Comprehensive Proteomic Analysis Research Articles

Comprehensive SUMO Proteomic Analyses Identify HIV Latency-Associated Proteins in Microglia

SUMOylation, the post-translational modification of proteins by small ubiquitin-like modifiers, plays a critical role in regulating various cellular processes, including innate immunity. This modification is essential for modulating immune responses and influencing signaling pathways that govern the activation and function of immune cells. Recent studies suggest that SUMOylation also contributes to the pathophysiology of central nervous system (CNS) viral infections, where it contributes to the host response and viral replication dynamics. Here, we explore the multifaceted role of SUMOylation in innate immune signaling and its implications for viral infections within the CNS. Notably, we present novel proteomic analyses aimed at elucidating the role of the small ubiquitin-related modifier (SUMO) in human immunodeficiency virus (HIV) latency in microglial cells. Our findings indicate that SUMOylation may regulate key proteins involved in maintaining viral latency, suggesting a potential mechanism by which HIV evades immune detection in the CNS. By integrating insights from proteomics with functional studies, we anticipate these findings to be the groundwork for future studies on HIV–host interactions and the mechanisms that underlie SUMOylation during latent and productive infection.

Deep, Unbiased, and Quantitative Mass Spectrometry-Based Plasma Proteome Analysis of Individual Responses to mRNA COVID-19 Vaccine.

Global campaign against COVID-19 have vaccinated a significant portion of the world population in recent years. Combating the COVID-19 pandemic with mRNA vaccines played a pivotal role in the global immunization effort. However, individual responses to a vaccine are diverse and lead to varying vaccination efficacy. Despite significant progress, a complete understanding of the molecular mechanisms driving the individual immune response to the COVID-19 vaccine remains elusive. To address this gap, we combined a novel nanoparticle-based proteomic workflow with tandem mass tag (TMT) labeling, to quantitatively assess the proteomic changes in a cohort of 12 volunteers following two doses of the Pfizer-BioNTech mRNA COVID-19 vaccine. This optimized protocol seamlessly integrates comprehensive proteome analysis with enhanced throughput by leveraging the enrichment of low-abundant plasma proteins by engineered nanoparticles. Our data demonstrate the ability of this workflow to quantify over 3,000 proteins, providing the deepest view into COVID-19 vaccine-related plasma proteome study. We identified 69 proteins with boosted responses post-second dose and 74 proteins differentially regulated between individuals who contracted COVID-19 despite vaccination and those who did not. These findings offer valuable insights into individual variability in response to vaccination, demonstrating the potential of personalized medicine approaches in vaccine development.

Comprehensive proteomic analysis and multidimensional model construction of peritoneal metastasis in gastric cancer

Comprehensive proteomic analysis and multidimensional model construction of peritoneal metastasis in gastric cancer

Proteomics of urinary exosomes for discovering novel non-invasive biomarkers of acute myocardial infarction patients.

Patients with acute myocardial infarction (AMI) can be identified by myocardial enzymes in peripheral blood, but no protein markers have been found in urinary exosomes derived from AMI patients. In the present study, a comprehensive proteomics analysis of urinary exosomes derived from patients with AMI was performed. Firstly, we employed the outstanding separation method known as EXODUS to isolate urinary exosomes from AMI patients and healthy controls. Then, we characterized urinary exosomes by nanoparticle tracking analysis (NTA), western blotting and transmission electron microscopy (TEM). After that, we identified the protein components of exosomes through label-free proteomics and conducted bioinformatics analysis. High-quality exosomes were obtained through separation using EXODUS, which could be demonstrated by NTA, Western blotting and TEM. NTA analysis showed that partilce amount in AMI patients was significantly higher than healthy controls. The equal-volume Western blotting experiment indicated that the expression level of classic exosomal markers Alix, heat shock protein90 (HSP90), CD63 and TSG101 (Tumor susceptibility gene101) in AMI patents was obviously stronger than healthy subjects. We first described the protein profiles of urinary exosomes in AMI patients through proteomics. In this study, We have identified 3194 proteins, among which a total of 30 differential proteins were detected between the urinary exosomes of AMI patients and healthy controls. We investigated F2 and OLR1 among identified exosomal proteins significantly elevated in AMI group, whereas F3 and APCS dysregulated in AMI development. F2, F3, OLR1 and APCS are able to distinguish individuals between the AMI group and the healthy controls, and the protein panel represent a novel prospective non-invasive biomarkers for the diagnostic process of acute myocardial infarction.

