Clinical proteomics最新文献

Background: Low-grade serous ovarian carcinoma (LGSOC) is a rare and largely chemoresistant subtype of epithelial ovarian cancer. Unlike treatment for high-grade serous ovarian cancer (HGSOC), management options for LGSOC patients are limited, in part, due to a lack of deep molecular characterization of this disease. To address this limitation, we aimed to define highly conserved proteome alterations in LGSOC by performing deep quantitative proteomic analysis of tumors collected from LGSOC and HGSOC patients or normal fallopian tube tissues and validating proteins within two independent proteomic datasets of LGSOC and HGSOC tumors.

Methods: Formalin-fixed, paraffin-embedded LGSOC (n = 12), HGSOC (n = 24), and FT (n = 12) tissues underwent pressure-assisted trypsin digestion followed by quantitative proteomic analyses using a multiplex, tandem-mass tag (TMT11) workflow and high-resolution mass spectrometry. Proteome alterations between LGSOC and HGSOC tumors were validated against two independent proteome datasets generated from LGSOC (n = 25) and HGSOC (n = 49) tumors. Mucin-16 (MUC16/CA125) was assessed in LGSOC and HGSOC tumors by immunohistochemistry and reviewed by three independent pathologists.

Results: Our efforts identified 275 protein alterations conserved between LGSOC and HGSOC tumors that exhibit high quantitative correlation between discovery and validation cohorts (Spearman Rho ≥ 0.82, P < 1E-4). Conserved proteins elevated in LGSOC tumors were enriched for pathways regulating cell adhesion and defective cellular apoptosis signaling and candidates mapping as putative drug targets included 5'-nucleotidase/ cluster of differentiation 73 (NT5E/CD73). We also identified MUC16 (CA125) as significantly elevated in LGSOC versus HGSOC tumors and confirmed this by immunohistochemistry analysis. We further find that MUC16 exhibits a more apical versus membrane-staining pattern in LGSOC tumors, suggesting unique regulation of MUC16 in this disease subtype.

Conclusion: Our efforts define highly conserved protein alterations distinguishing LGSOC from HGSOC tumors, including CD73, as well as the novel identification that MUC16 is elevated and exhibits more apical staining pattern in LGSOC tumor tissues. These findings deepen our molecular understanding of LGSOC and provide unique insights into highly conserved proteome alterations in LGSOC tumors.

Background/study objectives: Age-related macular degeneration (AMD), a degenerative disease of the photoreceptor support system of the macula, is a leading cause of vision loss in individuals over 60 years of age. In this exploratory longitudinal study, we studied VEGF-related proteins and other protein concentrations in the aqueous humor of patients with treatment naïve neovascular AMD (defined as patients with a previously untreated and recently diagnosed advanced neovascular form of AMD (NVAMD) who were eligible for an intra-vitreal administration of an anti-VEGF agent to treat choroidal neovascularization). The objectives of this small pilot study were: (1) To determine levels of VEGF-related proteins in the aqueous humor of treatment naïve NVAMD patients compared with control patients, (2) To determine whether levels of VEGF-related proteins change over time with anti-VEGF injections in NVAMD patients, (3) To put these differences into perspective relative to all protein targets and identify other off-target (non-VEGF) proteins that may be related to NVAMD or NVAMD treatment.

Methods: We used an aptamer-based proteomic technology to study protein concentrations. Cases had a sample of aqueous collected immediately prior to starting the anti-VEGF intra-vitreal injection and at two follow-up visits. Controls were cataract patients with no AMD. Aqueous was collected at the time of cataract surgery.

Results: Comparison between 9 cases and 11 controls revealed 56 proteins, out of 3,803 targets, with significant differences in baseline levels. After treatment, a decline in aqueous VEGF concentrations was indicated from two aptamer reagents, while a third recorded a significant increase. Interference studies demonstrated that the increase in levels observed for the latter reagent was due to measuring both drug-bound and free VEGF concentrations (total VEGF) while the others measured only free VEGF.

Conclusions: In this exploratory study, 56 proteins were identified that could potentially be linked with NVAMD. In interference studies free aqueous VEGF levels declined while total VEGF levels increased following anti-VEGF treatment. No large off-target effects on the proteome were observed with treatment. We illustrate how the protein interactome can mask or potentially unmask binding epitopes leading to signal changes not necessarily related to the absolute protein level.

Background: Sepsis is a critical condition characterized by a dysregulated immune response to infection. As sepsis develops to septic shock, its most severe form, morbidity and mortality increases. Hyaluronan is a key component of the extracellular matrix and the endothelial glycocalyx. In sepsis, plasma hyaluronan concentrations are increased and correlate with disease severity. In this study we aimed to explore and compare the proteomic profiles of hyaluronan-associated proteins in patients with the dysregulated immune response of septic shock and the sterile inflammation of acute alcohol-related pancreatitis.

