Biology subjects

Wühr, M.

Publications and source records attributed to Wühr, M..

6 recordsLinked to original sources

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↗

bioRxiv2023

Quantitative proteome dynamics across embryogenesis in a model chordate

The evolution of gene expression programs underlying the development of vertebrates remains poorly characterized. Here, we present a comprehensive proteome atlas of the model chordate Ciona, covering eight developmental stages and [~]7,000 translated genes, accompanied by a multi-omics analysis of co-evolution with the vertebrate Xenopus. Quantitative proteome comparisons argue against the widely held hourglass model, based solely on transcriptomic profiles, whereby peak conservation is observed during mid-developmental stages. Our analysis reveals maximal divergence at these stages, particularly gastrulation and neurulation. Together, our work provides a valuable resource for evaluating conservation and divergence of multi-omics profiles underlying the diversification of vertebrates.

developmental biology↗

bioRxiv2021

Glycogen-dependent demixing of frog egg cytoplasm at increased crowding

Crowding increases the tendency of macromolecules to aggregate and phase separate, and high crowding can induce glass-like states of cytoplasm. To explore the effect of crowding in a well-characterized model cytoplasm we developed methods to selectively concentrate components larger than 25 kDa from Xenopus egg extracts. When crowding was increased 1.4x, the egg cytoplasm demixed into two liquid phases of approximately equal volume. One of the phases was highly enriched in glycogen while the other had a higher protein concentration. Glycogen hydrolysis blocked or reversed demixing. Quantitative proteomics showed that the glycogen phase was enriched in proteins that bind glycogen, participate in carbohydrate metabolism, or are in complexes with especially high native molecular weight. The glycogen phase was depleted of ribosomes, ER and mitochondria. These results inform on the physical nature of a glycogen-rich cytoplasm and suggest a role of demixing in the localization of glycogen particles in tissue cells.

cell biology↗

bioRxiv2019

Precise temporal regulation of post-transcriptional repressors is required for an orderly Drosophila maternal-to-zygotic transition

In animal embryos the maternal-to-zygotic transition (MZT) hands developmental control from maternal to zygotic gene products. We show that the maternal proteome represents over half of the protein coding capacity of the Drosophila melanogaster genome and that 2% of this proteome is rapidly degraded during the MZT. Cleared proteins include the post-transcriptional repressors Cup, Trailer hitch (TRAL), Maternal expression at 31B (ME31B), and Smaug (SMG). While the ubiquitin-proteasome system is necessary for clearance of all four repressors, distinct E3 ligase complexes target them: the C-terminal to Lis1 Homology (CTLH) complex targets Cup, TRAL and ME31B for degradation early in the MZT; the Skp/Cullin/F-box-containing (SCF) complex targets SMG at the end of the MZT. Deleting the C-terminal 233 amino acids of SMG makes the protein immune to degradation. We show that artificially persistent SMG downregulates the zygotic re-expression of mRNAs whose maternal contribution is cleared by SMG. Thus, clearance of SMG permits an orderly MZT.

developmental biology↗

bioRxiv2017

Proteomics of phosphorylation and protein dynamics during fertilization and meiotic exit in the Xenopus egg

Fertilization triggers release from meiotic arrest and initiates events that prepare for the ensuing developmental program. Protein degradation and phosphorylation are known to regulate protein activity during this process. However, the full extent of protein loss and phospho-regulation is still unknown. We examined absolute protein and phospho-site dynamics after fertilization by mass spectrometry-based proteomics. To do this, we developed a new approach for calculating the stoichiometry of phospho-sites from multiplexed proteomics that is compatible with dynamic, stable and multi-site phosphorylation. Overall, the data suggest that degradation is limited to a few low abundance proteins. However, this degradation promotes extensive dephosphorylation that occurs over a wide range of abundances during meiotic exit. We also show that eggs release a large amount of protein into the medium just after fertilization, most likely related to the blocks to polyspermy. Concomitantly, there is a substantial increase in phosphorylation likely tied to calcium activated kinases. We identify putative degradation targets as well as new components of the block to polyspermy. The analytical approaches demonstrated here are broadly applicable to studies of dynamic biological systems.\n\nSignificance StatementProtein phosphorylation and degradation drive critical events in early embryogenesis and the cell cycle; however, comprehensive and accurate analysis of these changes is currently difficult. Using a mass spectrometric approach, we present a quantitative view of the protein and posttranslational economy of the fertilization response in the frog egg. Protein degradation affects a small but very important class of proteins, while regulatory phosphorylation and protein release occur on a far larger scale. We have developed new, broadly applicable analytical methods for phosphorylation that provide absolute quantification with confidence intervals for improved interpretability of post-translational modification analysis.

systems biology↗