Proteomic Characterization of NEDD4 Unveils Its Potential Novel Downstream Effectors in Gastric Cancer.

The E3 ubiquitin ligase neural precursor cell-expressed developmentally down-regulated 4 (NEDD4) is involved in various cancer signaling pathways, including PTEN/AKT. However, its role in promoting gastric cancer (GC) progression is unclear. This study was conducted to elucidate the role of NEDD4 in GC progression. We found that the inhibition of NEDD4 expression significantly reduced the migratory and proliferative abilities of GC cells, with minimal impact on the PTEN expression or p-AKT activation, suggesting that NEDD4 may exert its GC-promoting effects through alternative pathways. To gain novel insights into the role of NEDD4 in GC, we performed a comprehensive proteomic analysis to search for proteins with altered expression levels following NEDD4 gene knockdown, identifying a total of 3916 proteins. Pathway analysis of differentially expressed proteins (DEPs) indicated the potential involvement of NEDD4 in cancer-related metabolic pathways. Furthermore, the protein-protein interaction network of the DEPs revealed enriched core modules, highlighting key cellular processes and signaling pathways regulated by NEDD4 in GC. Additionally, we identified proteins whose expression was altered by NEDD4 inhibition, some of which were associated with poor prognosis in GC. These findings suggest that these proteins may act as downstream effectors that contribute to NEDD4-mediated GC progression.

Size-Dependent Separation of Extracellular Vesicle Subtypes with Exodisc Enabling Proteomic Analysis in Prostate Cancer.

Extracellular vesicles (EVs) are emerging as crucial biomarkers in cancer diagnostics and therapeutics with their heterogeneity presenting both challenges and opportunities in prostate cancer research. However, existing methods for isolating and characterizing EV subtypes have been limited by inefficient separation and inadequate proteomic analysis. Here we show an optimized centrifugal microfluidic device, Exodisc, that efficiently isolates large quantities of EV subtypes from particle-enriched medium, enabling comprehensive proteomic analysis of small (EV-S, 20-200 nm) and large (EV-L, >200 nm) EVs. Using this device, we successfully separated EV-S and EV-L from prostate cancer cell lines LNCaP and PC3. Mass spectrometry-based proteomics revealed that EV proteins reflect parental cell characteristics more than EV size, with EV-L demonstrating increased expression of PSMA-correlated proteins. Our optimized protocol addresses challenges in EV isolation and characterization, providing a more effective method for studying cellular and molecular mechanisms of specific EV subtypes. This study extends the potential use of EVs as a liquid biopsy for cancer theranostics, paving the way for more precise isolation of EV subtypes and potentially leading to improved biomarker discovery and the development of personalized treatments.

Structure-Based Approaches for Protein-Protein Interaction Prediction Using Machine Learning and Deep Learning.

Protein-Protein Interaction (PPI) prediction plays a pivotal role in understanding cellular processes and uncovering molecular mechanisms underlying health and disease. Structure-based PPI prediction has emerged as a robust alternative to sequence-based methods, offering greater biological accuracy by integrating three-dimensional spatial and biochemical features. This work summarizes the recent advances in computational approaches leveraging protein structure information for PPI prediction, focusing on machine learning (ML) and deep learning (DL) techniques. These methods not only improve predictive accuracy but also provide insights into functional sites, such as binding and catalytic residues. However, challenges such as limited high-resolution structural data and the need for effective negative sampling persist. Through the integration of experimental and computational tools, structure-based prediction paves the way for comprehensive proteomic network analysis, holding promise for advancements in drug discovery, biomarker identification, and personalized medicine. Future directions include enhancing scalability and dataset reliability to expand these approaches across diverse proteomes.

Senescent cell-derived extracellular vesicles inhibit cancer recurrence by coordinating immune surveillance.

