Metabolic Diversity in Human Non-Small Cell Lung Cancer Cells.

Intermediary metabolism in cancer cells is regulated by diverse cell-autonomous processes, including signal transduction and gene expression patterns, arising from specific oncogenotypes and cell lineages. Although it is well established that metabolic reprogramming is a hallmark of cancer, we lack a full view of the diversity of metabolic programs in cancer cells and an unbiased assessment of the associations between metabolic pathway preferences and other cell-autonomous processes. Here, we quantified metabolic features, mostly from the 13C enrichment of molecules from central carbon metabolism, in over 80 non-small cell lung cancer (NSCLC) cell lines cultured under identical conditions. Because these cell lines were extensively annotated for oncogenotype, gene expression, protein expression, and therapeutic sensitivity, the resulting database enables the user to uncover new relationships between metabolism and these orthogonal processes.

Derailed protein turnover in the aging mammalian brain.

Efficient protein turnover is essential for cellular homeostasis and organ function. Loss of proteostasis is a hallmark of aging culminating in severe dysfunction of protein turnover. To investigate protein turnover dynamics as a function of age, we performed continuous in vivo metabolic stable isotope labeling in mice along the aging continuum. First, we discovered that the brain proteome uniquely undergoes dynamic turnover fluctuations during aging compared to heart and liver tissue. Second, trends in protein turnover in the brain proteome during aging showed sex-specific differences that were tightly tied to cellular compartments. Next, parallel analyses of the insoluble proteome revealed that several cellular compartments experience hampered turnover, in part due to misfolding. Finally, we found that age-associated fluctuations in proteasome activity were associated with the turnover of core proteolytic subunits, which was recapitulated by pharmacological suppression of proteasome activity. Taken together, our study provides a proteome-wide atlas of protein turnover across the aging continuum and reveals a link between the turnover of individual proteasome subunits and the age-associated decline in proteasome activity.

Chemical isotope labeling for quantitative proteomics.

Advancements in liquid chromatography and mass spectrometry over the last decades have led to a significant development in mass spectrometry-based proteome quantification approaches. An increasingly attractive strategy is multiplex isotope labeling, which significantly improves the accuracy, precision and throughput of quantitative proteomics in the data-dependent acquisition mode. Isotope labeling-based approaches can be classified into MS1-based and MS2-based quantification. In this review, we give an overview of approaches based on chemical isotope labeling and discuss their principles, benefits, and limitations with the goal to give insights into fundamental questions and provide a useful reference for choosing a method for quantitative proteomics. As a perspective, we discuss the current possibilities and limitations of multiplex, isotope labeling approaches for the data-independent acquisition mode, which is increasing in popularity.

Metabolic regulatory network kinetic modeling with multiple isotopic tracers for iPSCs.

The rapidly expanding market for regenerative medicines and cell therapies highlights the need to advance the understanding of cellular metabolisms and improve the prediction of cultivation production process for human induced pluripotent stem cells (iPSCs). In this paper, a metabolic kinetic model was developed to characterize the underlying mechanisms of iPSC culture process, which can predict cell response to environmental perturbation and support process control. This model focuses on the central carbon metabolic network, including glycolysis, pentose phosphate pathway, tricarboxylic acid cycle, and amino acid metabolism, which plays a crucial role to support iPSC proliferation. Heterogeneous measures of extracellular metabolites and multiple isotopic tracers collected under multiple conditions were used to learn metabolic regulatory mechanisms. Systematic cross-validation confirmed the model's performance in terms of providing reliable predictions on cellular metabolism and culture process dynamics under various culture conditions. Thus, the developed mechanistic kinetic model can support process control strategies to strategically select optimal cell culture conditions at different times, ensure cell product functionality, and facilitate large-scale manufacturing of regenerative medicines and cell therapies.

A green-footprint approach for parallel multiclass analysis of contaminants in roasted coffee via LC-HRMS.

