Proteogenomics of Non-smoking Lung Cancer in East Asia Delineates Molecular Signatures of Pathogenesis and Progression.

Lung cancer in East Asia is characterized by a high percentage of never-smokers, early onset and predominant EGFR mutations. To illuminate the molecular phenotype of this demographically distinct disease, we performed a deep comprehensive proteogenomic study on a prospectively collected cohort in Taiwan, representing early stage, predominantly female, non-smoking lung adenocarcinoma. Integrated genomic, proteomic, and phosphoproteomic analysis delineated the demographically distinct molecular attributes and hallmarks of tumor progression. Mutational signature analysis revealed age- and gender-related mutagenesis mechanisms, characterized by high prevalence of APOBEC mutational signature in younger females and over-representation of environmental carcinogen-like mutational signatures in older females. A proteomics-informed classification distinguished the clinical characteristics of early stage patients with EGFR mutations. Furthermore, integrated protein network analysis revealed the cellular remodeling underpinning clinical trajectories and nominated candidate biomarkers for patient stratification and therapeutic intervention. This multi-omic molecular architecture may help develop strategies for management of early stage never-smoker lung adenocarcinoma.

Cerebrospinal Fluid Proteome Map Reveals Molecular Signatures of Reversible Cerebral Vasoconstriction Syndrome.

Reversible cerebral vasoconstriction syndrome (RCVS) is a complex neurovascular disorder characterized by repetitive thunderclap headaches and reversible cerebral vasoconstriction. The pathophysiological mechanism of this mysterious syndrome remains underexplored and there is no clinically available molecular biomarker. To provide insight into the pathogenesis of RCVS, this study reported the first landscape of dysregulated proteome of cerebrospinal fluid (CSF) in patients with RCVS (n = 21) compared to the age- and sex-matched controls (n  = 20) using data-independent acquisition mass spectrometry. Protein-protein interaction and functional enrichment analysis were employed to construct functional protein networks using the RCVS proteome. An RCVS-CSF proteome library resource of 1054 proteins was established, which illuminated large groups of upregulated proteins enriched in the brain and blood-brain barrier (BBB). Personalized RCVS-CSF proteomic profiles from 17 RCVS patients and 20 controls reveal proteomic changes involving the complement system, adhesion molecules, and extracellular matrix, which may contribute to the disruption of BBB and dysregulation of neurovascular units. Moreover, an additional validation cohort validated a panel of biomarker candidates and a two-protein signature predicted by machine learning model to discriminate RCVS patients from controls with an area under the curve of 0.997. This study reveals the first RCVS proteome and a potential pathogenetic mechanism of BBB and neurovascular unit dysfunction. It also nominates potential biomarker candidates that are mechanistically plausible for RCVS, which may offer potential diagnostic and therapeutic opportunities beyond the clinical manifestations.

Lessons learned: establishing a CLIA-equivalent laboratory for targeted mass spectrometry assays - navigating the transition from research to clinical practice.

Mass spectrometry (MS) assays offer exceptional capabilities in high multiplexity, specificity, and throughput. As proteomics technologies continue advancements to identify new disease biomarkers, transition of these innovations from research settings to clinical applications becomes imperative. To meet the rigorous regulatory standards of clinical laboratories, development of a clinical protein MS assay necessitates adherence to stringent criteria. To illustrate the process, this project focused on using thyroglobulin (Tg) as a biomarker and an immuno-multiple reaction monitoring (iMRM) MS-based assay as a model for establishing a Clinical Laboratory Improvement Amendments (CLIA) compliant laboratory within the Centers of Genomic and Precision Medicine, National Taiwan University. The chosen example also illustrates the clinical utility of MS assays to complement conventional immunoassay-based methods, particularly in cases where the presence of autoantibodies in 10-30% of patients hinders accuracy. The laboratory design entails a comprehensive coordination in spatial layout, workflow organization, equipment selection, ventilation systems, plumbing, electrical infrastructure, documentation procedures, and communication protocols. Practical aspects of the transformation process, including preparing laboratory facilities, testing environments, instrument validation, assay development and validation, quality management, sample testing, and personnel competency, are discussed. Finally, concordant results in proficiency testing demonstrate the harmonization with the University of Washington Medical Center and the quality assurance of the CLIA-equivalent Tg-iMRM MS assay established in Taiwan. The realization of this model protein MS assay in Taiwan highlights the feasibility of international joint development and provides a detailed reference map to expedite the implementation of more MS-based protein assays in clinical laboratories for patient care.