Senescence is a non-proliferative, survival state that cancer cells can enter to escape therapy. In addition to soluble factors, senescence cells secrete extracellular vesicles (EVs), which are important mediators of intercellular communication. To explore the role of senescent cell-derived EVs (senEVs) in inflammatory responses to senescence, we developed an engraftment-based senescence model in wild-type mice and genetically blocked senEV release in vivo, without significantly affecting soluble mediators. SenEVs were both necessary and sufficient to trigger immune-mediated clearance of senescent cells, thereby suppressing tumor growth. In the absence of senEVs, the recruitment of MHC-II+ antigen-presenting cells to the senescence microenvironment was markedly impaired. Blocking senEV release redirected the primary target of senescent cell signaling from antigen-presenting cells to neutrophils. Comprehensive transcriptional and proteomic analyses identified six ligands specific to senEVs, highlighting their role in promoting antigen-presenting cell-T cell adhesion and synapse formation. Antigen-presenting cells activated CCR2+CD4+ TH17 cells, which appeared to inhibit B cell activation, and CD4+ T cells were essential for preventing tumor recurrence. These findings suggest that senEVs complement the activity of secreted inflammatory mediators by recruiting and activating distinct immune cell subsets, thereby enhancing the efficient clearance of senescent cells. These conclusions may have implications not only for tumor recurrence but also for understanding senescence during de novo carcinogenesis. Consequently, this work could inform the development of early detection strategies for cancer based on the biology of cellular senescence.

Exploration of Novel Metabolic Mechanisms Underlying Primary Biliary Cholangitis Using Hepatic Metabolomics, Lipidomics, and Proteomics Analysis.

Metabolic reprogramming is important in primary biliary cholangitis (PBC) development. However, studies investigating the metabolic signature within the liver of PBC patients are limited. In this study, liver biopsies from 31 PBC patients and 15 healthy controls were collected, and comprehensive metabolomics, lipidomics, and proteomics analysis were conducted to characterize the metabolic landscape in PBC. We observed distinct lipidome remodeling in PBC with increased polyunsaturated fatty acid levels and augmented fatty acid β-oxidation (FAO), evidenced by the increased acylcarnitine levels and upregulated expression of proteins involved in FAO. Notably, PBC patients exhibited an increase in glucose-6-phosphate (G6P) and purines, alongside a reduction in pyruvate, suggesting impaired glycolysis and increased purines biosynthesis in PBC. Additionally, the accumulation of bile acids as well as a decrease in branched chain amino acids and aromatic amino acids were observed in PBC liver. We also observed an aberrant upregulation of proteins associated with ductular reaction, apoptosis, and autophagy. In conclusion, our study highlighted substantial metabolic reprogramming in glycolysis, fatty acid metabolism, and purine biosynthesis, coupled with aberrant upregulation of proteins associated with apoptosis and autophagy in PBC patients. Targeting the specific metabolic reprogramming may offer potential targets for the therapeutic intervention of PBC.

Exploring the molecular mechanisms of tirzepatide in alleviating metabolic dysfunction-associated fatty liver in mice through integration of metabolomics, lipidomics, and proteomics

Clinical studies have suggested that tirzepatide may also possess hepatoprotective effects; however, the molecular mechanisms underlying this association remain unclear. In our study, we performed biochemical analyses of serum and histopathological examinations of liver tissue in mice. To preliminarily explore the molecular mechanisms of tirzepatide on metabolic dysfunction-associated fatty liver disease (MAFLD), liquid chromatography-mass spectrometry (LC-MS) was employed for comprehensive metabolomic, lipidomic, and proteomic analyses in MAFLD mice fed a high-fat diet (HFD). The results demonstrated that tirzepatide significantly reduced serum levels of alanine transaminase (ALT) and aspartate transaminase (AST), as well as hepatic triglycerides (TG) and total cholesterol (TC), indicating its efficacy in treating MAFLD. Further findings revealed that tirzepatide reduced fatty acid uptake by downregulating Cd36 and Fabp2/4, as well as enhance the mitochondrial-lysosomal function by upregulating Lamp1/2. In addition, tirzepatide promoted cholesterol efflux and reduced cholesterol reabsorption by upregulating the expression of Hnf4a, Abcg5, and Abcg8. These results suggest that tirzepatide exerts its therapeutic effects on MAFLD by reducing fatty acid uptake, promoting cholesterol excretion, and enhancing mitochondrial-lysosomal function, providing a theoretical basis for a comprehensive understanding of tirzepatide.

