Biology subjects

Cobley, J. N.

Publications and source records attributed to Cobley, J. N..

4 recordsLinked to original sources

bioRxiv2026

CysNet: Theorem constrained inference of cysteine redox proteoform states from bottom-up mass spectrometry data

Here, we present CysNet, a theorem-constrained method designed to infer cysteine redox proteoforms, i.e.,oxiforms, from bottom-up, mass spectrometry (MS)-based proteomic data. This overcomes limitations with previous MS redox proteomic approaches, which can quantify residue-resolved cysteine redox states, but leave distinct oxiforms unresolved. CysNet treats each residue-resolved oxidation value as a binary redox-coordinate marginal, enabling theorem-constrained inference of the oxiforms that are necessary, impossible or bounded within the compatible protein-group ensemble. This collapses the vast theoretically possible set of oxiform states to a finite set of allowed values by extracting existence and exclusion constraints from the data, despite the incomplete proteome coverage typical for bottom-up MS datasets. Using CysNet to analyse human induced pluripotent stem cell lines (~6,300 cysteine-containing protein groups, ~22% cysteine coverage), resolved 519 exact oxiforms, inferring 7,000 oxiforms per line. Quantitatively, CysNet bounded the oxiform content to 6.36-8.24 x 1012 protein copies, corresponding to 14-19% of the measured cysteine proteome. These data define the deepest oxiform survey recorded. CysNet revealed a latent structural layer in redox variation between the cell lines, distinguishing changes in oxiform identity (composition) from changes in oxiform weighting (intensity). Hence, CysNet moves bottom-up redox proteomics beyond isolated site-level cataloguing by reconstructing copy-number-weighted oxiform maps, providing a scalable route to deep oxiform information from peptide-level data.

biochemistry↗

bioRxiv2026

ReCap enables deep, copy number-scaled cysteine redox proteomics with minimal exogenous oxidation

Cysteine oxidation analyses require the preservation of the redox state present at harvest and quantitative scaling to relate oxidation to protein copy numbers, rather than only providing fractional oxidation data. Here, we present ReCap, a Redox Capture workflow combining Oxi-DIA, an enrichment-free isotope-encoded DIA workflow, with Oxi-Stop, a simple oxygen-exclusion strategy for cryopreserved tissue. In mouse brains, Oxi-DIA quantified 17,809 cysteine sites belonging to 6,085 protein groups in every sample, enabling matched measurements of residue-resolved oxidation and protein abundance. Atmospheric oxygen exposure during 14 days of cryopreservation distorted the measured cysteine redox state. The resultant increase of an estimated 5.3176 x 1011 {micro}g-1 oxidised cysteine molecules was mitigated by Oxi-Stop, which minimised exogenous oxidation during cryopreservation. Copy-number scaling altered the interpretation of cysteine oxidation values. Although cysteine oxidation was detected across 2,371 sites and 1,439 proteins, 20 sites on abundant proteins accounted for 44% of the oxidised signal. ReCap advances redox proteomics from providing a site catalogue into a biologically weighted map of redox information, revealing cysteine oxidation as a sparse, ordered and quantitatively concentrated signal.

biochemistry↗

bioRxiv2024

Computational Analysis of Human Cysteine Redox Proteoforms Reveals Novel Insights

Since cysteine redox proteoforms (i) are virtually unstudied, we derived novel insights by computationally analysing the human proteome. Our analysis revealed a vast, effectively infinite, theoretical i space housing 3.02 x 10169 unique cysteine redox proteoforms. For >80% and 99% of the human proteome, the i space comprises 6.83 x 108 and 1.76 x 1031 unique proteoforms, respectively. The heterogenous distribution of the i space by gene ontology terms, suggests, but does not prove, functional speciation. To theoretically limit the number of cysteine redox proteoforms that can be "downloaded" from the abstract i "cloud", we implement novel equations. Protein copy numbers limit the i space by 161-logs to 4.04 x 107 unique cysteine redox proteoforms per HeLa cell. An immutable law: the number of cysteine redox proteoform molecules (Ni) must equal the number of cysteine-containing protein molecules. We compute an Ni value of 1.70 x 109 per HeLa cell. While Ni will be displaced from thermodynamic equilibrium towards the reduced state (e.g., {approx}90%-reduced), it is possible that the number of partially oxidised cysteine redox proteoform molecules is in the order of 106-8 per HeLa cell. Consistent with this, 100%-oxidised forms were observed in 60% of the proteins studied to date. Our analysis advances understanding of redox biology at the proteoform level.

biochemistry↗

bioRxiv2024

Cleland Immunoblotting Unmasks Unexpected Cysteine Redox Proteoforms

Cysteine redox proteoforms are virtually unstudied because they are extremely difficult to detect. They are difficult to detect by immunoblotting when the polyethylene glycol (PEG)-payloads used to mobility-shift proteoforms into distinct bands block antibody binding. Here, we synthesised a novel compound to reversibly crosslink the PEG-payloads to oxidised cysteines using disulfide bonds. To reductively release the PEG-payloads from mobility-shifted proteoforms, we soaked the gel in Clelands reagent: 1,4-dithiothreitol (DTT). Hence, Cleland immunoblotting. Cleland immunoblotting unmasked hitherto undetectable cysteine redox proteoforms. In Xenopus oocytes, we detected 2 cdc20-specific coordinates in an otherwise abstract space housing 1,024 theoretical proteoforms. The coordinates mapped to the fully, all 10 cysteine residues, 100%-reduced and 100%-oxidised proteoforms. The unexpected absence of partially oxidised molecules along the 10-reaction path to the 100%-oxidised proteoform implies novel biology. By providing the technological means to detect cysteine redox proteoforms, Cleland immunoblotting opens new avenues of discovery.

biochemistry↗