Downregulation of TAZ elicits a mitochondrial redox imbalance and ferroptosis in lung epithelial cells exposed to diesel exhaust particles.

Mitochondrial dysfunction was reported to be involved in the development of lung diseases including chronic obstructive pulmonary disease (COPD). However, molecular regulation underlying metabolic disorders in the airway epithelia exposed to air pollution remains unclear. In the present study, lung bronchial epithelial BEAS-2B and alveolar epithelial A549 cells were treated with diesel exhaust particles (DEPs), the primary representative of ambient particle matter. This treatment elicited cell death accompanied by induction of lipid reactive oxygen species (ROS) production and ferroptosis. Lipidomics analyses revealed that DEPs increased glycerophospholipid contents. Accordingly, DEPs upregulated expression of the electron transport chain (ETC) complex and induced mitochondrial ROS production. Mechanistically, DEP exposure downregulated the Hippo transducer transcriptional co-activator with PDZ-binding motif (TAZ), which was further identified to be crucial for the ferroptosis-associated antioxidant system, including glutathione peroxidase 4 (GPX4), the glutamate-cysteine ligase catalytic subunit (GCLC), and glutathione-disulfide reductase (GSR). Moreover, immunohistochemistry confirmed downregulation of GPX4 and upregulation of lipid peroxidation in the bronchial epithelium of COPD patients and Sprague-Dawley rats exposed to air pollution. Finally, proteomics analyses confirmed alterations of ETC-related proteins in bronchoalveolar lavage from COPD patients compared to healthy subjects. Together, our study discovered that involvement of mitochondrial redox dysregulation plays a vital role in pulmonary epithelial cell destruction after exposure to air pollution.

N-glycosylated GPNMB ligand independently activates mutated EGFR signaling and promotes metastasis in NSCLC.

Lung cancer is the leading cause of cancer-related death worldwide. As well as the identified role of epidermal growth factor receptor (EGFR), its association with driver mutations has improved the therapeutics for patients with lung cancer harboring EGFR mutations. These patients usually display shorter overall survival and a higher tendency to develop distant metastasis compared with those carrying the wild-type EGFR. Nevertheless, the way to control mutated EGFR signaling remains unclear. Here, we performed membrane proteomic analysis to determine potential components that may act with EGFR mutations to promote lung cancer malignancy. Expression of transmembrane glycoprotein non-metastatic melanoma protein B (GPNMB) was positively correlated with the status of mutated EGFR in non-small-cell lung cancer (NSCLC). This protein was not only overexpressed but also highly glycosylated in EGFR-mutated, especially EGFR-L858R mutated, NSCLC cells. Further examination showed that GPNMB could activate mutated EGFR without ligand stimulation and could bind to the C-terminus of EGFR, assist phosphorylation at Y845, turn on downstream STAT3 signaling, and promote cancer metastasis. Moreover, we also found that Asn134 (N134) glycosylation of GPNMB played a crucial role in this ligand-independent regulation. Depleting N134-glycosylation on GPNMB could dramatically inhibit binding of GPNMB to mutated EGFR, blocking its downstream signaling, and ultimately inhibiting cancer metastasis in NSCLC. Clarifying the role of N-glycosylated GPNMB in regulating the ligand-independent activation of mutated EGFR may soon give new insight into the development of novel therapeutics for NSCLC.

Prolonged exposure to traffic-related particulate matter and gaseous pollutants implicate distinct molecular mechanisms of lung injury in rats.

