Proteomics最新文献

To assess the potential for high resolution ion mobility (HRIM) as an alternative means of precursor isolation for mass spectrometry fragmentation analysis we performed a meta-analysis of predicted tryptic peptide features from the human proteome to measure the rate of chimeric spectrum generation relative to traditional quadrupole-based isolation. Results indicate that for proteomic mixtures, HRIM separation with a peak capacity of 100 produces chimeric spectra at a rate commensurate with a ∼5 Th quadrupole isolation window, while providing the additional benefit of generating non-chimeric spectra for many isobaric and isomeric peptides unresolvable with a quadrupole filter. This behavior was verified experimentally using a HRIM-QTOF mass spectrometry system. The ability to combine HRIM and MS isolation resulted in >10× increase in precursor isolation specificity as compared to either of the techniques independently.

The proteomic analysis of blood is routine for disease phenotyping and biomarker development. Blood is commonly separated into soluble and cellular fractions. However, this can introduce pre-analytical variability, and analysis of a single component (which is common) may ignore important pathophysiology. We have recently developed methods for the facile processing of dried blood for mass spectrometry-based quantification of the proteome, N-glycoproteome, and phosphoproteome. Here, we applied this approach to 38 patients in Tanzania who presented to the hospital with sepsis. Blood was collected on Mitra devices at presentation and 1, 3, and 28-42 days post-enrollment. Processing of 96 total samples was performed in plate-based formats and completed within 2 days. Approximately 2000 protein groups and 8000 post-translational modifications were quantified in 3 LC-MS/MS runs at ∼1.5 h per sample. Analysis of differential abundance revealed blood proteome signatures of acute phase response and neutrophilic inflammation that partially resolved at the 28-42 day timepoint. Numerous analytes correlated with clinical laboratory values for c-reactive protein and white blood cell counts, as well as the Universal Vital Assessment illness severity score. These datasets serve as proof-of-concept for large-scale MS-based (sub)phenotyping of disease using dried blood and are available via the ProteomeXchange consortium (PXD060377). SUMMARY: For the first time, we report the integrated quantitative analysis of proteins, N-glycopeptides, and phosphopeptides from dried blood specimens in a disease context. Sample collection on Mitra devices is easily incorporated into existing biobanking protocols and provides a convenient solution for sample storage and preparation for downstream mass spectrometry analysis. Signatures of sepsis are reflected in each of the analyzed proteomes and decline between presentation to the hospital and 1 month post. In addition to well-described markers, these analyses identify mediators of inflammation and innate immune signaling that would be missed in the more common analysis of cell-free plasma.

The β-secretase BACE1 (β-site amyloid precursor (APP) cleaving enzyme 1) is a major drug target for Alzheimer's disease (AD), as it catalyzes the first step in amyloid β (Aβ) generation, but has additional substrates and functions, in particular in the brain. Several advanced clinical trials with BACE1 inhibitors were stopped because of an adverse event, a mild cognitive worsening. The underlying mechanism is not yet known but may result from co-inhibition of the BACE1-homolog BACE2. While a cerebrospinal fluid (CSF) biomarker for measuring BACE2 activity is not yet established, VCAM-1 has been suggested as such a biomarker, but has not yet been tested upon prolonged dosing in vivo. Using CSF pharmacoproteomics and a subchronic dosing paradigm in non-human primates, we demonstrate that compound 89, a BACE inhibitor not yet tested in humans, and the clinically tested drug elenbecestat inhibit BACE1 in vivo, with little or no effect on BACE2, as seen with a reduction of substrates of BACE1, but not of the BACE2 substrate VCAM-1. As a control, verubecestat, which inhibits both BACE2 and BACE1, reduced CSF abundance of BACE1 substrates as well as of VCAM-1. This study demonstrates the suitability of VCAM-1 as a pharmacodynamic biomarker for measuring BACE2 target engagement in CSF.