Methods: The present study involved proteomic analysis of patients with septic shock (n = 13), pancreatitis (n = 8), and healthy controls (n = 8). LC-MS/MS was conducted for peptide analysis. Hyaluronan-associated proteins were identified using the UniProt REST API, followed by functional and pathway enrichment analyses with GOATOOLS and GSEApy. Statistical analyses, including ANOVA and post hoc tests, were performed using Python and SPSS, with significance set at p < 0.05.

Results: From a total sum of 663 detected unique plasma proteins, 15 were identified as hyaluronan-related proteins. Plasma levels of 11/15 proteins separated septic shock from pancreatitis in a statistically significant manner. Between the groups differences were apparent on day 1 (8 proteins in septic shock versus 3 in pancreatitis) and day 4 (6 proteins in septic shock versus 3 in pancreatitis) relative to controls. Functional enrichment analysis revealed associations with extracellular matrix organization, proteolytic enzyme regulation, and hyaluronan metabolism. Notably, members of the inter-alpha-inhibitor family demonstrated distinct patterns, with ITIH3 levels increasing and ITIH1, ITIH2, and ITIH4 levels decreasing in septic shock compared to controls. Additionally, plasma hyaluronidase inhibition correlated positively with ITIH3 levels.

Conclusion: The present study explored the role of hyaluronan-related proteins in septic shock pathophysiology, revealing potential dysregulation associated with sepsis severity. The decrease in ITIH1, ITIH2 and ITIH4, as compared to the increase in ITIH3, suggest a complex alteration in the protein balance of the IαI-family in sepsis. Overall, the altered proteomic profile of hyaluronan-related proteins as reflected by the GO terms indicates a complex dysregulation not only in hyaluronan metabolism and extracellular matrix, but also in the regulation of several proteolytic enzymes. Future studies on this area are warranted.

Background: Small cell lung cancer (SCLC) is an aggressive malignancy with a poor prognosis. This study aimed to analyze the urinary exosomal proteome of SCLC patients to identify and validate potential non-invasive biomarkers for improving diagnosis, treatment response monitoring, and prognosis prediction.

Methods: We analyzed 90 urine samples from SCLC patients, divided into training (n = 38) and validation (n = 52) sets, including untreated, partial/complete remission, and relapsed groups. Ten healthy controls were included. Urinary exosomes were isolated by ultracentrifugation. The proteomic analysis employed data-independent acquisition mass spectrometry (DIA-MS) and parallel reaction monitoring (PRM). Immunohistochemistry was performed on 30 pairs of SCLC and adjacent normal tissues.

Results: Proteomic analysis revealed distinct exosomal protein expression patterns across SCLC stages. RAB11A emerged as a key differentially expressed protein. PRM validation confirmed significant changes in RAB11A levels across disease stages. ROC curve analysis demonstrated excellent diagnostic performance of RAB11A in distinguishing SCLC patients from healthy controls (AUC = 0.91, 95% CI 0.79-1.00, P = 0.0004), with a sensitivity of 85% and specificity of 92%. RAB11A also showed significant potential in monitoring treatment response (AUC = 0.86, 95% CI 0.69-1.00, P = 0.0019) and disease relapse (AUC = 0.90, 95% CI 0.76-1.00, P = 0.0005). Immunohistochemistry showed significantly higher RAB11A expression in SCLC tissues compared to adjacent normal tissues (70% vs. 33% positive expression, P = 0.043).

Conclusion: Urinary exosomal RAB11A shows promise as a non-invasive biomarker for SCLC diagnosis, treatment response monitoring, and early detection of relapse, potentially improving clinical management of SCLC patients. The findings provide insights into SCLC pathogenesis and offer a non-invasive approach for patient monitoring, which could improve clinical management strategies.

Background: Plasma is the most used clinical specimen, yet diurnal variation in plasma proteins remains largely unexplored. We aimed to identify diurnally-regulated proteins in healthy individuals and assess their potential diagnostic implications, and highlight how diurnal awareness can advance future biomarker research.

Methods: Twenty-four healthy young individuals were studied under highly controlled conditions. Venous blood was drawn every three hours over a 24-h period, yielding 216 samples, of which 208 high-quality plasma samples were analyzed via high-throughput mass spectrometry. The missing data were filtered and imputed, and rhythmicity was assessed using Cosinor-based modeling with Benjamini-Hochberg correction. Tissue and pathway enrichment analyses were performed using the DAVID functional annotation tool.