BACKGROUND: Exposure to air pollution exerts direct effects on respiratory organs; however, molecular alterations underlying air pollution-induced pulmonary injury remain unclear. In this study, we investigated the effect of air pollution on the lung tissues of Sprague-Dawley rats with whole-body exposure to traffic-related PM1 (particulate matter < 1 µm in aerodynamic diameter) pollutants and compared it with that in rats exposed to high-efficiency particulate air-filtered gaseous pollutants and clean air controls for 3 and 6 months. Lung function and histological examinations were performed along with quantitative proteomics analysis and functional validation. RESULTS: Rats in the 6-month PM1-exposed group exhibited a significant decline in lung function, as determined by decreased FEF25-75% and FEV20/FVC; however, histological analysis revealed earlier lung damage, as evidenced by increased congestion and macrophage infiltration in 3-month PM1-exposed rat lungs. The lung tissue proteomics analysis identified 2673 proteins that highlighted the differential dysregulation of proteins involved in oxidative stress, cellular metabolism, calcium signalling, inflammatory responses, and actin dynamics under exposures to PM1 and gaseous pollutants. The presence of PM1 specifically enhanced oxidative stress and inflammatory reactions under subchronic exposure to traffic-related PM1 and suppressed glucose metabolism and actin cytoskeleton signalling. These factors might lead to repair failure and thus to lung function decline after chronic exposure to traffic-related PM1. A detailed pathogenic mechanism was proposed to depict temporal and dynamic molecular regulations associated with PM1- and gaseous pollutants-induced lung injury. CONCLUSION: This study explored several potential molecular features associated with early lung damage in response to traffic-related air pollution, which might be used to screen individuals more susceptible to air pollution.

Suppressor of variegation 3-9 homologue 1 impairment and neutrophil-skewed systemic inflammation are associated with comorbidities in COPD.

BACKGROUND: Systemic manifestations and comorbidities are characteristics of chronic obstructive pulmonary disease (COPD) and are probably due to systemic inflammation. The histone methyltransferase SUV39H1 controls the Th1/Th2 balance. We previously reported that reduced SUV39H1 expression contributed to abnormal inflammation in COPD. Here, we aimed to determine whether impaired SUV39H1 expression in COPD patients associated with neutrophilic/eosinophilic inflammation responses and comorbidities. METHODS: A total of 213 COPD patients and 13 healthy controls were recruited from the Shuang Ho Hospital, Taipei Medical University. SUV39H1 levels in peripheral blood mononuclear cells (PBMCs) from 13 healthy and 30 COPD participants were measured by immunoblotting. We classified the patients into two groups based on low (fold change, FC < 0.5) and high SUV39H1 expression (FC ≥ 0.5) compared to normal controls. Clinical outcomes including neutrophil or eosinophil counts associated with SUV39H1-related inflammation were evaluated by Chi square analyses or Mann-Whitney U test. The correlations between the percentage of neutrophils and number of COPD comorbidities or Charlson Comorbidity Index (CCI) scores were performed by Spearman's rank analysis. RESULTS: Low SUV39H1 expression group had high neutrophil counts relative to high SUV39H1expression group. In the COPD cohort, the high comorbidity group (≥ 2 comorbidities) had higher counts of whole white blood cell (WBC) and neutrophil, and lower proportion of eosinophil and eosinophil/neutrophil, as compared with low comorbidity group (0 and 1 comorbidities). The quantity of neutrophils was associated with COPD comorbidities (Spearman's r = 0.388, p < 0.001), but not with CCI scores. We also found that the high comorbidity group had more exacerbations per year compared with low comorbidity group (1.5 vs. 0.9 average exacerbations, p = 0.005). However, there were no significant differences between groups with these non-frequent (0-1 exacerbation) and frequent exacerbations per year (> 1 exacerbation) in numbers of WBC and proportion of neutrophils, eosinophils or eosinophil/neutrophil. Finally, patients with high comorbidities had lower SUV39H1 levels in their PBMCs than did those with low comorbidities. CONCLUSION: Blood neutrophil counts are associated with comorbidities in COPD patients. Impaired SUV39H1 expression in PBMCs from COPD patients are correlated with neutrophilic inflammation and comorbidities.