Characterizing the extractable proteins from tomato leaves - A proteomics study.

The ambition to utilize agricultural by-products has spotlighted tomato leaves as a promising source for plant-based proteins. High-yielding protein extractability is key for its industrial use, but previous studies reported decreased protein extractability at later stages of plant development. This study investigated the underlying factors in protein extractability through a comprehensive proteomics analysis across four plant developmental stages (vegetative, flowering, fruit-forming, mature-fruit). The findings linked reduced yields to a shift in leaf function, from anabolic to catabolic processes and (a)biotic stress responses. This functional shift is accompanied by decreased protein synthesis and increased protein degradation, leading to an overall decrease of the soluble protein fraction. Furthermore, incomplete extraction of soluble proteins from leaves of later developmental stages, suggested the presence of inhibitory molecules hindering the extraction process. These findings indicate that breeding strategies towards increased amounts of soluble proteins and reduced concentration of inhibitory molecules could enhance protein extraction yields.

An integrated proteomic and phosphoproteomic landscape of chronic kidney disease.

The prevalence of chronic kidney disease (CKD) is gradually rising worldwide. Patients often remain asymptomatic for an extended period, leaving them unaware of their condition, which can lead to progressing to end-stage renal disease and cause significant economic burden. Improved understanding of CKD pathogenesis can enhance early detection and facilitate advances in drug development. Here, we performed proteomic and phosphoproteomic analyses of the mouse unilateral ureteral obstruction model to explore the molecular mechanisms of chronic kidney injury. 474 significantly differentially expressed proteins and 96 significantly differentially expressed phosphoproteins were screened, respectively. Chronic kidney injury involves complex metabolic pathways such as citrate cycle and hematopoietic system in proteome, and mitochondrial oxidative phosphorylation suppression is a notable alteration. The phosphoproteomic analysis revealed a significant upregulation in epithelial mesenchymal transition and P53 pathways, with a corresponding increase in the phosphorylation of Jun at serine 73. Utilizing HK2 cells, we observed that the reduction oxidative phosphorylation was consistently associated with an augmentation in oxidative stress, which subsequently activated Jun and induced apoptosis. Proteins that act as hubs in these pathways may be candidate targets for CKD intervention. These findings contribute significantly to the current understanding of CKD and provide valuable insights for future studies. SIGNIFICANCE: Chronic kidney disease (CKD) incidence rising annually with varied etiologies, kidney often irreversibly fibrotic, the treatment options are limited and often ineffective due to deficient understanding of renal fibrosis mechanisms. Despite the extensive efforts and numerous omics studies conducted on renal fibrosis, to date, no study has been undertaken to investigate the role of phosphorylated proteins in UUO models. Previously, we performed a comprehensive transcriptome and proteome analysis based on the CKD model, but the potential alterations in the phosphoproteome were not addressed. Here, an integrated proteomic and phosphoproteomic landscape of CKD was completed, which was the the first phosphoproteomic profiles of UUO model. Phosphoproteomic profile suggests that the epithelial mesenchymal transition and P53 pathways is significantly activated in mouse models of kidney injury, and the core protein Jun played a key role in CKD. And a preliminary correlation between P-Jun and oxidative phosphorylation was found base on HK2 cells. Our work contributes to a deeper understanding of the disease characteristics and molecular mechanisms of CKD. Identifying potential CKD targets from proteome and phosphoproteome may provide valuable insights for early diagnosis and treatment of CKD.

Proteomics and transcriptomic analyses provide new insights into the pectin polysaccharide biosynthesis in Premna puberula Pamp

Premna puberula Pamp. is a plant with similarities to both food and medicine, which has attracted the attention of researchers because of the rich pectin in its leaves. However, the mechanism of efficient pectin accumulation remains unclear. Through transcriptomic and proteomic analyses, this study investigated the differentially expressed genes and proteins associated with pectin during various developmental stages of P. puberula. The combined omics approach revealed that the majority of differential genes are mainly involved in the anabolic metabolism of primary and secondary metabolites. Notable differential pathways include starch and sucrose metabolism, amino acid sugar metabolism, and nucleotide sugar metabolism. The SWEET gene family was identified and analyzed, leading to the cloning of PpSWEET15 and subcellular localization. Overexpression of PpSWEET15 resulted in a significant decrease in sucrose content in leaves from 134.44 μg·g−1 to <10 μg·g−1. Glucose content decreased from 407.75 μg·g−1 to 318.38 μg·g−1, while fructose levels showed no significant difference between the two groups of leaves. This comprehensive transcriptomic and proteomic analysis provides valuable insights into the molecular mechanisms underlying the efficient accumulation of pectin in P. puberula.

