Journal of proteomics最新文献

Predicting chemotherapy response in advanced non-small cell lung cancer (NSCLC) remains a clinical challenge, as baseline profiles often fail to capture dynamic molecular adaptations under treatment. This prospective study employed serial plasma proteomics to identify mechanistic pathways associated with chemotherapy resistance in 44 patients with stage IV NSCLC receiving platinum-based doublet chemotherapy. By analyzing blood samples collected immediately before the first and second cycles using liquid chromatography-tandem mass spectrometry, we demonstrated that a ratio-based proteomic model (early-treatment/pre-treatment) yielded superior separation between controlled and uncontrolled disease (UCD) compared to baseline-only assessment. Among 159 quantified proteins, 13 showed significant differential abundance, with UCD patients exhibiting marked upregulation of tetranectin, coagulation factor XIII A chain, and complement factor H-related protein 2. Ingenuity Pathway Analysis revealed that therapeutic resistance was characterized by three dominant axes: the activation of complement-coagulation-acute-phase signaling, the induction of lipid-nuclear receptor activity (LXR/RXR and DHCR24 signaling), and the relative attenuation of immune-regulatory pathways such as IL-12 signaling. These findings highlight the potential of serial proteomic profiling to uncover treatment-induced molecular adaptations, providing insights for therapeutic monitoring and hypothesis generation in precision oncology. SIGNIFICANCE: This study demonstrates the added value of prospective serial plasma proteomic profiling, compared with baseline-only approaches, for capturing early treatment-associated molecular adaptations in advanced non-small cell lung cancer (NSCLC) receiving chemotherapy. By quantifying proteomic changes between pre-treatment and early-treatment time points, we identified coordinated alterations involving the complement-coagulation-acute-phase axis and lipid-nuclear receptor signaling programs, including LXR/RXR and DHCR24, alongside relative attenuation of immune-regulatory pathways. Rather than reflecting isolated protein effects, these findings highlight interconnected host-tumor response programs that emerge under therapeutic pressure and may contribute to early adaptive resistance. Importantly, this work moves beyond static baseline markers by emphasizing dynamic, pathway-level changes and provides a hypothesis-generating framework for longitudinal therapeutic monitoring. Candidate proteins such as tetranectin and coagulation factor XIII A chain are proposed as molecular features associated with treatment response, warranting further validation in larger, prospective cohorts before translational application.

Ischemic stroke is a detrimental central nervous system (CNS) disorder with high morbidity and disability rates, caused by local cerebral ischemia. Extracellular matrix (ECM) is a complex network structure secreted by cells and located in the intercellular compartment, which is significant changed following ischemic stroke. Here, we quantified the proteomic profile of the ECM of brain from young (2-month-old) and aged (18-month-old) mice underwent cerebral ischemia/reperfusion (I/R) at 24 h and 60 d post I/R. The proteomics results indicated that proteins associated with the tricarboxylic acid (TCA) cycle and neutrophil extracellular trap (NET) formation were significantly up-regulated in the brain ECM from mice underwent cerebral I/R during acute stage, while those associated with synaptic vesicle cycle were significantly down-regulated in all stage post cerebral I/R. Differently, the brain ECM from aged mice underwent I/R expressed higher levels of lysosomal proteins and lower levels of autophagy and synaptic vesicle cycle associated proteins than the brain ECM from young mice underwent I/R. Furtherly, the proteomics identified that Hematopoietic Cell Kinase (HCK) is a regulator for NET formation. Inhibition of HCK could down-regulate LPS-induced phosphorylation of ERK1/2 and IKKα/β, as well as blocking the LPS plus nigericin induced activation of NLRP3 inflammasome and NET-like trap formation in vitro. In addition, inhibition of HCK significantly ameliorated cerebral I/R-induced brain injury and NET formation in vivo, suggesting HCK is a therapeutic target for ischemic stroke treatment. SIGNIFICANCE: Our results systemically analyzed the protein profiles of ECM in the brain post- acute and chronic ischemic stroke, and identified upregulation of NET-associated proteins was a common feature of the cerebral ECM during the acute phase, while down-regulation of synaptic vesicle cycle associated proteins was a common character of the brain ECM in all stage. What's more, HCK was identified as a regulator for NET formation, inhibition of HCK could block the formation of NET by inhibiting the activation of ERK1/2, IKKα/β and NLRP3 inflammasome, suggesting HCK is a therapeutic target for ischemic stroke treatment.

