Molecular & Cellular Proteomics最新文献

Nitric oxide (NO) is a crucial signaling molecule involved in various developmental processes and stress responses through post-translational protein modification and modulation of gene expression. Despite significant advances in understanding the mechanism of NO-mediated protein modifications, how NO regulates gene expression remains largely unclear. Here, we show that the energy sensor KIN10, a catalytic α-subunit of sucrose non-fermenting 1-related kinase 1, plays a vital role in NO-mediated regulation of gene expression in Arabidopsis. NO-mediated S-nitrosylation at Cys-177 of KIN10 inhibits its degradation, leading to protein stabilization. A non-nitrosylatable mutation of Cys-177 to serine results in NO insensitivity and functional deficiencies. Quantitative phosphoproteomic analysis reveals that S-nitrosylation at Cys-177 of KIN10 modulates the phosphorylation of splicing factors within the spliceosome. We propose that NO regulates RNA splicing through the enhancement of KIN10 activity via S-nitrosylation, thereby establishing a molecular link between NO signaling and gene expression.

Achieving high-resolution spatial tissue proteomes requires careful balancing and integration of optimized sample processing, chromatography, and MS acquisition. Here, we present an advanced cellenONE protocol for loss-reduced tissue processing and compare all Evosep ONE Whisper Zoom gradients (20, 40, 80, and 120 samples per day), along with three common data-independent acquisition schemes on a timsUltra athena ion processor mass spectrometer. We found that tissue type was as important as gradient length and sample amount in determining proteome coverage. Moreover, the benefit of increased tissue sampling was gradient- and dynamic range-dependent. Analyzing mouse liver, a high dynamic range tissue, over tenfold more tissue sampling led to only ∼30% gain in protein identification for short gradients (120 samples per day (SPD) and 80 SPD). However, even the lowest tested tissue amount (0.04 nl) yielded 3200 reproducibly quantified proteins for the 120 SPD method. Longer gradients (40 SPD and 20 SPD) instead significantly benefited from more tissue sampling, quantifying over 7500 proteins from 0.5 nl of tonsil T-cell niches. Finally, we applied our workflow to a rare squamous cell carcinoma of the oral cavity, uncovering disease-associated pathways and region-specific protein level changes. Our study demonstrates that more than 100 high-quality spatial tissue proteomes can be prepared and acquired daily, laying a strong foundation for cohort-size spatial tissue proteomics in translational research.

Single-cell omics technologies, such as single-cell RNA-Seq and single-cell proteomics, offer unprecedented insights into cellular heterogeneity and dynamic regulatory processes. However, integrating these data types to construct comprehensive transcription-translation profiles remains challenging because of their distinct and complex behaviors. This study presents a novel approach using pseudotemporal cell ordering to integrate single-cell RNA-Seq and single-cell proteomics by mass spectrometry data, facilitating the analysis of transcription-translation expression dynamics. We collected longitudinal single-cell samples following hypoxia. By leveraging key markers, we constructed pseudotemporal trajectories for each data type, revealing transcriptional and translational responses to hypoxia. This profile of unified single-cell mRNA and protein expression uncovers distinct regulatory mechanisms, including an immediate transcriptomic response, followed by delayed proteomic expression. It illustrates the use of pseudotemporal integration to integrate single-cell transcriptomic and proteomic datasets to understand the cellular phenotypes under hypoxic stress and provides a framework for future investigations into transcription-translation dynamics.

Hair follicle development is a complex, highly regulated process involving interactions between epithelial and mesenchymal cells. However, the specific molecular mechanisms and important biological processes of hair follicle development remain poorly understood. How the extracellular matrix is involved in the hair follicle formation from hair germs remains to be investigated. In this study, we applied spatially resolved proteomic mapping to investigate the process of hair follicle development in skin organoids at different stages: D55, D75, D90, D140, D150, and D170, which corresponds to that from hair germ formation to hair follicle aging. Our analysis identified dynamic changes in protein expression and active protein synthesis during hair follicle appearance. We observed stage-specific protein expression patterns, with hair germ and hair peg formation, enriched in proteins involved in RNA processing and lipid metabolism. Meanwhile, hair follicle initial and full maturation highlighted proteins related to keratinization and extracellular matrix organization. Notably, trend proteins involved in keratinization and neuron-neuron synaptic transmission were upregulated from hair germ formation to the hair follicle appearance. We also found that CSNK1A1 and SFN exhibit abnormal expression in the hair follicles of patients with cicatricial alopecia, which further proves the role of CSNK1A1 and SFN in the normal development of hair follicles. The results provide a comprehensive spatial proteomic map of hair follicle development and offer new insights into the biological process driving hair follicle formation and maturation. These findings may guide future therapeutic strategies for hair regeneration and the treatment of hair disorders.

