

In the last several years, we have studied the role of lipid peroxidation primarily in skeletal muscle (Anderson et al., JCI, 2018, Eshima et al., JAP, 2020, Ferrara et al., FASEB J, 2021, Miranda et al., Nature Aging, 2022, Eshima et al., eLife, 2023, Miranda et al., Antiox Redox Signal, 2023, Eshima et al., biorxiv, 2023, Shahtout et al., JCSM, 2024, Miranda et al., biorχiv, 2025). In these studies, we have utilized genetic and pharmacologic approaches to suppress different components of lipid peroxidation pathway to show that it can prevent decline in muscle function associated with aging, disuse, and cancer. In particular, carnosine and N-acetylcarnosine are both a member of the histidine-containing dipeptide (HCD) family of metabolites that have high affinity to neutralize lipid carbonyls generated from lipid hydroperoxides. Both of these metabolites are endogenously present, safe for human consumption, and sold as nutraceuticals in stores. Leveraging its safety and accessibility, we are in a process of starting three clinical trials:

1. a unilateral limb immobilization in healthy young subjects,
2. patients undergoing ACL reconstruction, and
3. lung cancer patients experiencing cachexia. We continue to complement these studies with a variety of preclinical and mechanistic studies.

One of these efforts involve an extensive use of data-independent acquisition (DIA) mass spectrometry to study protein post-translational modification known as carbonylation. As a relatively newly discovered pathway, downstream mechanisms by which lipid peroxidation promote cellular dysfunctions are largely unknown. One highly likely downstream effectors are the highly reactive lipid carbonyls produced from lipid hydroperoxides that exhibit their effects by covalently modifying Lys, Cys, His, and Arg residues of the cellular proteome. Studying their identifies have been highly challenging because these carbonyls can in principle bind to any of these amino acid residues in multiple ways (Michael addition and/or Schiff base), such that protein carbonylation has complex mass shifts with low abundance.

We have recently overcome this barrier by utilizing dinitrophenylhydrazine (DNPH) to “tag” all carbonylated residues and use it to affinity purify and enrich them. We then combine this with the advent of ultra-sensitive/fast trapped state-of-the-art trapped ion mobility spectrometry with quadrupole time-of-flight (timsToF) to perform DIA mass spectrometry. We coined our platform as carbonylated proteome with DNPH derivatization (CAPDD). We then run our CAPDD datasets through the Fragpipe software to analyze spectra for protein carbonylation, and perform secondary and tertiary analyses using a software we generated (termed Pipecleaner) to perform functional annotation of the carbonylated proteome, automatically mapping all our carbonylation sites on the proteome using UnitProt and Alphafold, and discover motifs using ProteinBERT, ESM, and K-Means. Our carbonylated proteome pipeline is still in its early development, but we are excited about the potential that these tools will have in understanding the mechanisms of lipid peroxidation.