Molecular & Cellular Proteomics最新文献

Dynamic environments require cyanobacteria to rapidly respond to fluctuating light conditions on timescales faster than transcription-translation processes allow, which is possible through immediate regulation of protein function via molecular and conformational adjustments. Traditional abundance-based proteomics cannot capture these rapid structural changes, creating a critical gap in understanding cellular adaptation mechanisms. We hypothesized that application of alternative structural proteomics approaches would enable identification of immediate and extensive structural remodeling across the cyanobacterial proteome triggered by environmental perturbations, potentially driving functional adaptations invisible to conventional abundance-based methods. We interrogated three complementary techniques-limited proteolysis mass spectrometry (LiP-MS), thermal proteome profiling (TPP-MS), and redox proteomics-for their capacity to unveil structural reorganization within the model cyanobacterium Synechococcus elongatus PCC 7942 during physiologically relevant light transitions. Within 30 minutes of increased light exposure, we detected structural changes in 753 proteins (LiP-MS), thermal stability shifts in 600 proteins (TPP-MS), and cysteine oxidation in 1,887 sites, while only 145 proteins changed in abundance. All three techniques consistently revealed coordinated remodeling of photosynthetic machinery, ribosomal complexes, and carbon metabolism, exemplified by cytochrome f stabilization modulating electron transport efficiency. Remarkably, <10% of proteins overlapped between methods, demonstrating that each technique captures distinct molecular dimensions of environmental adaptation. This structural proteomics framework demonstrates how alternative techniques can reveal hidden facets of proteome dynamics underlying cellular processes, offering new methodological approaches for understanding environmental responses and informing biotechnological applications.

The precise structure of glycosaminoglycans is critical for their bioactivity and the development of glycopharmaceuticals. Herein, cellular and animal experiments were conducted to assess the differences in the activities of heparin (HP) and heparan sulfate (HS) against liver cancer and drug-induced liver injury. Label-free quantitative proteomics, bioinformatics, biolayer interferometry, and immunohistochemical analyses were used to determine key proteins with differential expression. As a result, HP demonstrated superior antiliver cancer activity compared with HS, whereas HS exhibited strong potential in resisting acetaminophen-induced liver injury. DIRAS family GTPase 2 (DIRAS2) was identified as a key HS-binding protein that was strongly associated with cell proliferation, and its expression levels in cells and tissues showed opposite trends following HP and HS administration. HP significantly reduced the abundance of DIRAS2 in the tumor tissue, thereby inhibiting tumor cell proliferation, whereas HS promoted proliferation by increasing DIRAS2 expression. Cluster sequencing revealed that consecutive GlcNS6S-IdoA2S domains in HP and IdoA2S-GlcNS6S, GlcA-GlcNS6S, and IdoA-GlcNAc domains in HS were required for affinity binding within the decasaccharide region. Molecular docking suggested that differences in the binding modes of HP and HS chains to DIRAS2 underlie their functional diversity. These findings indicate that HP and HS oligosaccharides with well-defined structures may serve as potential therapeutic agents for liver-related diseases.

Protein glycosylation plays a pivotal role in various biological processes, and the analysis of intact glycopeptides (IGPs) has emerged as a powerful approach for characterizing alterations in protein glycosylation associated with diseases. Despite the critical insights gained from IGP analysis, dedicated databases and specialized tools for comprehensive glycoproteomics remain scarce. In response to this deficiency, we developed "Glycoprotein-Notebook," an online resource that consolidates the mass spectrometry evidence for IGPs identified from pan-cancer types studied in the Clinical Proteomic Tumor Analysis Consortium projects and provides analytical tools for in-depth glycopeptide characterization. Using pancreatic ductal adenocarcinoma as a case study, we validated and showcased the toolkit's analytical capabilities. Our results underscore the promise of IGPs as cancer-specific diagnostic and therapeutic targets. Accordingly, Glycoprotein-Notebook emerges as a valuable resource for cancer researchers exploring the intricate relationship between protein glycosylation and cancer phenotypes.

Toxic polyglutamine (polyQ) expansions in ataxin-2 (ATXN2) trigger neurodegenerative processes, causing spinocerebellar ataxia type 2, and enhancing TAR DNA-binding protein 43-dependent pathology in amyotrophic lateral sclerosis/frontotemporal dementia. Primary disease events can be compensated transiently, delaying disease manifestation. To define potential therapy targets, here we studied how cells modify phosphoprotein signals, using preferentially affected nervous tissue from end-stage Atxn2-CAG100-Knockin mice. The spinal cord phosphoproteome revealed massive hyperphosphorylations flanking the polyQ expansion in ATXN2 and for SQSTM1 and moderate hyperphosphorylations also for amyotrophic lateral sclerosis proteins, OPTN (optineurin), UBQLN2 (ubiquilin-2), TNIP1 (TNFAIP3 interacting protein 1), and TBK1-targeted TAX1BP1. Conversely, strong hypophosphorylations of WNK1 (protein kinase with no lysine 1), SPARCL1 (secreted protein acidic and cysteine rich-like 1), and PSMD9 (proteasome 19S regulator non-ATPase assembly chaperone P27) were found. Significant enrichments of SRC-homology domain type 3-containing proteins, autophagy/endocytosis factors, and actin modulators could be explained by N-terminal, polyQ-adjacent, proline-rich motifs in ATXN2, suggesting that spinocerebellar ataxia type 2 pathogenesis is highly similar to Huntington's disease, where neurotoxicity is mediated by abnormal polyQ-proline-rich motif-SRC-homology domain type 3 interactions. Validation of protein and mRNA levels was done in mouse spinal cord and embryonic fibroblasts or patient fibroblasts after bafilomycin or arsenite treatment, observing polyQ-dependent OPTN deficiency and SQSTM1 induction impairment. Overall, this phosphoproteome profile identified and quantified the main cellular efforts in adapting autophagy pathways to the aggregation propensity of the ATXN2-N-term.