Subcellular Proteome Landscape of Human Embryonic Stem Cells Revealed Missing Membrane Proteins.

Human embryonic stem cells (hESCs) have the capacity for self-renewal and multilineage differentiation, which are of clinical importance for regeneration medicine. Despite the significant progress of hESC study, the complete hESC proteome atlas, especially the surface protein composition, awaits delineation. According to the latest release of neXtProt database (January 17, 2018; 19 658 PE1, 2, 3, and 4 human proteins), membrane proteins present the major category (1047; 48%) among all 2186 missing proteins (MPs). We conducted a deep subcellular proteomics analysis of hESCs to identify the nuclear, cytoplasmic, and membrane proteins in hESCs and to mine missing membrane proteins in the very early cell status. To our knowledge, our study achieved the largest data set with confident identification of 11 970 unique proteins (1% false discovery rate at peptide, protein, and PSM levels), including the most-comprehensive description of 6 138 annotated membrane proteins in hESCs. Following the HPP guideline, we identified 26 gold (neXtProt PE2, 3, and 4 MPs) and 87 silver (potential MP candidates with a single unique peptide detected) MPs, of which 69 were membrane proteins, and the expression of 21 gold MPs was further verified either by multiple reaction monitoring mass spectrometry or by matching synthetic peptides in the Peptide Atlas database. Functional analysis of the MPs revealed their potential roles in the pluripotency-related pathways and the lineage- and tissue-specific differentiation processes. Our proteome map of hESCs may provide a rich resource not only for the identification of MPs in the human proteome but also for the investigation on self-renewal and differentiation of hESC. All mass spectrometry data were deposited in ProteomeXchange via jPOST with identifier PXD009840.

Surface markers of human embryonic stem cells: a meta analysis of membrane proteomics reports.

INTRODUCTION: Human embryonic stem cells (hESCs) have unique biological features and attributes that make them attractive in various areas of biomedical research. With heightened applications, there is an ever increasing need for advancement of proteome analysis. Membrane proteins are one of the most important subset of hESC proteins as they can be used as surface markers. Areas covered: This review discusses commonly used surface markers of hESCs, and provides in-depth analysis of available hESC membrane proteome reports and the existence of these markers in many other cell types, especially cancer cells. Appreciating, existing ambiguity in the definition of a membrane protein, we have attempted a meta analysis of the published membrane protein reports of hESCs by using a combination of protein databases and prediction tools to find the most confident plasma membrane proteins in hESCs. Furthermore, responsiveness of plasma membrane proteins to differentiation has been discussed based on available transcriptome profiling data bank. Expert commentary: Combined transcriptome and membrane proteome analysis highlighted additional proteins that may eventually find utility as new cell surface markers.

Membrane Proteomics of Impaired Energetics and Cytoskeletal Disorganization in Elderly Diet-Induced Diabetic Mice.

Diabetic cardiomyopathy is a well-recognized complication of diabetes, but its pathophysiology is unclear. We aimed to investigate the mechanisms underlying cardiac dysfunction in an elderly type 2 diabetic (T2DM) mouse model, using membrane proteomic analyses. Elderly mice were fed a high fat diet for 12 weeks to induce T2DM, and myocardial structure and function were assessed by echocardiography. Cardiomyocytes were isolated by Langendorff perfusion and subjected to iTRAQ-based quantitative membrane proteomic profiling, immunoblotting, and real-time quantitative reverse-transcriptase polymerase chain reaction. Compared to controls, elderly T2DM mice showed worse systolic function, more myocardial fibrosis and up-regulation of several heart failure markers (all p < 0.05). Cardiomyocyte membrane proteomic profiling revealed that 417 proteins had differential expressions related to perturbations in several biological processes in T2DM mice compared with the control. The most up-regulated proteins were involved in oxidative phosphorylation, whereas many down-regulated proteins were involved in cytoskeletal regulation. Differential protein expression correlated with myocardial systolic velocity by tissue Doppler. In addition, cardiomyocyte immunofluorescence staining showed greater disorganization of thick/parallel F-actin stress fibers and marked reduction in F-to-G-actin ratio in T2DM vs control (p < 0.05), which paralleled worsened myocardial systolic velocity. We concluded that cardiac contractile dysfunction in elderly T2DM mice was associated with impaired energetics and cytoskeletal disorganization.

