Molecular & Cellular Proteomics最新文献

Pathogenic variants in the CACNA1F gene are linked to congenital stationary night blindness type 2 though their specific molecular effects remain elusive. This study examines the retinal impact of two variants: a truncation (RX) and a gain-of-function (IT) to explore variant-specific retinal proteome changes. Electroretinography showed that RX primarily affects rod pathways, while IT disrupts both rod and cone signaling, consistent with morphological findings. Comprehensive quantitative proteomic analysis using mass spectrometry identified approximately 4000 proteins across wild-type control and mutant retinas, including also low-abundant membrane proteins. IT retinas exhibited widespread proteomic remodeling suggesting broad cellular responses and also compensatory molecular adaptations. In contrast, RX retinas displayed a more restricted profile. Similar to IT retinas, we found reduced Cav1.4 protein levels but without transcriptional downregulation in RX, alongside selective changes in synaptic proteins such as Erc1, Lrfn2, vGlut1, and Rab3a. These findings suggest selective molecular changes in synaptic organization and calcium-related pathways in RX retinas, offering insights into the mechanisms of Cav1.4 dysfunction in retinal disease. Deep proteomic analysis demonstrates how retinal cells reorganize their molecular architecture in response to calcium channel defects and highlights the utility of comprehensive proteomics to characterize adaptive cellular responses to genetic perturbations in retinal synaptic organization.

Total N-glycome in blood serum or plasma provides information about all serum/plasma protein enzymatic glycosylation, a tightly regulated cotranslational and post-translational modification. Total plasma/serum N-glycome has shown specific patterns (signatures) in patients with high-mortality pathologies, such as cancer and cardiovascular diseases; thus, we explored the capacity of total serum N-glycome to predict mortality in a general adult population. This prospective cohort study was performed in a municipality in Spain including a random sample of 1516 adults. Participants were profiled for total serum N-glycome at baseline. Serum enzymatic N-glycan release was performed on a robotic platform followed by hydrophilic interaction chromatography-ultraperformance liquid chromatography glycan separation. The computerized medical records were checked at a median follow-up of 7.52 years to collect the date and cause of all deaths. N-glycan groups from total serum were used to develop mortality prediction models. Total serum N-glycome peak (GP) 16, mainly composed of A2[3]BG1S[3]1, predisposed to all-cause mortality; GP 22, mainly composed of FA2G2S[6]1, protected from all-cause mortality. The balance between them predicted all-cause mortality incidence over time (area under the curve [AUC], 0.810 [0.773-0.847]). Similar results were obtained for cancer mortality, with GPs 16, 17, 22, and 23 (AUC, 0.786 [0.728-0.843]); and for cardiovascular mortality, with GPs 7 and 9 (AUC, 0.747 [0.645-0.850]). Their predictive powers had an independent and additive effect on classical prediction factors. The balances between specific GPs are independent predictors of all-cause, cancer, and cardiovascular mortality and could contribute significantly to improving prognostic tools.

The overarching symptom of myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is post-exertional malaise (PEM), an exacerbation of symptoms following physical or mental exertion. To investigate the molecular underpinnings of PEM, we performed longitudinal plasma proteomics using the Somascan 7K aptamer-based assay to monitor 6361 unique plasma proteins in 132 individuals (96 females and 36 males) subjected to two maximal cardiopulmonary exercise tests separated by a 24-h recovery period. The cohort included 79 ME/CFS cases compared to 53 age- and BMI-matched sedentary controls, allowing us to distinguish disease-specific molecular alterations from those due to physical deconditioning. Longitudinal profiling revealed widespread proteomic changes following exertion, with the most pronounced alterations observed in ME/CFS participants during the recovery phase, coinciding with the onset of PEM. Compared to controls, patients with ME/CFS showed persistent dysregulation of immune, metabolic, and neuromuscular pathways. Key findings included suppression of T and B cell signaling, downregulation of IL-17 and cell-cell communication pathways, and upregulation of glycolysis/gluconeogenesis, suggestive of mitochondrial stress and impaired immune recovery from exercise. Proteomic associations with physiological performance (VO₂max, anaerobic threshold) revealed disruptions between protein abundance and exercise capacity in ME/CFS versus controls. Correlations with symptom severity linked changes in immune-related proteins and ME/CFS symptoms, including muscle pain, recurrent sore throat, and lymph node tenderness. Sex-stratified analyses revealed distinct molecular responses between females and males, emphasizing the importance of considering sex as a biological variable in ME/CFS research. Finally, our analysis of sedentary controls contributes new data on molecular responses to acute exertion in a predominantly female sedentary cohort, a population historically underrepresented in exercise physiology studies. Together, these findings underscore the value of dynamic, proteomic profiling over time for characterizing maladaptive responses to exertion in ME/CFS and provide a foundation for deeper mechanistic investigation into PEM.

