Identification of 4‐hydroxynonenal protein targets in rat, mouse and human liver microsomes by two‐dimensional liquid chromatography/tandem mass spectrometry
Bibliographic record
Abstract
RATIONALE: 4-Hydroxynonenal (HNE), endogenously generated through peroxidation and breakdown of polyunsaturated fatty acids, has been linked to a number of adverse biological effects through carbonylation of essential biomolecules. Covalent binding of HNE to proteins can alter their structure and functions, causing cell damage as well as adverse immune responses. The liver plays a predominant role in metabolic transformations and hepatic proteins are often targeted by reactive metabolites. METHODS: Rat, mouse and human liver microsomes were incubated with HNE, enzymatically digested, and subjected to strong cation-exchange peptide fractionation prior to liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis coupled to electrospray ionization quadrupole time-of-flight (QqTOF) mass spectrometry. HNE-modified peptides were detected by probability-driven peptide spectral matching and comparative analysis between treated and control samples, and confirmed based on accurate mass and high-resolution MS/MS spectra. RESULTS: A total of 99, 123 and 51 HNE-modified peptides were identified in rat, mouse and human liver microsomes related to 76, 103 and 44 target proteins, respectively. Eight proteins were found to be adducted by HNE in all three species, including ATP synthase, carbamoyl phosphate synthase, cytochrome P450 1A2, glutamate dehydrogenase 1, protein ERGIC-53, protein disulfide-isomerase, and voltage-dependent anion-selective channel protein 1. These proteins play crucial roles in cellular processes and their covalent modification could potentially alter their function and lead to cytotoxicity. CONCLUSIONS: An analytical approach was developed for the identification of in vitro HNE protein targets in rat, mouse and human liver microsomes using two-dimensional (2D) LC/MS/MS. This approach can be applied to study HNE modification of proteins in vitro and in vivo, providing insight into the toxicology of HNE protein adduction. Copyright © 2016 John Wiley & Sons, Ltd.
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.001 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.001 | 0.001 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.001 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one teacher head, not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".