USP44 regulates HEXIM1 stability to inhibit tumorigenesis and metastasis of oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is the most frequent type of oral malignancy with high metastasis and poor prognosis. The deubiquitinating enzyme Ubiquitin Specific Peptidase 44 (USP44) regulates the mitotic checkpoint, and its deficiency leads to aneuploidy and increases tumor incidence. However, the role of USP44 in OSCC is not well understood. Herein, we analyzed mRNA sequencing data of OSCC samples downloaded from the TCGA and GEO databases and found that USP44 was decreased in human OSCC tissues and was positively correlated to the survival of OSCC patients. To investigate the biological impact of USP44, we used recombinant lentiviruses to overexpress or knockdown USP44 expression in OSCC cell lines, which were also injected subcutaneously or into the lateral tail vein of Male BALB/c nude mice to model tumorigenesis or lung metastasis in vivo, respectively. The results showed that overexpression of USP44 inhibited malignant cell phenotypes in vitro and suppressed tumor growth and lung metastasis in vivo, while its downregulation had the opposite effects. Comprehensive proteomic analyses through Co-IP mass spectrometry and label-free quantitative LC-MS/MS methods identified 112 differentially expressed proteins positively regulated by USP44, among which 13 were involved in cancer-related pathways including apoptotic signaling and cell cycle regulation. PPI analysis identified Hexamethylene Bis-Acetamide-Inducible Protein 1 (HEXIM1) as the hub protein. Upregulation of USP44 enhanced HEXIM1 protein stability, leading to its higher expression in OSCC cells. Silencing of HEXIM1 further enhanced the malignant phenotype of OSCC cells. At the same time, HEXIM1 knockdown reversed the antitumor effects of USP44. These findings demonstrated that USP44 acted as a critical tumor suppressor in OSCC by inhibiting cell proliferation and metastasis through the stabilization of HEXIM1 protein, suggesting that USP44-HEXIM1 axis is a promising target for OSCC therapy.

Proteomic profiling reveals the significance of lipid metabolism in small cell lung cancer recurrence and metastasis

BackgroundSmall cell lung cancer (SCLC) is a lethal and recalcitrant malignancy with early metastases. However, the molecular and cellular mechanisms underlying its aggressive characteristics remain relatively elusive.MethodsIn this study, we conducted a comprehensive proteomic analysis of 90 primary tumors, 15 patient-matched lymph node metastatic tumors, and 15 brain metastatic tumors derived from a cohort of 105 SCLC patients. The potential mechanism for SCLC metastasis was investigated based on the variety of protein expression profiles.ResultsPrimary tumors were divided into two categories according to the their different protein expression profiles, using metastatic tumors as reference. Proteomic comparisons across different groups revealed that lipid metabolism, especially phospholipid metabolism, and immune response had a critical role in SCLC metastasis. Additionally, it was shown that high- and low-density lipoprotein cholesterol were both independent prognostic factors for disease free survival of SCLC patients. To identify critical regulators of metastasis in SCLC, support vector machine was adopted to generate a biomarker combination of ten proteins, all of which significantly correlated with the infiltration of immune cells. Furthermore, it was demonstrated that high expression of phospholipase A2 group IIA in stroma was associated with delayed disease recurrence in limited stage SCLC.ConclusionsThis study highlighted the critical significance of lipid metabolism, especially phospholipid metabolism in the disease recurrence and metastasis of SCLC.