The central dogma of molecular biology, traditionally focused on nucleic acids and proteins, has historically overlooked the crucial involvement of carbohydrates (sugars/glycans) in cellular processes. Carbohydrates combine with lipids, proteins, and RNA to form glycoconjugates-glycolipids, glycoproteins, and glycoRNAs-that mediate signaling, recognition, and dynamic cellular responses. A glycomics-centered perspective within the paracentral dogma extends the central dogma by incorporating additional biomacromolecular modifications, illustrating how glycans function as context-dependent molecular signals synthesized through non-template enzymatic processes that are partly genetically constrained yet structurally unpredictable. Recognized as the "third alphabet of life" after nucleic acids (the first) and amino acids (the second), the glycan encodes rich information that regulates cellular communication, immunity, and disease processes.Glycoconjugates with the recently discovered glycoRNAs demonstrate how glycan modifications integrate with nucleic acids, while O-GlcNAcylation in DNA synthesis and DNA damage response, exemplified by O-GlcNAc transferase and O-GlcNAcase, modulates genome stability and cellular homeostasis. Advances in glycomics, framed within the concept of paracentral dogma, provide deep insights into glycoconjugates and their sugar-encoded information, offering novel avenues for vaccination, targeted interventions and glycomedicine.

Prostate cancer (PCa) is a leading male malignancy worldwide, with metabolic reprogramming being a critical hallmark of its progression. Extracellular vesicles (EVs) derived from tissues directly reflect the tumor microenvironment, offering unique insights into cancer pathophysiology that are unattainable through cell line or biofluid-derived EVs. However, the functional roles of tissue-derived EVs in PCa metabolism remain poorly understood. Leveraging our expertise in murine PCa model establishment and EV isolation from prostate tissue, this study aimed to characterize functional differences between PCa and normal prostate tissue via proteomic analysis of tissue-derived sEVs. We orthotopically implanted luciferase-labeled PCa cells into nude mice to establish an in situ PCa model, confirmed tumor formation via in vivo imaging, and harvested tissues after 4 weeks. sEVs were isolated using ultracentrifugation combined with an iodixanol density cushion and characterized by transmission electron microscopy, nanoparticle tracking analysis, and protein marker profiling. Proteomic analysis identified 28 upregulated and 24 downregulated proteins in PCa-derived sEVs compared to normal controls. Subcellular localization revealed enrichment in the cytoplasm, while pathway analysis highlighted significant involvement in metabolic processes, particularly glycolysis, amino acid biogenesis, carbon metabolism, and pyruvate metabolism. Our study establishes a robust method for isolating prostate tissue sEVs and provides the first evidence that PCa tissue-derived sEVs exhibit profound metabolic pathway alterations. These findings enhance our understanding of PCa progression mechanisms and may facilitate the development of novel diagnostic biomarkers and therapeutic strategies targeting metabolic dysregulation in PCa. SUMMARY: In this study, we created a method to isolate prostate tissue small EVs, based on our knowledge of the murine prostate cancer model building. Our data suggested that prostate tissue small EVs proteins significantly changed in many metabolism pathways, such as Glycolysis, Biogenesis of amino acids, Carbon metabolism and Pyruvate metabolism. In this study, we are the first to report prostate tissue-derived EVs proteins enriched in alterations of cancer metabolism. These differential proteins in PCa tissue EVs reflect metabolic changes in PCa and may provide insights into the development of early diagnostic biomarkers or novel therapeutic strategies.

Myofiber regeneration and membrane repair play crucial roles in maintaining the continuous physiological functioning of the neuromuscular system. A swift and efficient repair mechanism enables the rapid restoration of sarcolemmal integrity following cellular impairment in damaged skeletal muscles. Members of the annexin family of proteins, which are characterized by the peripheral binding to acidic phospholipid membranes, are intrinsically involved in this myofiber repair process. The biochemical and proteomic profiling of dystrophinopathy, a severe and highly progressive neuromuscular disorder of early childhood, is outlined in this article with special focus on skeletal muscle-associated annexins and their role in membrane repair, myofiber regeneration and the cellular pathogenesis of Duchenne muscular dystrophy. Findings from comparative mass spectrometry-based surveys are described, and dystrophinopathy-related alterations in annexin expression patterns are discussed regarding the establishment of improved biomarker signatures of skeletal muscle wasting disorders. Mass spectrometry-based proteomic profiling is highly suitable for the systematic study of complex pathobiochemical alterations and inherent adaptations in dystrophinopathy. Disease-specific changes in annexins and related proteins of the membrane repair machinery can now be used to improve diagnosis, evaluation of disease severity, prognosis and therapeutic monitoring, and identify novel therapeutic targets to treat X-linked muscular dystrophy.