The development and validation of a simple, comprehensive, and environment-friendly procedure to determine pesticide residues, naturally occurring and processing contaminants in roasted coffee is presented. A solid-liquid extraction of pesticides and mycotoxins with ethyl acetate and the concurrent partition of acrylamide to an aqueous phase follows a parallel analytical strategy that requires a single analytical portion to determine contaminants that are typically analyzed by dedicated single residue methods. The partition rules the lipids out of the aqueous extract before an "in-tube" dispersive solid phase microextraction (dSPME) for acrylamide retention. This is followed by the elution with buffer prior to injection. This extract is independently introduced into the system front end followed by the injection of the compounds from the organic phase, yet all spotted in the same run. A novel liquid chromatography high-resolution mass spectrometry (LC-HRMS) method setup enables the quantification of 186 compounds at 10 µg/kg, 226 at 5 µg/kg, and the acrylamide at 200 µg/kg for a total of 414 molecules, with acceptable recoveries (70-120%) and precision (RSD < 20%) making this strategy significantly faster and cost-effective than the dedicated single residue methods. Even though the presence of chlorpyrifos, acrylamide, and ochratoxin A was confirmed on samples of different origins, the findings were below the limit of quantification. During the storage of raw coffee, no proof of masking of OTA was found; however, condensation with glucose was evidenced during thermal processing experiments with sucrose by using stable isotope labeling (SIL). No detected conjugates were found in roasted nor in commercial sugar-added torrefacto samples, an industrial processing usually carried out above the decomposition temperature of the disaccharide.

A Mechanism to Transform Complex Salicinoids with Caffeoylquinic Acids in Lepidopteran Specialist Herbivores (Notodontidae).

Larvae of the Salicaceae-adapted Notodontidae have developed a unique mechanism to metabolize the chemical defenses of their Salicaceae host plants. Salicinoids and salicortinoids are enzymatically transformed into salicyloyl, benzoyl and mixed salicyloyl-benzoyl quinates. The source of quinates and benzoates was previously unknown. To elucidate the origin of quinate and benzoate in the metabolic end-products, we fed Cerura vinula caterpillars with 13C-labelled poplar defense compounds. Caffeoylquinic acids (CQAs), such as chlorogenic acid, neochlorogenic acid and their methyl esters, were identified as the source of quinates in the caterpillar's metabolism. Benzoyl substituents in the quinate end-products were found to originate from compounds such as tremulacin or trichocarpin. Salicaceae-adapted Notodontidae caterpillars have the ability to overcome their host plant's chemical defense by metabolizing CQAs and salicinoids, both abundant defense compounds in Salicacea plants, by a strategy of transformation and recombination. We believe that our study opens up avenues for understanding salicortinoid biotransformation at the enzymatic level.

Harmonizing Labeling and Analytical Strategies to Obtain Protein Turnover Rates in Intact Adult Animals.

Changes in the abundance of individual proteins in the proteome can be elicited by modulation of protein synthesis (the rate of input of newly synthesized proteins into the protein pool) or degradation (the rate of removal of protein molecules from the pool). A full understanding of proteome changes therefore requires a definition of the roles of these two processes in proteostasis, collectively known as protein turnover. Because protein turnover occurs even in the absence of overt changes in pool abundance, turnover measurements necessitate monitoring the flux of stable isotope-labeled precursors through the protein pool such as labeled amino acids or metabolic precursors such as ammonium chloride or heavy water. In cells in culture, the ability to manipulate precursor pools by rapid medium changes is simple, but for more complex systems such as intact animals, the approach becomes more convoluted. Individual methods bring specific complications, and the suitability of different methods has not been comprehensively explored. In this study, we compare the turnover rates of proteins across four mouse tissues, obtained from the same inbred mouse strain maintained under identical husbandry conditions, measured using either [13C6]lysine or [2H2]O as the labeling precursor. We show that for long-lived proteins, the two approaches yield essentially identical measures of the first-order rate constant for degradation. For short-lived proteins, there is a need to compensate for the slower equilibration of lysine through the precursor pools. We evaluate different approaches to provide that compensation. We conclude that both labels are suitable, but careful determination of precursor enrichment kinetics in amino acid labeling is critical and has a considerable influence on the numerical values of the derived protein turnover rates.

Cardiac Myosin Filaments are Maintained by Stochastic Protein Replacement.

