Retrospective detection for V-type OPNAs exposure via phosphonylation and disulfide adducts in albumin.

Organophosphorus nerve agents (OPNAs) that damage the central nervous system by inhibiting acetylcholinesterase activity, pose severe threats to human health and life security. Reliable biomarkers that quickly and accurately detect OPNAs exposure are urgently needed to help diagnose quickly and treat in time. Albumins that covalently bind to OPNAs could serve as important targets for retrospective verification of OPNAs exposure. The goal of this study is to explore the potential biomarkers in albumins with high reactivity and good stability and expand the group of potential biomarkers in different species for detecting the exposure of V-type OPNAs including O-ethyl S-(2-(diisopropylamino)ethyl) methylphosphonothioate (VX), O-isobutyl S-(2(diethylamino)ethyl) methylphosphonothioate (VR), and O-butyl S-(2-(diethylamino)ethyl) methylphosphonothioate (Vs). Taking human serum albumin (HSA), bovine serum albumin (BSA) and rabbit serum albumin (RSA) as the research objectives, multiple active sites including phosphonylation and disulfide adduct sites were observed in albumins from different species. Numerous phosphonylation sites labeled by all agents in one type of albumin were found. Among the different species, four shared phosphonylation sites with high reactivity include K499, K549, K249, and Y108. In addition, Y108 on ETY*GEMADCCAK, Y287 on Y*ICENQDSISSK, Y377 on TY*ETTLEK and Y164 on YLY*EIAR in HSA were stably phosphonylated by all agents in gradient concentration, making them stable and suitable potential biomarkers for V-type OPNAs exposure. Notably, Y108 on ETY*GEMADCCAK in HSA, on DTY*GDVADCCEK in RSA, and on ETY*GDMADCCEK in BSA were highly reactive to all V-type agents, regardless of species. It was also successfully labeled in HSA exposed to class V agents in gradient concentration. Y108 is expected to be used to screen and identify the exposure of V-type agents in the retrospective research. Disulfide adducts sites, consisted of four sites in HSA and two sites in BSA were also successfully labeled by V-type agents, and characteristic ion fragments from these disulfide adducts were also identified by secondary mass spectrometry. Molecular simulation of the stably modified sites were conducted to discover the promoting factors of covalent adduct formation, which help further clarify formation mechanism of albumin adducts at active sites.

A Novel Potential Biomarker on Y263 Site in Human Serum Albumin Poisoned by Six Nerve Agents.

Albumin is a new biomarker of organophosphorus compounds (OPs) and nerve agents (OPNAs) for retrospective verification. Recent studies on OPs adducts show that amino acid residues can covalently bind to OPs and OPNAs. In this article, after being incubated with soman, sarin, cyclosarin, VX, ethyl tabun, and propyl tabun, human serum albumin (HSA) is analyzed by quadrupole-Orbitrap mass spectrometer (Q Exactive LC-MS/MS). In addition to the three known phosphonylated sites, six new sites modified by OPNAs are detected. To identify the most reactive residue, we calculate the area ratio of the modified peptides to the whole peptides. The result demonstrates that tyrosine 263 (Y263) in peptide Y263ICENQDSISSK, which has been poisoned with six kinds of nerve agents, possesses the highest reactivity. The structure characteristics based on molecular simulation provide a theoretical evidence for the reactivity of the nine binding sites. It suggests that Y263 also has the potential to be used as a biomarker to detect OPNAs exposure, and the presented Q Exactive LC-MS/MS method might be of relevance for the verification of new phosphonylated sites.

Mass spectral characterization of tabun-labeled lysine biomarkers in albumin.

Tabun has been shown to form phosphylated adducts on tyrosine residues in albumin in vivo and in vitro. However, in this work, tabun-labeled lysine adducts were found in albumin. Three types of albumin were treated with overdose of tabun in vitro and 17 tabun-labeled lysine residues were found: K4, K12, K224, K377, and K524 in bovine albumin, K186, K188, K212, K329, K414, and K525 in leporine albumin, and K79, K186, K188, K212, K376, and K525 in rat albumin. To investigate the modification of tabun in vivo, three leporines were injected with 0.8×LD50 dose of tabun. The results showed that the labeled lysine residues in vivo, were consistent with modified lysines in vitro. Structure characteristics and the binding mode of 6 tabun-labeled lysines of leporine albumin were further analyzed using theory simulation and molecular docking in Discovery Studio. For the first time, we show that tabun-labeled lysine peptides are found in vivo and in vitro. These modified lysine peptides are good biomarkers for exposure to tabun in albumin of leporine and rat.

Identification of new binding sites of human transferrin incubated with organophosphorus agents via Q Exactive LC-MS/MS.

Organophosphorus agents (OPs) like sarin, VX, or soman could inhibit acetylcholinesterase activity and cause poisoning. OPs could bind many proteins, such as butyrylcholinesterase and albumin, and the adducts formed could identify the exposure. In this paper, we studied human transferrin, which was one of the proteins that could be labeled by OPs. Pure human transferrin was incubated with an overdose of organophosphorus agents, including sarin, soman, VX, tabun, cyclosarin, ethyl tabun, and propyl tabun, and then additional OPs was removed through dialysis. Trypsin was used to cleave the OP-treated proteins and Q Exactive liquid chromatography tandem mass spectrometry (Q Exactive LC-MS/MS) was used to identify them. The present study set out to accomplish two goals. The first goal was to find a good method for identifying multiple binding sites on a given protein through Q Exactive LC-MS/MS. The second goal was to investigate the labeled peptides when transferrin was incubated with a numerous molar excess of OPs. Results showed that tyrosine, lysine, and serine formed covalent bonds with OPs. Twenty OP-labeled sites were found: ten tyrosine sites (including two reported sites), seven lysine sites, and three serine sites. Characteristic fragments for labeled-tyrosine and labeled-lysine adducts were summarized in detail. In conclusion, the method by Q Exactive LC-MS/MS using in this present work is a good way to diagnose exposure to OPs accurately when the binding sites of OPs are uncertain. Novel modified peptides and the characteristic ions found in this work could help investigators assess exposure to OPs.

Development and characterization of a novel GHR antibody antagonist, GF185.

Here, we describe the development of a panel of monoclonal antibodies targeting the growth hormone receptor (GHR). Of these monoclonal antibodies (Mabs), GF185 was selected for further characterization due to its activities. Competitive receptor-binding assays and Western blotting analyses were used to demonstrate that GF185's epitopes are localized within subdomain 1 of the growth hormone receptor extracellular domain (GHR-ECD). Subsequently, we evaluated GF185's antagonistic activities in vivo and in vitro and showed that GF185 was able to neutralize growth hormone (GH) signalling and inhibit GH-induced Ba/F3-GHR proliferation. Our findings suggest that GF185 may serve as an attractive tool for GHR-related research and has a potential future application for the treatment of GH-dependent disease.