Proteomics最新文献

Proteomic experiments, particularly those addressing dynamic proteome properties, time series, or genetic diversity, require the analysis of large sample numbers. Despite significant advancements in proteomic technologies in recent years, further improvements are needed to accelerate measurement and enhance proteome coverage and quantitative performance. Previously, we demonstrated that incorporating a scanning MS2 dimension into data-independent acquisition (DIA) methods (Scanning SWATH, or more generally scanning DIA), but also ion trapping, improves analytical depth and quantitative performance, especially in proteomic methods using fast chromatography. Here, we evaluate the scanning DIA approach combined with ion trapping via the Zeno trap in a method termed ZT Scan DIA, using a ZenoTOF 7600+ instrument (SCIEX). Applying this method to established proteome standards across various analytical setups, enabling intermediate to high sample throughput, we observed a 30%–40% increase in identified precursors. This enhancement extended to overall protein identification and precise quantification. Furthermore, ZT Scan DIA effectively eliminated quantitative bias, as demonstrated by its ability to deconvolute proteomes in multi-species mixtures. We propose that ZT Scan DIA can be used for a broad range of applications in proteomics, particularly in studies requiring high quantitative precision with low sample input and high-throughput workflows.

Fibrosis is characterised by inappropriate wound healing and the buildup of excessive fibrous connective tissue, in particular within the extracellular matrix (ECM). This can occur in multiple organs, ultimately leading to organ failure. Despite the high burden of fibrosis, treatment options only delay disease progression. Therefore, leveraging publicly available proteomics data, we investigated whether common fibrotic proteins and pathways in different organs could be found, to define potential core changes related to fibrosis. We identified 124 significantly differentially expressed proteins in heart fibrosis, four in early-versus-mild liver fibrosis, 135 in mild-versus-severe liver fibrosis and 160 in early-versus-severe liver fibrosis. Functional annotation of each of these groups of proteins demonstrated a consistent upregulation of ECM proteins and a consistent downregulation of proteins associated with mitochondrial activity. Using these data for drug repurposing, 26 compounds were proposed for further investigation, with 20 of them having demonstrated a promising anti-fibrotic effect. A core set of 18 proteins were shared between heart and liver fibrosis, and are associated with increased ECM deposition and fibroblast activation. This approach can be generalised for other pathologies, improving the knowledge on the affected molecular pathways, and based on this, identifying potential drug candidates/compounds.

Seminal extracellular vesicles (SEVs) carry a diverse array of bioactive molecules, including proteins, lipids, and nucleic acids, which influence sperm function and have potential to modulate the female reproductive tract immune response after intromission. However, the full spectrum of SEV cargo involved in these processes remains incompletely defined. Here, we employed label-free quantitative high-resolution mass spectrometry to characterize the human SEV proteome, identifying 5079 associated proteins. These proteins were shown to likely originate from multiple regions of the male reproductive tract, notably the seminal vesicles and prostate, providing evidence for heterogeneous tissue origins of SEVs. Bioinformatic analysis revealed enrichment in sperm- and immune-related functions, as well as functions linked to protein translation. Notably, we identified several proteins with established roles in sperm physiology and immune signaling that had not previously been linked with SEV function. These included; Adenylate kinase isoenzyme (AK)2/9, and Calcium-binding tyrosine-phosphorylation regulated protein (CABYR), implicated in sperm motility, and immune regulators such as the Toll-like receptor 4 ligand, High mobility group protein B1 (HMGB1), and the Nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) inhibitor epsilon (NFκBIE). Altogether, these findings expand the known SEV proteome and highlight proteins that may influence both male and female reproductive capacity.

Misfolding and aggregation of α-Synuclein (α-Syn) play a central role in Parkinson's disease (PD), with oligomeric intermediates implicated as key toxic species. Here, we investigate the aggregation of two α-Syn segments, the NACore (₆₈GAVVTGVTAVA₇₈, WT-PD1) and the preNAC region (₄₇GVVHGVATVA₅₆, WT-PD2), using high-resolution trapped ion-mobility mass spectrometry (TIMS-Qq-ToF) and Thioflavin T fluorescence spectroscopy. The NACore is the minimal sequence required for α-Syn aggregation, whereas most mutations affecting the onset of PD appear to be in the preNAC region, therefore modulating aggregation dynamics and toxicity. Our results demonstrate that TIMS-Qq-ToF effectively resolves and identifies oligomeric species, revealing distinct aggregation pathways for both peptide segments. Fluorescence assays confirm differences in aggregation kinetics and morphology, highlighting the different oligomer formation pathways observed with ion-mobility mass spectrometry. Despite the oligomer-preserving nature of the TIMS itself, ion transmission remains too harsh for the fragile oligomers, leading to fragmentation of fragile non-covalent assemblies. However, collisional cross-section (^N2CCS_TIMS) values support the presence of large oligomers. This study highlights the potential of TIMS-Qq-ToF for mapping α-Syn aggregation and underscores the need for optimised, soft ion transmission to better preserve fragile transient intermediates, ultimately contributing to a deeper understanding of PD. SUMMARY: Understanding early-stage protein aggregation is essential for unravelling the molecular mechanisms of Parkinson's disease, a neurodegenerative disorder that currently lacks effective therapeutic solutions. This study employs high-resolution Trapped Ion Mobility Mass Spectrometry combined with fluorescence spectroscopy to elucidate the oligomerisation of two α-synuclein segments, revealing distinct aggregation pathways and associated structural characteristics. This study underscores the value of peptide models in advancing our understanding of protein aggregation behaviour. This multifaceted approach provides detailed structural insights into the unexplored, transient early-stage oligomers of key α-Syn segments, contributing to a deeper understanding of aggregation mechanisms and providing valuable insights for therapeutic strategies against Parkinson's disease.