Selecting the optimal biofluid for accurate biomarker assessment is vital to an informative clinical assay. However, in the initial stages of candidate biomarker discovery, the biologically appropriate biofluid might be unclear. To resolve this dilemma, we demonstrate a mass spectrometry-based workflow where paired urine, plasma, and serum samples are processed in parallel, creating biofluid-specific peptide libraries. These libraries are then harmonized to monitor consistent peptides and transitions, enabling cross-fluid normalization and quantitative comparisons. We also present a reference dataset, "CATalog," to aid in determining which biofluid to pursue based on protein relative abundance in healthy feline urine, plasma, and serum. Using this workflow and database, we explore the interchangeability of blood biofluid proteins compared to urinary proteins relating to sample processing, relative protein quantification, and clinical application. Our results suggest that when processed correctly, urine could sometimes represent blood biofluid proteins without requiring venipuncture or sample depletion of highly abundant proteins.

Crosslinking mass spectrometry (MS) is a powerful approach for probing protein structures. However, most widely used crosslinkers rely on N-hydroxysuccinimide (NHS) esters, restricting reactivity primarily to lysine residues and protein N-termini, and rendering them incompatible with many amine-containing buffers (e.g., Tris) and key biochemical cofactors (e.g., ATP). To address these limitations, we introduce two novel vinyl-sulfone-based crosslinkers. Alkyne-BVSC is an enrichable, homobifunctional crosslinker featuring an acid-cleavable alkyne handle for downstream peptide enrichment. VSD is a heterobifunctional crosslinker combining a vinyl sulfone with a diazirine moiety for UV-activated photo-crosslinking. Both reagents are synthetically accessible from inexpensive precursors and retain reactivity in amine-rich biochemical environments. We show that vinyl sulfones react with cysteine, histidine, and lysine residues, thereby expanding crosslinkable residues beyond those accessible to NHS-esters. Moreover, we develop a stub-based post-search filtering strategy that leverages the MS-cleavable nature of vinyl sulfone linkages to improve crosslink identification sensitivity. Together, these advances establish vinyl-sulfone-based crosslinkers as versatile and complementary tools for structural proteomics.

The extracellular matrix (ECM) plays a crucial role in tissue structure and function and serves as an integral component of diverse biological systems. However, the comprehensive characterization of ECM components is challenging because of the insoluble nature of highly cross-linked and glycosylated ECM proteins. This study introduces a chemical digestion-assisted proteomic approach to overcome challenges in ECM profiling, offering an in-depth analysis of the human periodontal ligament (PDL), a tissue critical for oral function and regeneration. Furthermore, we investigated alterations in the ECM composition of cultured PDL cells to provide insights relevant to tissue engineering applications. Our protocol combined chemical digestion and deglycosylation to enhance the identification of ECM proteins. Chemical digestion by hydroxylamine improved protein extraction efficiency by approximately two-fold compared to conventional chaotropic extraction, and deglycosylation increased the number of identified ECM proteins without substantially altering the ECM profile, offering robust quantification of ECM components. A sequential protein extraction approach revealed that proteins insoluble by conventional methods are primarily composed of highly cross-linked fibrillar collagen. Through the application of this technique to human PDL tissue, we present a comprehensive ECM profile for the first time, revealing a high collagen content (>80%) and identifying dominant non-collagenous ECM proteins, such as periostin, dermatopontin, and lumican. Our findings highlight the significant differences between the native ECM of PDL tissue and that produced by cultured PDL cells, emphasizing the importance of considering these variations in regenerative strategies. This study offers a robust tool for analyzing the ECM and its dynamics across diverse tissues and under various physiological and pathological conditions. The results enhance our understanding of periodontal tissue and will inform future approaches for periodontal tissue regeneration.