Neutrophils respond rapidly to inflammation and infection via defense mechanisms, including degranulation, reactive oxygen species production, and neutrophil extracellular trap formation (known as "NETosis"). As the most abundant neutrophil components, granule proteins constitute the major mediators of neutrophil effector functions and likely orchestrate their functional diversity. However, a systematic profile of these proteins, particularly their temporal release dynamics during inflammatory responses, remains uncharacterized. Here, we performed a "multistate" proteomic study to explore circulating neutrophils' dynamic responses to diverse infectious and inflammatory signals over time. Circulating neutrophils exhibited both conserved and stimulus-specific protein expression programs. Through integrated characterization of the cellular and secretory proteome landscapes, we delineated the release patterns of canonical granule proteins and identified inflammatory mediators, including soluble membrane receptors. Notably, granule membrane receptors were translocated to the cell surface and shed via proteolytic cleavage, highlighting their dynamic regulation and diversity. These findings revealed the complexity of the neutrophil degranulation program, demonstrating its stimulus-dependent and temporally layered nature. Our study provides a functional atlas of neutrophil degranulation upon inflammation, which would strengthen our understanding of neutrophil activation in inflammation and facilitate the exploration of inflammation management therapies.

N-glycosylation is an abundant and essential co/post-translational modification that is preserved across all eukaryotes. N-glycans have important functions in protein stability and protein-protein interactions. N-glycans exhibit a high degree of heterogeneity, even within an individual site on the same protein, a phenomenon that is termed "microheterogeneity," which is the focus of this review. Traditional analytical approaches with released glycans are limited in their usefulness in studying microheterogeneity because of most glycoproteins having more than one site of N-glycosylation. Since specific N-glycans at specific sites can confer important functions to glycoproteins, this presents a significant gap between the information content of glycomics and glycoproteomics experiments. More recently, tandem mass spectrometry of intact glycopeptides has been used to obtain site-specific information on N-glycan microheterogeneity. The microheterogeneity of glycoproteins presents a significant analytical challenge not only during mass spectrometry analyses but also in downstream data processing. Use of specialized search engines followed by extensive manual validation is often required for accurate and in-depth glycoproteomics. Overall, recent advances in analytical technology and data processing present exciting new opportunities to analyze N-glycans in a site-specific manner. Being able to define, understand functional consequences of, and even predict and direct N-glycan microheterogeneity has implications across many fields, including the manipulation and production of glycoprotein biologics.

In Alzheimer's disease (AD) and other tauopathies, tau dissociates from microtubules and forms toxic aggregates that contribute to neurodegeneration. Although some of the pathological interactions of tau have been identified from postmortem brain tissue, these studies are limited by their inability to capture transient interactions. To investigate the interactome of aggregate-prone fragments of tau, we applied an in vitro proximity labeling technique using split TurboID biotin ligase (sTurbo) fused with the tau microtubule repeat domain (TauRD), a core region implicated in tau aggregation. We characterized this sTurbo TauRD tagging interactors with the requirement for both ligase fragment co-expression for robust enzyme activity and nuclear and cytoplasmic localization of the recombinant proteins. Following enrichment of biotinylated proteins and mass spectrometry, we identified over 700 TauRD interactors. Gene ontology analysis of enriched TauRD interactors highlighted processes often dysregulated in tauopathies, including spliceosome complexes, RNA-binding proteins, and nuclear speckles. The disease relevance of these interactors was supported by integrating recombinant TauRD interactome data with human AD tau interactome datasets and protein co-expression networks from individuals with AD and related tauopathies. This revealed an overlap with the TauRD interactome and several modules enriched with RNA-binding proteins and increased in AD and progressive supranuclear palsy. These findings emphasize the importance of nuclear pathways in tau pathology, such as mRNA surveillance and RNA splicing, establishing the sTurbo TauRD system as a valuable tool for exploring the tau interactome.

Cystic echinococcosis (CE), a parasitic disease caused by Echinococcus granulosus (Eg), remains prevalent in underdeveloped pastoral regions. Current diagnostic methods for CE primarily rely on imaging techniques, whereas serological tests still require significant improvement. To address this challenge, we have developed an immunoproteomics workflow to identify novel diagnostic Eg antigens. Our approach involved extracting proteins from CE surgical tissues, which were then recognized by patient plasma through immunoblotting and subsequently identified using mass spectrometry. Applying stringent criteria to evaluate Eg protein antigenicity, we selected 25 candidates for expression, and 18 recombinant proteins demonstrated enhanced immunoreactivity with CE patient plasma. Further validation led to the identification of a novel panel comprising eight Eg recombinant antigens, which exhibited superior diagnostic capabilities with sensitivities ranging from 91.26% to 99.09% and specificities ranging from 95% to 97%. This panel was tested in a large-scale study involving 1068 plasma samples collected from patients with ultrasound-confirmed CE (+) and healthy controls. Our findings introduce a set of novel Eg antigens with significant potential for improving CE clinical diagnosis, particularly in its early stages. This research not only advances our understanding of CE immunology but also offers promising tools for enhancing disease detection and management in affected populations.

Malaria transmission from humans to mosquitoes is essential to the parasite life cycle. In the human malaria parasite, Plasmodium falciparum, the rate of commitment to produce the sexual transmission stages, or gametocytes varies and is governed by genetic, epigenetic and environmental factors. The sexually committed parasite has so far remained elusive due to the lack of markers to efficiently isolate these parasites for subsequent functional studies including proteomic analysis of the isolated population. Here, we demonstrate that MSRP1 is a highly specific sexual commitment marker. Using this marker, we generated and validated reporter parasite lines for subsequent FACS-based isolation of sexually and asexually committed parasites. Proteomics of isolated parasites defined distinct protein signatures, including several merozoite surface proteins, indicating functional differences between the two parasite populations. This study provides a blueprint for systematic characterization of the parasite stage at this crucial juncture in the life cycle.