Identification of Potential Plasma Biomarkers for Nonalcoholic Fatty Liver Disease by Integrating Transcriptomics and Proteomics in Laying Hens.

Background: Prevalent worldwide obesity is associated with increased incidence of nonalcoholic fatty liver disease (NAFLD) and metabolic syndrome. The identification of noninvasive biomarkers for NAFLD is of recent interest. Because primary de novo lipogenesis occurs in chicken liver as in human liver, adult chickens with age-associated steatosis resembling human NAFLD is an appealing animal model.Objective: The objective of this study was to screen potential biomarkers in the chicken model for NAFLD by transcriptomic and proteomic analysis.Methods: Hy-Line W-36 laying hens were fed standard feed from 25 to 45 wk of age to induce fatty liver. They were killed every 4 wk, and liver and plasma were collected at each time point to assess fatty liver development and for transcriptomic and proteomic analysis. Next, selected biomarkers were confirmed in additional experiments by providing supplements of the hepatoprotective nutrients betaine [300, 600, or 900 parts per million (ppm) in vivo; 2 mM in vitro] or docosahexaenoic acid (DHA; 1% in vivo; 100 µM in vitro) to 30-wk-old Hy-Line W-36 laying hens for 4 mo and to Hy-Line W-36 chicken primary hepatocytes with oleic acid-induced steatosis. Liver or hepatocyte lipid contents and the expression of biomarkers were then examined.Results: Plasma acetoacetyl-CoA synthetase (AACS), dipeptidyl-peptidase 4 (DPP4), glutamine synthetase (GLUL), and glutathione S-transferase (GST) concentrations are well-established biomarkers for NAFLD. Selected biomarkers had significant positive associations with hepatic lipid deposition (P < 0.001). Betaine (900 ppm in vivo; 2 mM in vitro) and DHA (1% in vivo; 100 µM in vitro) supplementation both resulted in lower steatosis accompanied by the reduced expression of selected biomarkers in vivo and in vitro (P < 0.05).Conclusion: This study used adult laying hens to identify biomarkers for NAFLD and indicated that AACS, DPP4, GLUL, and GST could be considered to be potential diagnostic indicators for NAFLD in the future.

Identification of downstream components of ubiquitin-conjugating enzyme PHOSPHATE2 by quantitative membrane proteomics in Arabidopsis roots.

MicroRNA399-mediated regulation of the ubiquitin-conjugating enzyme UBC24/phosphate2 (PHO2) is crucial for Pi acquisition and translocation in plants. Because of a potential role for PHO2 in protein degradation and its association with membranes, an iTRAQ (for isobaric tags for relative and absolute quantitation)- based quantitative membrane proteomic method was employed to search for components downstream of PHO2. A total of 7491 proteins were identified from Arabidopsis thaliana roots by mass spectrometry, 35.2% of which were predicted to contain at least one transmembrane helix. Among the quantifiable proteins, five were significantly differentially expressed between the wild type and pho2 mutant under two growth conditions. Using immunoblot analysis, we validated the upregulation of several members in phosphate transporter1 (PHT1) family and phosphate transporter traffic facilitator1 (PHF1) in pho2 and demonstrated that PHO2 mediates the degradation of PHT1 proteins. Genetic evidence that loss of PHF1 or PHT1;1 alleviated Pi toxicity in pho2 further suggests that they play roles as downstream components of PHO2. Moreover, we showed that PHO2 interacts with PHT1s in the postendoplasmic reticulum compartments and mediates the ubiquitination of endomembrane-localized PHT1;1. This study not only uncovers a mechanism by which PHO2 modulates Pi acquisition by regulating the abundance of PHT1s in the secretory pathway destined for plasma membranes, but also provides a database of the membrane proteome that will be widely applicable in root biology research.