Transcriptomic and proteomic sequencing unveils the role of vitamin D and metabolic flux shifts in the induction of human hepatic organoids

BackgroundHepatic organoids (HOs), validated through comparative sequencing with human liver tissues, are reliable models for liver research. Comprehensive transcriptomic and proteomic sequencing of HOs throughout their induction period will enhance the platform’s utility, aiding in the elucidation of liver development’s molecular mechanisms.MethodsWe developed hepatic organoids (HOs) from embryonic stem cells (ESCs) through a de novo induction protocol, mimicking the stages of fetal liver development: ESCs to definitive endoderm (DE), then to foregut (FG), hepatoblasts (HB), and finally to HOs stage 1 (HO1), culminating in self-organizing HOs stage 2 (HO2) via dissociation and re-inoculation. The successful establishment of HOs was validated by immunofluorescence staining and RT-qPCR for specific markers. Comprehensive transcriptomic and proteomic sequencing and analysis were conducted on FG, HB, HO1, and HO2.ResultsOur data suggest that several transcription factors (TFs) activated during the HB stage share overlapping target genes with the vitamin D receptor (VDR). Calcitriol, a direct activator of VDR, notably facilitated the FG to HB stage transition by activating VDR and enhancing key TFs, thereby promoting hepatic progenitor cell maturation. Furthermore, our findings revealed a significant transition towards glycolytic energy metabolism at the HO2 stage, characterized by increased glycolytic flux and reduced oxidative phosphorylation. Inhibition of glycolysis using 2-deoxy-D-glucose (2-DG) led to suppressed growth and differentiation at the HO2 stage. Analysis of signaling pathways indicated upregulation of the HIF-1 pathway, which is associated with glycolysis activation, as well as the MAPK and PI3K-AKT pathways, which regulate HIF-1α protein translation.ConclusionsWe elucidated a pivotal role for calcitriol in facilitating the transition from FG to HB by activating VDR and augmenting the expression of critical transcription factors (TFs). Besides, our research underscores a shift in metabolic pathways toward glycolytic energy metabolism in HO2 organoids. Overall, our multiomics approach reveals the intricate molecular regulation during the development of HOs.

Senescence of endothelial cells increases susceptibility to Kaposi's sarcoma-associated herpesvirus infection via CD109-mediated viral entry.

The aging process is characterized by cellular functional decline and increased susceptibility to infections. Understanding the association between virus infection and aging is crucial for developing effective strategies against viral infections in older individuals. However, the relationship between Kaposi's sarcoma-associated herpesvirus (KSHV) infection, a cause of Kaposi's sarcoma prevalent among the elderly without HIV infection, and cellular senescence remains enigmatic. This study uncovers a fascinating link between cellular senescence and enhanced KSHV infectivity in human endothelial cells. Through a comprehensive proteomic analysis, we identified caveolin-1 and CD109 as novel host factors significantly upregulated in senescent cells that promote KSHV infection. Remarkably, CRISPR-Cas9-mediated knockout of these factors reduced KSHV binding and entry, leading to decreased viral infectivity. Furthermore, surface plasmon resonance analysis and confocal microscopy revealed a direct interaction between KSHV virions and CD109 on the cell surface during entry, with recombinant CD109 protein exhibiting an intriguing ability to inhibit infection by blocking virion binding. These findings uncover a previously unrecognized role of cellular senescence in enhancing KSHV infection through upregulation of specific host factors and provide novel insights into the complex interplay between aging and viral pathogenesis.

Protein “purity,” proteoforms, and the albuminome: critical observations on proteome and systems complexity

IntroductionThe identification of effective, selective biomarkers and therapeutics is dependent on truly deep, comprehensive analysis of proteomes at the proteoform level.MethodsBovine serum albumin (BSA) isolated by two different protocols, cold ethanol fractionation and heat shock fractionation, was resolved and identified using Integrative Top-down Proteomics, the tight coupling of two-dimensional gel electrophoresis (2DE) with liquid chromatography and tandem mass spectrometry (LC-MS/MS).Results and discussionNumerous proteoforms were identified in both “purified” samples, across a broad range of isoelectric points and molecular weights. The data highlight several concerns regarding proteome analyses using currently popular analytical approaches and what it means to (i) purify a “protein” if the isolate consists of a wide variety of proteoforms and/or co-purifying species; and (ii) use these preparations as analytical standards or therapeutics. Failure to widely recognize and accept proteome complexity has likely delayed the identification of effective biomarkers and new, more selective drug targets. iTDP is the most logical available analytical technique to effectively provide the necessary critical depth and breadth for complex proteome analyses. Routine analyses at the level of proteoforms will provide the much-needed data for the development and validation of selective biomarkers and drugs, including biologics.