Myosin and myosin-binding protein C are exquisitely organized into giant filamentous macromolecular complexes within cardiac muscle sarcomeres, yet these proteins must be continually replaced to maintain contractile fidelity. The overall hypothesis that myosin filament structure is dynamic and allows for the stochastic replacement of individual components was tested in vivo, using a combination of mass spectrometry- and fluorescence-based proteomic techniques. Adult mice were fed a diet that marked all newly synthesized proteins with a stable isotope-labeled amino acid. The abundance of unlabeled and labeled proteins was quantified by high-resolution mass spectrometry over an 8-week period. The rates of change in the abundance of these proteins were well described by analytical models in which protein synthesis defined stoichiometry and protein degradation was governed by the stochastic selection of individual molecules. To test whether the whole myosin filaments or the individual components were selected for replacement, cardiac muscle was chemically skinned to remove the cellular membrane and myosin filaments were solubilized with ionic solutions. The composition of the filamentous and soluble fractions was quantified by mass spectrometry, and filament depolymerization was visualized by real-time fluorescence microscopy. Myosin molecules were preferentially extracted from ends of the filaments in the presence of the ionic solutions, and there was only a slight bias in the abundance of unlabeled molecules toward the innermost region on the myosin filaments. These data demonstrate for the first time that the newly synthesized myosin and myosin-binding protein C molecules are randomly mixed into preexisting thick filaments in vivo and the rate of mixing may not be equivalent along the length of the thick filament. These data collectively support a new model of cardiac myosin filament structure, with the filaments being dynamic macromolecular assemblies that allow for replacement of their components, rather than rigid bodies.

Elevated Temperature Effects on Protein Turnover Dynamics in Arabidopsis thaliana Seedlings Revealed by 15N-Stable Isotope Labeling and ProteinTurnover Algorithm.

Global warming poses a threat to plant survival, impacting growth and agricultural yield. Protein turnover, a critical regulatory mechanism balancing protein synthesis and degradation, is crucial for the cellular response to environmental changes. We investigated the effects of elevated temperature on proteome dynamics in Arabidopsis thaliana seedlings using 15N-stable isotope labeling and ultra-performance liquid chromatography-high resolution mass spectrometry, coupled with the ProteinTurnover algorithm. Analyzing different cellular fractions from plants grown under 22 °C and 30 °C growth conditions, we found significant changes in the turnover rates of 571 proteins, with a median 1.4-fold increase, indicating accelerated protein dynamics under thermal stress. Notably, soluble root fraction proteins exhibited smaller turnover changes, suggesting tissue-specific adaptations. Significant turnover alterations occurred with redox signaling, stress response, protein folding, secondary metabolism, and photorespiration, indicating complex responses enhancing plant thermal resilience. Conversely, proteins involved in carbohydrate metabolism and mitochondrial ATP synthesis showed minimal changes, highlighting their stability. This analysis highlights the intricate balance between proteome stability and adaptability, advancing our understanding of plant responses to heat stress and supporting the development of improved thermotolerant crops.

Feed Components and Timing to Improve the Feed Conversion Ratio for Sustainable Aquaculture Using Starch.

Aquaculture contributes to the sustainable development of food security, marine resource conservation, and economy. Shifting aquaculture feed from fish meal and oil to terrestrial plant derivatives may result in cost savings. However, many carnivorous fish cannot be sustained on plant-derived materials, necessitating the need for the identification of important factors for farmed fish growth and the identification of whether components derived from terrestrial plants can be used in feed. Herein, we focused on the carnivorous fish leopard coral grouper (P. leopardus) to identify the essential growth factors and clarify their intake timing from feeds. Furthermore, we evaluated the functionality of starch, which are easily produced by terrestrial plants. Results reveal that carbohydrates, which are not considered essential for carnivorous fish, can be introduced as a major part of an artificial diet. The development of artificial feed using starch offers the possibility of increasing the growth of carnivorous fish in aquaculture.

Retention Time Alignment for Protein Turnover Studies Using Heavy Water Metabolic Labeling.

Retention time (RT) alignment has been important for robust protein identification and quantification in proteomics. In data-dependent acquisition mode, whereby the precursor ions are semistochastically chosen for fragmentation in MS/MS, the alignment is used in an approach termed matched between runs (MBR). MBR transfers peptides, which were fragmented and identified in one experiment, to a replicate experiment where they were not identified. Before the MBR transfer, the RTs of experiments are aligned to reduce the chance of erroneous transfers. Despite its widespread use in other areas of quantitative proteomics, RT alignment has not been applied in data analyses for protein turnover using an atom-based stable isotope-labeling agent such as metabolic labeling with deuterium oxide, D2O. Deuterium incorporation changes isotope profiles of intact peptides in full scans and their fragment ions in tandem mass spectra. It reduces the peptide identification rates in current database search engines. Therefore, the MBR becomes more important. Here, we report on an approach to incorporate RT alignment with peptide quantification in studies of proteome turnover using heavy water metabolic labeling and LC-MS. The RT alignment uses correlation-optimized time warping. The alignment, followed by the MBR, improves labeling time point coverage, especially for long labeling durations.