Proteomics of human platelet lysates and insight from animal studies on platelet protein diffusion to hippocampus upon intranasal administration.

Human platelet lysates (HPLs) from allogeneic platelet concentrates (PCs) are biomaterials, which are rich in various trophic factors, increasingly used in regenerative medicine and biotherapy. Understanding how preparation methods influence the HPL protein profile, biological function, and clinical outcomes is crucial. Our study sheds light on the proteomes and functionality of different HPLs, with the aim of advancing their scientifically grounded clinical applications. To achieve this, PCs suspended in plasma underwent three distinct processing methods, resulting in seven HPL types. We used three characterization techniques: label-free proteomics and tandem mass tag (TMT)-based quantitative proteomics, both before and after the immunodepletion of abundant plasma proteins. Bioinformatic tools assessed the proteome, and western blotting validated our quantitative proteomics data. Subsequent pre-clinical studies with fluorescent labeling and label-free proteomics were used as a proof of concept for brain diffusion. Our findings revealed 1441 proteins detected using the label-free method, 952 proteins from the TMT experiment before and after depletion, and 1114 proteins from the subsequent TMT experiment on depleted HPLs. Most detected proteins were cytoplasmic, playing key roles in catalysis, hemostasis, and immune responses. Notably, the processing methodologies significantly influenced HPL compositions, their canonical pathways, and, consequently, their functionality. Each HPL exhibited specific abundant proteins, providing valuable insight for tailored clinical applications. Immunoblotting results for selected proteins corroborated our quantitative proteomics data. The diffusion and differential effects to the hippocampus of a neuroprotective HPL administered intranasally to mice were demonstrated. This proteomics study advances our understanding of HPLs, suggesting ways to standardize and customize their production for better clinical efficacy in regenerative medicine and biotherapy. Proteomic analyses also offered objective evidence that HPPL, upon intranasal delivery, not only effectively diffuses to the hippocampus but also alters protein expression in mice, bolstering its potential as a treatment for memory impairments.

Proteomic profiling of tumor microenvironment and prognosis risk prediction in stage I lung adenocarcinoma.

OBJECTIVES: With the increasing popularity of CT screening, more cases of early-stage lung cancer are being diagnosed. However, 24.5% of stage I non-small-cell lung cancer (NSCLC) patients still experience treatment failure post-surgery. Biomarkers to predict lung cancer patients at high risk of recurrence are needed. MATERIALS AND METHODS: We collected protein mass spectrometry data from the Taiwan Lung Cancer Moonshot Project and performed bioinformatics analysis on proteins with differential expressions between tumor and adjacent normal tissues in 74 stage I lung adenocarcinoma (LUAD) cases, aiming to explore the tumor microenvironment related prognostic biomarkers. Findings were further validated in 6 external cohorts. RESULTS: The analysis of differentially expressed proteins revealed that the most enriched categories of diseases and biological functions were cellular movement, immune cell trafficking, and cancer. Utilizing proteomic profiling of the tumor microenvironment, we identified five prognostic biomarkers (ADAM10, MIF, TEK, THBS2, MAOA). We then developed a risk score model, which independently predicted recurrence-free survival and overall survival in stage I LUAD. Patients with high risk scores experienced worse recurrence-free survival (adjusted hazard ratio = 8.28, p < 0.001) and overall survival (adjusted hazard ratio = 6.88, p = 0.013). Findings had been also validated in the external cohorts. CONCLUSION: The risk score model derived from proteomic profiling of tumor microenvironment can be used to predict recurrence risk and prognosis of stage I LUAD.

An iTRAQ proteomic study reveals an association between diet-induced enhanced fatty acid metabolism and the development of glucose intolerance in prediabetic mice.