Findings: Of 523 proteins that passed quality thresholds, 138 (~ 26%) exhibited significant diurnal oscillations. Tissue enrichment analysis revealed that most rhythmic proteins originated from the liver and platelets, with additional enrichment in a variety of tissue types. Pathway enrichment showed diurnal regulation of hemostasis, immune signaling, integrin-mediated processes, glucose metabolism, and protein synthesis. Notably, 36 clinically utilized biomarkers, including albumin, amylase, and cystatin C exhibited diurnal variation, suggesting that failing to account for temporal fluctuations may reduce diagnostic precision.

Interpretation: These findings demonstrate that over one-quarter of the human plasma proteome is under diurnal control. Such oscillations might have direct clinical implications, as the time-of-day may alter biomarker accuracy. Incorporating diurnal timing into diagnostic and research protocols, through standardized sampling or time-sensitive reference intervals, could improve patient care and inform future biomarker discoveries. Further research in larger, more diverse populations is needed to generalize these results and streamline practices in a way that takes diurnal variation into account.

Colorectal cancer (CRC) has emerged as the second most prevalent cause of cancer-related mortality globally. Early identification of precancerous lesions prone to malignant transformation is pivotal in CRC prevention. Proteins, as microscopic reflections of cellular functional states, offer insights into pathological alterations within precancerous lesions through changes in their expression and function. Our review summarizes the protein research on colorectal adenomas under different sample conditions, including traditional adenomas, serrated lesions, LST, FAP and IBD. It highlights the changes in the expression patterns of key proteins and their potential mechanisms underlying the transition from precancerous to cancerous states. Additionally, it summarizes the research on post-translational modifications of characteristic protein families and associated signaling pathways, while discussing current techniques for studying protein expression and function in colorectal cancer, such as proteomics and artificial intelligence. However, current research limitations, such as small sample sizes, limited sample types, and insufficient in-depth mechanistic analysis, hinder comprehensive understanding. Future research should expand study cohorts, diversify sample types, and leverage machine learning and multi-omics approaches to develop predictive models. By doing so, a more comprehensive understanding of protein profiles during the progression from colorectal precancerous to cancerous lesions can be obtained, facilitating early CRC diagnosis and the development of targeted therapeutic interventions.

Background: Bacterial meningitis complicated by neurological complications (BMN) is a major cause of poor outcomes and mortality among children with bacterial meningitis. So far, the host-related mechanisms and diagnostic biomarkers of BMN and bacterial meningitis without neurological complications (BM) remain poorly understood and limited.

Methods: We implemented a two-stage cerebrospinal fluid (CSF) quantitative proteomics study involving three groups: children with BMN, BM, and diseases not involving the central nervous system(Ctrl). Initially, in the discovery cohort, data-independent acquisition (DIA) mass spectrometry was used for proteomic profiling on 242 CSF samples (77 BMN, 52 BM, and 113 Ctrl). Differentially expressed proteins (DEPs) were identified among the BMN/Ctrl, BM/Ctrl, and BMN/BM groups, followed by an analysis of their functional enrichment. Next, the parallel reaction monitoring (PRM) method was used to validate the essential DEPs identified during the DIA phase in a validation cohort of 196 subjects (94 BMN, 47 BM, and 55 Ctrl). Subsequently, the validated DEPs were further filtered to construct a PRM-based machine learning model that distinguishes between BMN and BM.

Results: A total of 1376 DEPs were identified in BM/Ctrl (757 upregulated, 619 downregulated), 1295 in BMN/Ctrl (745 upregulated, 550 downregulated), and 356 in BMN/BM(60 upregulated, 296 downregulated), respectively. The functional results indicate that the upregulated DEPs were primarily enriched in immunity, inflammation, complement, and phagocytosis in BMN/Ctrl and BM/Ctrl. Immunoglobulin production, phagocytosis, and the classical pathway of complement activation were further upregulated in BMN/BM. The downregulated DEPs are primarily enriched in cell adhesion, nervous system function, and synapses in BMN/Ctrl and BM/Ctrl; some were further enriched in BMN/BM. In addition, some of the innate immunity, translation, signal transduction, nervous system, and redox processes were mainly downregulated in BMN/BM. The PRM successfully verified 22 significant DEPs. Among them, UAB1, MTPN, ARHGDIB, IGHG3, and AMBP could be combined to distinguish BMN from BM, achieving an AUC of 0.998 in the training set and 0.824 in the validation set.