TurnoveR: A Skyline External Tool for Analysis of Protein Turnover in Metabolic Labeling Studies.

In spite of its central role in biology and disease, protein turnover is a largely understudied aspect of most proteomic studies due to the complexity of computational workflows that analyze in vivo turnover rates. To address this need, we developed a new computational tool, TurnoveR, to accurately calculate protein turnover rates from mass spectrometric analysis of metabolic labeling experiments in Skyline, a free and open-source proteomics software platform. TurnoveR is a straightforward graphical interface that enables seamless integration of protein turnover analysis into a traditional proteomics workflow in Skyline, allowing users to take advantage of the advanced and flexible data visualization and curation features built into the software. The computational pipeline of TurnoveR performs critical steps to determine protein turnover rates, including isotopologue demultiplexing, precursor-pool correction, statistical analysis, and generation of data reports and visualizations. This workflow is compatible with many mass spectrometric platforms and recapitulates turnover rates and differential changes in turnover rates between treatment groups calculated in previous studies. We expect that the addition of TurnoveR to the widely used Skyline proteomics software will facilitate wider utilization of protein turnover analysis in highly relevant biological models, including aging, neurodegeneration, and skeletal muscle atrophy.

Conditional Fragment Ion Probabilities Improve Database Searching for Nonmonoisotopic Precursors.

Stochastic, intensity-based precursor isolation can result in isotopically enriched fragment ions. This problem is exacerbated for large peptides and stable isotope labeling experiments using deuterium or 15N. For stable isotope labeling experiments, incomplete and ubiquitous labeling strategies result in the isolation of peptide ions composed of many distinct structural isomers. Unfortunately, existing proteomics search algorithms do not account for this variability in isotopic incorporation, and thus often yield poor peptide and protein identification rates. We sought to resolve this shortcoming by deriving the expected isotopic distributions of each fragment ion and incorporating them into the theoretical mass spectra used for peptide-spectrum-matching. We adapted the Comet search platform to integrate a modified spectral prediction algorithm we term Conditional fragment Ion Distribution Search (CIDS). Comet-CIDS uses a traditional database searching strategy, but for each candidate peptide we compute the isotopic distribution of each fragment to better match the observed m/z distributions. Evaluating previously generated D2O and 15N labeled data sets, we found that Comet-CIDS identified more confident peptide spectral matches and higher protein sequence coverage compared to traditional theoretical spectra generation, with the magnitude of improvement largely determined by the amount of labeling in the sample.

Quantitative Proteomics-Based Substrate Screening Revealed Cyclophilin Stabilization Regulated by Deubiquitinase Ubp7.

Quantitative proteomics has emerged as a crucial approach to identifying ubiquitinated substrates to investigate the functions of ubiquitination in cells. In this regard, although the substrate screening of certain enzymes in the ubiquitin system has been based on proteome or ubiquitinome level measurements, the direct comparison of these two approaches has not been determined to date. To quantitatively compare the efficiency and effectiveness of substrate screening from the entire proteomics to the ubiquitinomics filter, we used yeast deubiquitinating enzyme, Ubp7, as an example to evaluate it in this study. A total of 112 potential ubiquitinated substrates were identified from the ubiquitinomics level, whereas only 27 regulated substrates were identified from the entire proteomic screening, demonstrating the increased efficiency of ubiquitinomics quantitative analysis. Subsequently, we selected cyclophilin A (Cpr1) protein as an example, which was filtered out at the proteomics level but was a promising candidate according to the ubiquitinomics filter. Additional investigations revealed that Cpr1 possessed a K48-linked ubiquitin chain regulated by Ubp7, which may affect its homeostasis and, consequently, sensitivity to the therapeutic drug cyclosporine (CsA).

Phosphoproteomics Profiling Defines a Target Landscape of the Basophilic Protein Kinases AKT, S6K, and RSK in Skeletal Myotubes.