The early transition to cold-induced browning in mouse subcutaneous white adipose tissue (scWAT) involves proteins related to nerve remodeling, cytoskeleton, mitochondria, and immune cells

ABSTRACT White adipose tissue (WAT) is a dynamic organ capable of remodelling in response to metabolic state. For example, in response to stimuli such as cold exposure, WAT can develop inducible brown adipocytes (‘browning’) capable of non-shivering thermogenesis, through concurrent changes to mitochondrial content and function. This is aided by increased neurite outgrowth and angiogenesis across the tissue, providing the needed neurovascular supply for uncoupling protein 1 activation. While several RNA-sequencing studies have been performed in WAT, including newer single cell and single nuclei studies, little work has been done to investigate changes to the adipose proteome, particularly during dynamic periods of tissue remodelling such as cold stimulation. Here, we conducted a comprehensive proteomic analysis of inguinal subcutaneous (sc) WAT during the initial ‘browning’ period of 24 or 72hrs of cold exposure in mice. We identified four significant pathways impacted by cold stimulation that are involved in tissue remodelling, which included mitochondrial function and metabolism, cytoskeletal remodelling, the immune response, and the nervous system. Taken together, we found that early changes in the proteome of WAT with cold stimulation predicted later structural and functional changes in the tissue that are important for tissue and whole-body remodelling to meet energetic and metabolic needs.

A Comprehensive Proteomic and Bioinformatic Analysis of Human Spinal Cord Injury Plasma Identifies Proteins Associated with the Complement Cascade and Liver Function as Potential Prognostic Indicators of Neurological Outcome.

Spinal cord injury (SCI) is a major cause of disability, with complications postinjury often leading to lifelong health issues with the need for extensive treatment. Neurological outcome post-SCI can be variable and difficult to predict, particularly in incompletely injured patients. The identification of specific SCI biomarkers in blood may be able to improve prognostics in the field. This study has utilized proteomic and bioinformatic methodologies to investigate differentially expressed proteins in plasma samples across human SCI cohorts with the aim of identifying candidate prognostic biomarkers and biological pathway alterations that relate to neurological outcome. Blood samples were taken, following informed consent, from American Spinal Injury Association impairment scale (AIS) grade C "improvers" (those who experienced an AIS grade improvement) and "nonimprovers" (no AIS change) and AIS grade A and D at <2 weeks ("acute") and ∼3 months ("subacute") postinjury. The total protein concentration from each sample was extracted, with pooled samples being labeled and nonpooled samples treated with ProteoMiner™ beads. Samples were then analyzed using two 4-plex isobaric tag for relative and absolute quantification (iTRAQ) analyses and a label-free experiment for comparison before quantifying with mass spectrometry. Data are available via ProteomeXchange with identifiers PXD035025 and PXD035072 for the iTRAQ and label-free experiments, respectively. Proteomic datasets were analyzed using OpenMS (version 2.6.0). R (version 4.1.4) and, in particular, the R packages MSstats (version 4.0.1) and pathview (version 1.32.0) were used for downstream analysis. Proteins of interest identified from this analysis were further validated by enzyme-linked immunosorbent assay. The data demonstrated proteomic differences between the cohorts, with the results from the iTRAQ approach supporting those of the label-free analysis. A total of 79 and 87 differentially abundant proteins across AIS and longitudinal groups were identified from the iTRAQ and label-free analyses, respectively. Alpha-2-macroglobulin, retinol-binding protein 4 (RBP4), serum amyloid A1, peroxiredoxin 2 (PRX-2), apolipoprotein A1, and several immunoglobulins were identified as biologically relevant and differentially abundant, with potential as individual candidate prognostic biomarkers of neurological outcome. Bioinformatics analyses revealed that the majority of differentially abundant proteins were components of the complement cascade and most interacted directly with the liver. Many of the proteins of interest identified using proteomics were detected only in a single group and therefore have potential as binary (present or absent) biomarkers, RBP4 and PRX-2 in particular. Additional investigations into the chronology of these proteins and their levels in other tissues (cerebrospinal fluid in particular) are needed to better understand the underlying pathophysiology, including any potentially modifiable targets. Pathway analysis highlighted the complement cascade as being significant across groups of differential functional recovery.