Cell surface proteins (CSPs) regulate key cellular functions and represent valuable targets for diagnostics and therapeutics. Despite advances in proteomic workflows, CSP analysis from cryopreserved or low-input clinical samples remains limited by technical constraints, including reduced membrane integrity, inefficient labeling, and high background. To address these challenges, we optimized and benchmarked two complementary surface enrichment strategies compatible with low-input applications (fewer than 1 million cells) and real-world sample types, including viably cryopreserved cells and dissociated solid tissues. We systematically compared oxidation-based N-glycopeptide capture and wheat germ agglutinin-horseradish peroxidase-mediated proximity labeling across a range of input amounts using both solid tumor (A549) and hematologic cancer (KMS-12-BM) cell lines. The N-glycopeptide method yielded superior specificity in low-input contexts, while the wheat germ agglutinin-horseradish peroxidase method captured complementary CSP subsets. Together, the methods identified more than 700 CSPs, with approximately 175 unique identifications per protocol. Both workflows detected dynamic epidermal growth factor receptor internalization following epidermal growth factor stimulation and maintained high reproducibility (Pearson correlation greater than 0.9) between fresh and cryopreserved preparations. To extend these findings to tissue-derived samples, we optimized dissociation protocols for healthy endometrium and applied the N-glycopeptide method to cryopreserved dissociated endometrium from three healthy donors. Enzymatic dissociation enabled accurate CSP profiling from less than 1 to 2 million cells. This study provides a systematic comparison of two leading surface proteomics approaches, validates their performance on cryopreserved and low-input specimens, and demonstrates applicability to clinically relevant tissues. Our optimized workflows enable robust surfaceome characterization in translational settings where sample quantity and preservation methods are often limiting, opening new avenues for biomarker discovery and patient stratification.

Metformin, a first-line therapy for type 2 diabetes, has also been implicated in regulating diverse physiological and pathological processes, including lifespan extension, cancer, and other disease-related conditions. However, its mechanisms of action remain incompletely understood, with many effects still unexplained. In this study, we investigated the impact of metformin on the cellular ubiquitinome and associated protein turnover. Through an integrated analysis combining ubiquitinome profiling with pulsed metabolic labeling, we found that metformin markedly suppresses global protein ubiquitination, including various types of ubiquitin chain linkages, and concurrently inhibits both protein synthesis and degradation. Notably, metformin induces a marked reduction in the ubiquitination of histone H4, a modification closely associated with DNA damage repair. We further establish a mechanistic link whereby metformin regulates DNA damage repair and cell cycle progression through downregulating ubiquitination. Together, our findings demonstrate that metformin modulates ubiquitination and proteostasis, central processes that regulate numerous cellular functions. By identifying histone H4 ubiquitination as a key target, we elucidate a potential mechanism through which metformin influences DNA repair and cell cycle progression. This comprehensive dataset advances understanding of the drug's multifaceted pharmacological activities and provides a valuable resource for future drug development.

The MIRAGE (Minimum Information Required for A Glycomics Experiment) guidelines for mass spectrometry (MS) data were initially developed to standardize the reporting of instrumentation, data acquisition and analytical details of the MS-based identification of released glycans. However, the growing interest in the study of intact glycoproteins and recent advances in MS-based glycoproteomics now necessitate a revision and expansion of these guidelines. This update includes an enhanced section focused on glycan structure analysis (glycomics) and introduces a new component tailored to the specific requirements of glycoproteomics. It addresses both shared and unique aspects of each approach and highlights glycoinformatics resources designed to facilitate data submission in compliance with the updated standards.

The E3 ligase substrate receptor ankyrin and suppressor of cytokine signaling box protein 9 (ASB9) was shown to bind over 10 different proteins including metabolic enzymes such as creatine kinase, filament proteins such as vimentin, and histones. In previous work, we characterized the ASB9-Cullin 5 E3 ligase (ASB9-CRL5) ubiquitylation of creatine kinase and showed that ubiquitylation required the ring-between-ring ligase, ARIH2. Here, we characterized the ASB9-CRL5 ubiquitylation of histones and show that histones histone 3 (H3) and histone 4 (H4) are polyubiquitylated by the ASB9-CRL5 whereas histones Histone 2A and Histone 2B are much poorer substrates. Many, but not all lysines in the histones are ubiquitylated suggesting some substrate specificity. Binding experiments show that the ligase-histone interaction is highly electrostatic and the neddylated ASB9-CRL5 binds with the highest affinity. Histones in nucleosomes or in complex with the chaperone Asf1, are not ubiquitylated. Only K48 and K63 polyubiquitin chains were observed, suggesting that the ubiquitylation probably drives histone degradation. The presence of ASB9 in specific cell types correlates with situations in which free histones H3 and H4 need to be degraded. In this work, we demonstrate that the ASB9-CRL5 is the ligase that facilitates degradation of histones H3 and H4. In addition, this work represents the first example of Cullin-5 mediated ubiquitylation that does not require a ring-between-ring "helper" ligase.