Phosphorylation-dependent signal transduction plays an important role in regulating the functions and fate of skeletal muscle cells. Central players in the phospho-signaling network are the protein kinases AKT, S6K, and RSK as part of the PI3K-AKT-mTOR-S6K and RAF-MEK-ERK-RSK pathways. However, despite their functional importance, knowledge about their specific targets is incomplete because these kinases share the same basophilic substrate motif RxRxxp[ST]. To address this, we performed a multifaceted quantitative phosphoproteomics study of skeletal myotubes following kinase inhibition. Our data corroborate a cross talk between AKT and RAF, a negative feedback loop of RSK on ERK, and a putative connection between RSK and PI3K signaling. Altogether, we report a kinase target landscape containing 49 so far unknown target sites. AKT, S6K, and RSK phosphorylate numerous proteins involved in muscle development, integrity, and functions, and signaling converges on factors that are central for the skeletal muscle cytoskeleton. Whereas AKT controls insulin signaling and impinges on GTPase signaling, nuclear signaling is characteristic for RSK. Our data further support a role of RSK in glucose metabolism. Shared targets have functions in RNA maturation, stability, and translation, which suggests that these basophilic kinases establish an intricate signaling network to orchestrate and regulate processes involved in translation.

Recent progress in quantitative phosphoproteomics.

INTRODUCTION: Protein phosphorylation is a critical post-translational modification involved in the regulation of numerous cellular processes from signal transduction to modulation of enzyme activities. Knowledge of dynamic changes of phosphorylation levels during biological processes, under various treatments or between healthy and disease models is fundamental for understanding the role of each phosphorylation event. Thereby, LC-MS/MS based technologies in combination with quantitative proteomics strategies evolved as a powerful strategy to investigate the function of individual protein phosphorylation events. AREAS COVERED: State-of-the-art labeling techniques including stable isotope and isobaric labeling provide precise and accurate quantification of phosphorylation events. Here, we review the strengths and limitations of recent quantification methods and provide examples based on current studies, how quantitative phosphoproteomics can be further optimized for enhanced analytic depth, dynamic range, site localization, and data integrity. Specifically, reducing the input material demands is key to a broader implementation of quantitative phosphoproteomics, not least for clinical samples. EXPERT OPINION: Despite quantitative phosphoproteomics is one of the most thriving fields in the proteomics world, many challenges still have to be overcome to facilitate even deeper and more comprehensive analyses as required in the current research, especially at single cell levels and in clinical diagnostics.

Mechanistic Insights into the Biodegradation of Carbon Dots by Fungal Laccase.

While carbon dots (CDs) have the potential to support the agricultural revolution, it remains obscure about their environmental fate and bioavailability by plants. Fungal laccase-mediated biotransformation of carbon nanomaterials has received little attention despite its known capacity to eliminate recalcitrant contaminants. Herein, we presented the initial investigation into the transformation of CDs by fungal laccase. The degradation rates of CDs were determined to be first-order in both substrate and enzyme. Computational docking studies showed that CDs preferentially bonded to the pocket of laccase on the basal plane rather than the edge through hydrogen bonds and hydrophobic interactions. Electrospray ionization-Fourier transform-ion cyclotron resonance mass spectrometry (ESI-FT-ICR MS) and other characterizations revealed that the phenolic/amino lignins and tannins portions in CDs are susceptible to laccase transformation, resulting in graphitic structure damage and smaller-sized fragments. By using the 13C stable isotope labeling technique, we quantified the uptake and translocation of 13C-CDs by mung bean plants. 13C-CDs (10 mg L-1) accumulated in the root, stem, and leaf were estimated to be 291, 239, and 152 µg g-1 at day 5. We also evidenced that laccase treatment alters the particle size and surface chemistry of CDs, which could facilitate the uptake of CDs by plants and reduce their nanotoxicity to plants.

Choline supplementation for preterm infants: metabolism of four Deuterium-labeled choline compounds.