A long-standing limitation of Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) has been the difficulty in accurately measuring amide exchange with single amide resolution. Excitation of peptides or proteins during ionization, ion transmission, or collisional activation rapidly induces intermolecular hydrogen migration, leading to a loss of the deuterium-labeled state; a term commonly known as "scrambling." Electron-based fragmentation methods in conjunction with gentle ion transmission settings can minimize scrambling but often not completely. Levels of scrambling have been shown to vary with ion transmission settings, peptide charge, and size, but the general properties that govern the susceptibility of peptides to scrambling are not well understood. Furthermore, it remains unclear whether scrambling is generally a global process or if local scrambling networks commonly exist within peptides. Here, we examine a panel of peptides using gentle electron transfer dissociation and map the activation thresholds of scrambling to define a relationship between peptide charge density and scrambling propensity. This study suggests that by and large, the scrambling process has a single activation threshold and involves all exchangeable sites within a peptide. For some peptides, the activation energy required for scrambling is surprisingly close to that of amide bond dissociation.

Across the globe, fungi are impacting the lives of millions of people through the development of infections ranging from superficial to systemic with limited treatment options. To effectively combat fungal disease, rapid and reliable diagnostic methods are required, including current methodologies using antigen detection, culturing, microscopy, and molecular tools. However, the flexibility of these platforms to diagnose infection using non-invasive methods and predict the outcome of disease are limited. In this study, we apply state-of-the-art mass spectrometry-based proteomics to perform dual perspective (i.e., host and pathogen) profiling of cryptococcal infection. Whole blood collected over a temporal scale following murine model challenged with the human fungal pathogen, Cryptococcus neoformans, detected >3000 host proteins and 160 fungal proteins. From the host perspective, temporal regulation of known immune-associated proteins, including eosinophil peroxidase and lipocalin-2, along with suppression of lipoproteins, demonstrated infection- and time-dependent host remodeling. Conversely, from the pathogen perspective, known and putative virulence-associated proteins were detected, including proteins associated with fungal extracellular vesicles and host immune modulation. We also observed and validated a new mechanism of immune system response to C. neoformans through modulation of haptoglobin. Furthermore, we assessed the predictive power of dual perspective proteome profiling toward prognostics of cryptococcal infection and report a previously undisclosed integration among virulence factor production, immune system modulation, and individual model survival. Together, our findings pose novel biomarkers of cryptococcal infection from whole blood and highlight the potential of personal proteome profiles to determine the prognosis of cryptococcal infection, a new parameter in fungal disease management.

Myopia is a growing global public health concern. Recent studies have revealed that the regulation of eye growth occurs via a complex signaling cascade, which originates in the retina and across the choroid to the sclera. Identifying key proteins and specific biological processes (BPs) in the retina, choroid, and sclera is crucial for understanding the molecular mechanisms underlying myopia development. We conducted comprehensive proteomic and phosphoproteomic analyses of the retina, choroid, and sclera from form-deprivation myopia guinea pigs using liquid chromatography-tandem mass spectrometry. Differentially expressed proteins and phosphosites were identified, followed by functional annotation and signaling pathway enrichment analyses. The expression of key proteins was assessed using Western blotting and enzyme-linked immunosorbent assay. Distinct proteomic and phosphoproteomic profiles were observed across the three tissues, with 6470, 6708, and 3236 proteins and 9613, 9416, and 3685 phosphosites in the retina, choroid, and sclera, respectively. Proteomic analysis showed that neural signal transduction was enriched in the retina, with downregulation of NTRK2, suggesting impaired neurotrophic signaling. The upregulation of SYK and BTK, along with increased NF-κB, p65, and IL-1β levels in the choroid, indicated enhanced inflammatory responses. TNNT3, TPM2, and ACTN3 were upregulated in the sclera, reflecting cytoskeletal remodeling associated with scleral expansion. Phosphoproteomic analysis indicated key roles of phosphoproteins in BPs, particularly the spliceosome signaling pathway, which was broadly involved across all three tissues. Kinase network analysis revealed PRPF4B as a key kinase for SF3B1, suggesting the potential regulation roles of RNA splicing in myopia progression. The present study systematically elucidates the proteomic and phosphoproteomic characteristics of the retina, choroid, and sclera of form-deprivation myopia in guinea pigs, highlighting significant tissue-specific BPs to myopia. The findings provide a theoretical foundation for understanding that different tissues exhibit distinct biological reactions to myopia, each through specific signaling pathways and regulatory mechanisms.