Cerebral ischemia-reperfusion injury is categorized as "stroke" in traditional Chinese medicine. For thousands of years, traditional Chinese medicine has accumulated rich experience in the treatment of stroke and other diseases, and with remarkable curative effects. Currently, Xingnaojing injection and its component musk are commonly used in the treatment of acute stroke, and muscone is the main active ingredient of musk. In this study, a rat model of transient middle cerebral artery occlusion was established, and the neuroprotective effects of Xingnaojing and muscone on transient middle cerebral artery occlusion rats were validated by Zea-Longa neurological function score, behavioral test and 2,3,5-triphenyltetrazolium chloride staining. Quantitative proteomics analysis was then performed on the brain tissues from different groups to investigate the mechanisms by which Xingnaojing and muscone act on cerebral ischemia-reperfusion injury. Our data indicate that Xingnaojing and muscone significantly affect proteins related to oxidative phosphorylation in CIRI rats, highlighting mitochondrial energy metabolism as a potentially important pathway contributing to their neuroprotective effects. Furthermore, the limited proteolysis-coupled mass spectrometry, target-responsive accessibility profiling, and lysine reactivity profiling methods were used to identify the direct protein targets of muscone in rat brain tissue lysate. A total of 36 potential target proteins were commonly identified by all the three methods. Bioinformatics analysis suggested that muscone was more significantly enriched in glycolysis/gluconeogenesis related pathways and closely associated with oxidative phosphorylation. Finally, the glycolytic key enzyme phosphoglycerate kinase 1, one of the binding proteins with muscone, was selected and verified by drug affinity responsive target stability. The molecular docking and dynamics simulation analysis further confirmed the interaction of glycolytic key enzyme phosphoglycerate kinase 1 and muscone. This study provides evidences for the clinical application and mechanisms of Xingnaojing and muscone in treating cerebral ischemia-reperfusion injury, and identifies candidate protein targets of muscone.

Sugar beet M14 line is a diploid cultivated sugar beet (Beta vulgaris L.) that carries a monosomic addition of chromosome 9 from the wild white-flowered beet (B. corolliflora Zoss.), developed through distant hybridization. It exhibits enhanced salt and drought tolerance compared to the diploid cultivated beets. In this study, the M14 line exhibited superior water retention capacity under dehydration conditions compared with five major diploid cultivated varieties grown in northern China. Through integrated analysis of phenotype, photosynthetic parameters, physiological and biochemical indicators, and the expression of key drought-responsive genes, 3 days and 5 days of 20% PEG-6000 treatment were identified as two critical time points for the drought stress response of the M14 line. Through label-free quantitative proteomics, 903 and 526 DAPs were identified at 3 and 5 days, respectively. PPI network analysis further revealed key protein interaction modules in the M14 line under drought stress. Furthermore, qRT-PCR analysis of 12 key DAP-encoding genes revealed that their transcript levels generally corresponded to the protein expression trends. This study helped to produce molecular network maps of drought tolerance in the M14 line, uncovering the mechanisms underlying its drought tolerance. SIGNIFICANCE: The drought tolerance of the sugar beet M14 line and ive major diploid sugar beet varieties cultivated in northern China was evaluated, revealing that the M14 line showed the strongest drought resistance. This study uncovered the dynamic regulatory network responsible for drought tolerance in the M14 line at the proteomic level, highlighting the main response pathways and key functional proteins at 3 and 5 days after stress exposure. These results not only deepen our understanding of the molecular mechanisms behind the sugar beet drought tolerance but also identify important candidate proteins and key regulatory modules for molecular breeding drought-tolerant varieties.

Monoclonal antibodies are a class of biotherapeutic proteins that have been developed over the past decade, leading to improved standards of care for the treatment of multiple diseases. Multi-attribute methods have emerged as powerful tools for critical quality attributes (CQAs). They leverage high-resolution accurate mass spectrometry and automated computational pipelines to identify pre-established modifications using DDA. In this study, we describe the development of a mass spectrometry-based workflow capable of processing up to 96 samples simultaneously while monitoring a broad panel of PTMs. We evaluated microwave-assisted digestion under different buffers and pHs, assessing sequence coverage, missed cleavages, and the occurrence of chemical artifacts. Analyses were performed using both DDA and DIA. Raw data were processed in dependent-peptide search(DDA) and PTM-probing search(DIA), enabling PTM discovery without prior knowledge. Our results demonstrate that microwave-assisted digestion, combined with control of temperature and pH, provides a fast and reliable alternative for efficiently digesting biotherapeutic proteins. It achieves high sequence coverage while minimizing artificial PTM formation. We also show that DIA combined with MW digestion improved peptide identification, highlighting its potential for comprehensive characterization of antibodies. Among the tested buffers, sodium acetate under MW conditions was the most effective in reducing deamidation and oxidation levels. SIGNIFICANCE: This study presents a detailed and optimized protocol for microwave-assisted (MW) protein digestion, enabling simultaneous reduction and alkylation for antibody samples. The method is rapid and minimizes chemical artifacts typically introduced during sample preparation. By combining MW-assisted digestion with both data-dependent (DDA) and data-independent acquisition (DIA), we performed a comprehensive and unbiased multi-attribute analysis (MAM). Notably, the use of DIA alongside MW digestion allowed for higher reproducibility and more complete peptide and post-translational modification (PTM) detection compared to DDA alone. Compared to conventional overnight digestion, MW-assisted digestion significantly reduced deamidation levels, with evident influences of buffer composition and pH on PTM identification. Although the levels of protein oxidation persisted, indicating that further optimization is necessary, this approach substantially decreased other artifacts, particularly deamidation, highlighting its potential as a fast, reliable, and highly informative strategy for antibody characterization.