BACKGROUND: Supply of choline is not guaranteed in current preterm infant nutrition. Choline serves in parenchyma formation by membrane phosphatidylcholine (PC), plasma transport of poly-unsaturated fatty acids (PUFA) via PC, and methylation processes via betaine. PUFA-PC concentrations are high in brain, liver and lung, and deficiency may result in developmental disorders. We compared different deuterated (D9-) choline components for kinetics of D9-choline, D9-betaine and D9-PC. METHODS: Prospective study (1/2021-12/2021) in 32 enterally fed preterm infants (28 0/7-32 0/7 weeks gestation). Patients were randomized to receive enterally a single dose of 2.7 mg/kg D9-choline-equivalent as D9-choline chloride, D9-phosphoryl-choline, D9-glycerophosphorylcholine (D9-GPC) or D9-1-palmitoyl-2-oleoyl-PC(D9-POPC), followed by blood sampling at 1 + 24 h or 12 + 60 h after administration. Plasma concentrations were analyzed by tandem mass spectrometry. Results are expressed as median (25th/75th percentile). RESULTS: At 1 h, plasma D9-choline was 1.8 (0.9/2.2) µmol/L, 1.3 (0.9/1.5) µmol/L and 1.2 (0.7/1.4) µmol/L for D9-choline chloride, D9-GPC and D9-phosphoryl-choline, respectively. D9-POPC did not result in plasma D9-choline. Plasma D9-betaine was maximal at 12 h, with lowest concentrations after D9-POPC. Maximum plasma D9-PC values at 12 h were the highest after D9-POPC (14.4 (9.1/18.9) µmol/L), compared to the other components (D9-choline chloride: 8.1 [5.6/9.9] µmol/L; D9-GPC: 8.4 (6.2/10.3) µmol/L; D9-phosphoryl-choline: 9.8 (8.6/14.5) µmol/L). Predominance of D9-PC comprising linoleic, rather than oleic acid, indicated fatty-acyl remodeling of administered D9-POPC prior to systemic delivery. CONCLUSION: D9-Choline chloride, D9-GPC and D9-phosphoryl-choline equally increased plasma D9-choline and D9-betaine. D9-POPC shifted metabolism from D9-betaine to D9-PC. Combined supplementation of GPC and (PO) PC may be best suited to optimize choline supply in preterm infants. Due to fatty acid remodeling of (PO) PC during its assimilation, PUFA co-supplementation with (PO) PC may increase PUFA-delivery to critical organs. This study was registered (22.01.2020) at the Deutsches Register Klinischer Studien (DRKS) (German Register for Clinical Studies), DRKS00020502. STUDY REGISTRATION: This study was registered at the Deutsches Register Klinischer Studien (DRKS) (German Register for Clinical Studies), DRKS00020502.

Reductive Conversion Leads to Detoxification of Salicortin-like Chemical Defenses (Salicortinoids) in Lepidopteran Specialist Herbivores (Notodontidae).

Lepidopteran specialist herbivores of the Notodontidae family have adapted to thrive on poplar and willow species (Salicaceae). Previous research showed that Cerura vinula, a member of the Notodontidae family occurring throughout Europe and Asia, uses a unique mechanism to transform salicortinoids, the host plant's defense compounds, into quinic acid-salicylate conjugates. However, how the production of this conjugates relates to the detoxification of salicortinoids and how this transformation proceeds mechanistically have remained unknown. To find the mechanisms, we conducted gut homogenate incubation experiments with C. vinula and re-examined its metabolism by analyzing the constituents of its frass. To estimate the contribution of spontaneous degradation, we examined the chemical stability of salicortinoids and found that salicortinoids were degraded very quickly by midgut homogenates and that spontaneous degradation plays only a marginal role in the metabolism. We learned how salicortinoids are transformed into salicylate after we discovered reductively transformed derivatives, which were revealed to play key roles in the metabolism. Unless they have undergone the process of reduction, salicortinoids produce toxic catechol. We also studied constituents in the frass of the Notodontidae species Cerura erminea, Clostera anachoreta, Furcula furcula, Notodonta ziczac, and Pheosia tremula, and found the same metabolites as those described for C. vinula. We conclude that the process whereby salicortinoids are reductively transformed represents an important adaption of the Notodontidae to their Salicaceae host species.

Revealing mechanisms of mating plug function under sexual selection.

Mating plugs are produced by many sexually reproducing animals and are hypothesized to promote male fertilization success under promiscuous mating. However, tests of this hypothesis have been constrained by an inability to discriminate ejaculates of different males in direct competition. Here, we use stable isotope labeling in vivo and proteomics to achieve this in a promiscuous rodent, Myodes glareolus We show that, although the first male's plug is usually dislodged, it can be retained throughout the second male's copulation. Retained plugs did not completely block rival sperm but did significantly limit their numbers. Differences in the number of each male's sperm progressing through the female reproductive tract were also explained by natural variation in the size of mating plugs and reproductive accessory glands from which major plug proteins originate. Relative sperm numbers in turn predicted the relative fertilization success of rival males. Our application of stable isotopes to label ejaculates resolves a longstanding debate by revealing how rodent mating plugs promote fertilization success under competitive conditions. This approach opens new opportunities to reveal cryptic mechanisms of postcopulatory sexual selection among diverse animal taxa.