PKD1 and PKD2 are the most commonly mutated genes in autosomal dominant polycystic kidney disease (ADPKD). However, the precise roles of the encoded polycystin 1/2 (PC1 and PC2) proteins, and how their functions are disrupted in ADPKD, remain unclear. Here, we characterize the protein interaction networks of PC1 and PC2 in cycling and ciliated cells using proximity-dependent biotinylation (BioID), identifying a common set of 172 proteins that interact with the C terminus of PC1 and the full-length PC2 protein, enriched in autophagy regulators, endoplasmic reticulum tethers, endoplasmic reticulum stress proteins, and other proteins previously linked to ADPKD. Notably, we also find that PC1 specifically interacts with ciliary and lysosomal proteins, including components of the biogenesis of lysosome-related organelles complex (BLOC-1) and BLOC-one-related-complex (BORC). BLOC-1/BORC colocalizes with PC1 at lysosomes and cilia and is required for proper ciliary PC1 localization. In addition, PC1 mutant kidney cells derived from an ADPKD patient display defects in BLOC-1/BORC distribution. Renal cells depleted of PC1 exhibit abnormal lysosomal distribution, similar to those depleted of BLOC-1/BORC components. Finally, shRNA knockdown of BLOC-1/BORC components promoted cystogenesis in a 3D in vitro cyst model, and this could be attenuated by heterologous expression of the C terminus of PC1. This rich dataset thus links the BLOC-1/BORC complex to PC1 function and can be further mined for additional mechanistic insights into the PC1/2 ADPKD proteins.

Histone post-translational modifications (PTMs) play a crucial role in regulating gene expression and maintaining DNA integrity, and their aberrations are linked to various diseases, including cancer. While lysine acetylation and methylation have been extensively studied, recent research has uncovered additional PTMs that significantly contribute to chromatin structure and function. Mass spectrometry is the most effective analytical method for studying histone PTMs; however, computational limitations often restrict the analysis to common modifications. Unrestrictive search strategies have the potential to enable a more comprehensive characterization of the histone modification landscape. In this work, we systematically assess the application of unrestrictive search approaches to histone data. After evaluating the limitations of these methods, we develop a novel bioinformatics workflow, named HiP-Frag (histone PTM analysis with FragPipe), which enables the identification of 96 sites decorated with uncommon PTMs on core histones-60 of which were previously unreported-as well as 55 histone marks on linker histones, including 13 novel ones, purified from human cell lines and primary samples. The expanded histone PTM analysis enabled by this strategy is among the first to extract previously unexplored epigenetic information from mass spectrometry raw data. This approach paves the way for a facilitated and more streamlined identification of uncommon and yet unannotated histone modifications, supporting a deeper dissection of the histone code and the understanding of the potential biological role of the novel epigenetic marks.

Cholangiocarcinoma (CCA) comprises intrahepatic (iCCA) and extrahepatic (eCCA) subtypes, each exhibiting distinct molecular characteristics. Understanding these differences is critical for identifying subtype-specific therapeutic targets and advancing precision medicine. Protein glycosylation, a key post-translational modification, regulates immune evasion and metastasis, yet the glycoproteomic difference between iCCA and eCCA remains unexplored. Here we presented the first comprehensive N-glycoproteomic profile of eCCA and compared it with iCCA using a publicly available dataset. Our N-glycoproteomic analysis of paired eCCA tumors and normal adjacent tissues (NATs) identified 8372 N-glycopeptides, 3467 N-glycosites, and 2627 N-glycoproteins. Comparative analysis revealed distinct N-glycosylation signature, with eCCA exhibiting higher fucosylated glycans and iCCA showing increased sialylation. Pathway enrichment analysis of N-glycoproteins revealed a more prominent lysosome-related enrichment in eCCA, whereas pathways related to immune modulation, cytoskeletal components, and the extracellular matrix were significantly enriched in both subtypes. Immune profiling revealed an immunosuppressive microenvironment in both eCCA and iCCA, characterized by reduced natural killer cell infiltration and subtype-specific fibroblast and endothelial cell remodeling. DPM1, a glycosylation enzyme highly expressed in eCCA, was associated with tumor-specific N-glycopeptides and reduced immune cell infiltration. Its knockdown impaired cell migration, and glycoproteomic analysis implicated DPM1 in regulating adhesion, proteostasis, and immune pathways, highlighting its potential as a therapeutic target in eCCA. Our findings provide insights into N-glycosylation alterations in CCA subtypes, underscoring N-glycosylation-related mechanisms as potential biomarkers and therapeutic targets, particularly in eCCA.