Biology subjects

Pujari, V.

Publications and source records attributed to Pujari, V..

4 recordsLinked to original sources

bioRxiv2026

Proteome-wide quantification of protein turnover in frog and fly embryos reveals divergent strategies of maternal inheritance

Every embryo inherits a maternal proteome that it must remodel with zygotic proteins to build its many cell types. The fate of the maternal proteome remains contested because indirect measurements cannot resolve it. Here, we combine 18O-water labeling with multiplexed proteomics to quantify protein turnover proteome-wide in frog and fly embryos. Through hatching, the frog preserves the bulk of its maternal proteome, confining rapid degradation to a small regulatory module. The fly cannot meet its synthesis demand from yolk alone and instead degrades nearly all maternal proteins, including housekeeping proteins long assumed stable, recycling them into new protein. Yet the turnover hierarchy is conserved, with disordered and regulatory proteins degrading fastest, while the fly rescales the whole proteome ~eightfold faster. These results recast the developmental proteome as both informational inheritance and metabolic reserve, establish 18O-water labeling as a turnover method for non-feeding organisms, and provide a resource of embryonic half-lives.

developmental biology↗

bioRxiv2026

Menaquinone depletion resensitises bedaquiline-resistant tuberculosis

Tuberculosis remains a leading cause of global mortality, and rising bedaquiline resistance threatens the effectiveness of current drug-resistant treatment regimens. Bedaquiline resistance typically arises through mutations in Rv0678 that upregulate drug efflux and confer cross-resistance to multiple drug classes. Here, we identify and optimise a chemical series targeting MenG, a central enzyme in the menaquinone biosynthesis pathway, yielding potent bactericidal inhibitors with in vivo efficacy. Strikingly, MenG inhibition restored bedaquiline susceptibility in efflux-mediated resistant strains, an effect confirmed in vivo where combination therapy achieved a 99.8% reduction in bacterial burden compared with bedaquiline alone. Potentiation also extended to pretomanid and other key agents. Disruption of upstream menaquinone and shikimate pathway enzymes produced similar resensitisation, establishing these pathways as tractable targets for restoring drug susceptibility in Mycobacterium tuberculosis. These findings provide a novel strategy to overcome bedaquiline resistance and strengthen future regimens for efflux-mediated drug-resistant tuberculosis.

microbiology↗

bioRxiv2025

Synergistic Arg-C Ultra and Lys-C Digestion for Quantitative Proteomics

Shotgun proteomics hinges on complete enzymatic digestion of proteins into peptides. Incomplete digestion narrows proteome coverage and inflates variability in quantitative workflows, whether label-free DIA or multiplexing with isobaric tags. Sequential Lys-C/Trypsin digestions mitigate missed cleavages at lysine residues, but arginine sites remain a persistent challenge. Arg-C Ultra, a recently released cysteine protease, efficiently targets arginine residues but requires reducing conditions that inactivate Lys-C activity and compromise NHS- ester labeling in multiplexed workflows. Here, we systematically characterized Arg-C Ultra and Lys-C with chromogenic substrates that mimic arginine- and lysine-containing peptides, as well as shotgun proteomics. Arg-C Ultra operates optimally at room temperature, pH 7.5-8.5, under reducing conditions, whereas Lys-C is most active at 37 {degrees}C, pH 7.5-8.5, yet rapidly loses activity when exposed to common reductants. Among tested reducing agents, 1 mM TCEP uniquely preserved TMTpro integrity while sustaining Arg-C Ultra activity. Guided by these insights, we established a sequential digestion workflow that is fully compatible with both label- free DIA and TMTpro multiplexing. Proteins are first digested overnight with Lys-C at 37 {degrees}C (pH 8.5), then treated with 1 mM TCEP and Arg-C Ultra at room temperature (pH 8.5). The resulting peptides can be analyzed directly by label-free DIA or subjected to TMTpro labeling for multiplexed quantification. Applied to HeLa cell lysates, this protocol achieved >99% arginine and 95% lysine cleavage efficiencies, boosting the number of quantified proteins by 6% in label- free DIA and 11% in TMTproC experiments. Replicate measurements displayed reproducibility that approached the limits set by ion statistics. Thus, the introduced synergistic Lys-C/Arg-C Ultra digestion strategy enhances proteome coverage with excellent quantitative reproducibility across both label-free and multiplexed platforms.

systems biology↗

bioRxiv2025

Expanding Targeted Instrumentation for Discovery Applications: Complement Reporter Ion Quantification with a Quadrupole-Ion Trap Instrument

Proteomics workflows have traditionally been divided into discovery-based and targeted approaches, with instrumentation optimized specifically for each. Discovery experiments typically utilize high-resolution analyzers while targeted workflows rely on the sensitivity and specificity of triple quadrupole systems. Recently, a quadrupole-ion trap instrument (Stellar MS) demonstrated superior performance for targeted applications compared to conventional triple quadrupoles. In this study, we expand the capabilities of this platform to multiplexed shotgun proteomics using complement reporter ion quantification in an ion trap (iTMTproC). Benchmarking experiments with defined standards show that iTMTproC achieves quantification accuracy and interference reduction comparable to MultiNotch MS3 on the Orbitrap Fusion Lumos, a dedicated quadrupole-ion trap-Orbitrap tribrid instrument optimized for this purpose. Notably, iTMTproC quantifies slightly more proteins than MultiNotch MS3. We further validate this approach through a developmental time-series analysis of frog embryos, obtaining proteomic data nearly indistinguishable from MultiNotch MS3, with slightly increased protein quantification depth. These findings significantly extend the functionality of targeted instrumentation, underscoring the versatility of quadrupole-ion trap systems and providing cost-effective access to highly accurate, multiplexed quantitative shotgun proteomics.

systems biology↗