Conclusions: This study identified distinct CSF protein expression profiles in children with BMN, BM, and Ctrl, potentially enhancing our understanding of the molecular mechanisms underlying bacterial meningitis. These protein signatures help us distinguish bacterial meningitis from the Ctrl group. Furthermore, selected biomarkers could support the differentiation between BMN and BM.

Background: Four-week dawn-to-dusk dry fasting (DDDF) was previously shown to have a potent anti-inflammatory effect and induce an anti-tumorigenic proteome in the serum and peripheral blood mononuclear cells in subjects without cancer. The study goal was to determine if serum obtained from these subjects without cancer who underwent 4-week DDDF has an anti-tumorigenic effect.

Methods: HepG2 cells were treated with serum collected from four individuals with metabolic syndrome and metabolic dysfunction-associated steatotic liver disease (MASLD) and four healthy individuals who performed 4-week DDDF. The objective was to assess cell proliferation/viability in HepG2 cells treated with non-fasted and dry-fasted serum and determine proteomic changes in human serum. We comparatively performed 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) cell proliferation assay and untargeted proteomic analysis using nano ultra-high performance liquid chromatography coupled with tandem mass spectrometry.

Results: Serum collected from 3 out of 4 subjects with metabolic syndrome and MASLD at the end of 4-week DDDF (dry-fasted serum/V2) significantly reduced proliferation/viability in HepG2 cells compared with the serum collected before 4-week DDDF (non-fasted serum/V1). A similar reduction effect on cell proliferation was not observed when HepG2 cells were treated with dry-fasted serum collected from healthy subjects. In addition to the in vitro changes observed, the following circulating gene protein products (GP) demonstrated significant increases or decreases in subjects with metabolic syndrome and MASLD after a 4-week DDDF regimen, compared with their GP levels before the 4-week DDDF: CD248 molecule (mean log2 fold = 8.124, P = 0.001), dipeptidyl peptidase 4 (mean log2 fold = 0.937, P = 0.027), lymphatic vessel endothelial hyaluronan receptor 1 (mean log2 fold = 1.054, P = 0.029), LDL receptor related protein 1 (mean log2 fold = 1.401, P = 0.031), and beta-2-microglobulin (mean log2 fold= -0.977, P = 0.033) at the end of 4-week DDDF compared with the GP levels before 4-week DDDF.

Conclusion: This study demonstrated that dry-fasted serum collected from subjects with metabolic syndrome and MASLD decreased HepG2 cell proliferation in vitro and showed that proteomic changes occurred in vivo. These findings suggest that DDDF may be an effective intervention for inducing proteomic responses that could assist in the prevention and adjunct treatment of cancers associated with metabolic syndrome.

Background: The treatment of advanced or metastatic colorectal cancer (CRC) poses a global challenge. Mendelian Randomization (MR) has been primarily applied for repurposing licensed drugs and uncovering new therapeutic targets.

Objective: This study aims to systematically identify potential plasma protein targets for CRC using proteome-wide Mendelian randomization and evaluate their potential side effects through phenome-wide association studies (Phe-WAS).

Methods: We conducted a comprehensive proteome-wide MR study to assess the causal relationships between plasma proteins and the risk of CRC and evaluate their potential side effects through Phe-WAS. The plasma proteins were sourced from the Finland and Iceland decode database, encompassing GWAS data for plasma proteins (Olink-619 samples across 2925 proteins, SomaScan -828 samples across 7596 proteins and Iceland decode database across 4907 proteins). Additionally, GWAS data for CRC were extracted from the UK Biobank-SAIGE database, including 3051 cases and 382,756 controls. Subsequently, colocalization analysis was performed to identify shared causal variants between plasma proteins and CRC. Finally, a phenome-wide association study (Phe-WAS) was conducted to examine the potential adverse effects of druggable proteins for CRC, utilizing the extensive UK Biobank-SAIGE database, encompassing 783 phenotypes.

Results: The MR analysis identified GREM1, DKKL1, and CHRDL2 as plasma proteins whose genetically predicted levels were positively associated with CRC risk, whereas TMEM132A was inversely associated with CRC risk (P_fdr < 0.05). The colocalization analysis identified these four proteins as shared variation with CRC (PPH3 + PPH4 > 0.7), suggesting that these proteins represent potential direct targets for CRC intervention. Further phenotype-wide association studies showed no significant potential side effects of these targets (P_fdr > 0.05).

Conclusion: This proteome-wide Mendelian randomization study offers a comprehensive molecular landscape of CRC, identifying GREM1, DKKL1, CHRDL2, and TMEM132A as potential therapeutic targets. Our research provides a critical foundation for future experimental validation and therapeutic development in colorectal cancer management.