High-fat diet (HFD)-induced glucose intolerance and insulin resistance increases the chances of developing type-2 diabetes and cardiovascular disease. To study the mechanism(s) by which a HFD impairs glucose tolerance, we used a quantitative proteomic platform that integrated pI-based OFFGEL fractionation and iTRAQ labeling to profile the temporal changes in adipose membrane protein expression in mice fed a HFD for up to 8 months. Within 2 months of starting the diet, the mice adipose and liver tissues accumulated fat droplets, which contributed to subsequent insulin resistance and glucose intolerance within 6 months. The membrane proteomic delineation of such phenotypic expression resulted in quantification of 1713 proteins with 266, 343, and 125 differentially expressed proteins in 2-, 6-, and 8-month HFD-fed versus control mice, respectively. Pathway analysis of these differentially expressed proteins revealed the interplay between upregulation of fatty acid metabolism and downregulation of glucose metabolism. Substantial upregulation of adipose and liver carnitine palmitoyltransferase (Cpt) 1, the rate-limiting enzyme in the transport of long-chain fatty acids into mitochondria, occurred by 2 months. The increase in hepatic Cpt 1a expression was associated with a progressive decrease in glucose uptake as evidenced by downregulation of the liver glucose transporter protein (Glut) 2. Loss of glycogen storage was found in those hepatocytes full of fat droplets. Intriguingly, skeletal muscle Cpt 1b expression was unaltered by the HFD, whereas skeletal muscle Glut 4 and tyrosine phosphoryated insulin receptor substrate 1 (p-IRS1) were substantially upregulated at the same time as abnormal glucose metabolism developed in adipose and liver tissues. This study defines some of the molecular mechanisms as well as the relationship among adipose tissue, liver and skeletal muscle during development of HFD-induced glucose intolerance in vivo and identifies Cpt 1 as a potential drug target for the control or prevention of diabetes.

BAD-lectins: boronic acid-decorated lectins with enhanced binding affinity for the selective enrichment of glycoproteins.

The weak and variable binding affinities exhibited by lectin-carbohydrate interactions have often compromised the practical utility of lectin in capturing glycoproteins for glycoproteomic applications. We report here the development and applications of a new type of hybrid biomaterial, namely a boronic acid-decorated lectin (BAD-lectin), for efficient bifunctional glycoprotein labeling and enrichment. Our binding studies showed an enhanced affinity by BAD-lectin, likely to be mediated via the formation of boronate ester linkages between the lectin and glycan subsequent to the initial recognition process and thus preserving its glycan-specificity. Moreover, when attached to magnetic nanoparticles (BAD-lectin@MNPs), 2 to 60-fold improvement on detection sensitivity and enrichment efficiency for specific glycoproteins was observed over the independent use of either lectin or BA. Tested at the level of whole cell lysates for glycoproteomic applications, three different types of BAD-lectin@MNPs exhibited excellent specificities with only 6% overlapping among the 295 N-linked glycopeptides identified. As many as 236 N-linked glycopeptides (80%) were uniquely identified by one of the BAD-lectin@MNPs. These results indicated that the enhanced glycan-selective recognition and binding affinity of BAD-lectin@MNPs will facilitate a complementary identification of the under-explored glycoproteome.

A chemically functionalized magnetic nanoplatform for rapid and specific biomolecular recognition and separation.

We have developed a target-molecule-functionalized magnetic nanoparticle (MNP)-based method to facilitate the study of biomolecular recognition and separation. The superparamagnetic property of MNPs allows the corresponding biomolecules to be rapidly separated from crude biofluids with a significant improvement in recovery yield and specificity. Various MNPs functionalized with tag molecules (chitin, heparin, and amylose) were synthesized for recombinant protein purification, and several probe-functionalized MNPs, such as nitrilotriacetic acid (NTA)@MNP and P(k)@MNP, exhibited excellent extraction efficiency for proteins. In a cell recognition study, mannose-functionalized MNPs allowed specific purification of Escherichia coli with FimH adhesin on the surface. In an immunoprecipitation assay, the antibody-conjugated MNPs reduced the incubation time from 12 to 1 h while maintaining a comparable efficiency. The functionalized MNPs were also used in a membrane proteomic study that utilized the interaction between streptavidin-functionalized MNPs and biotinylated cell membrane proteins. Overall, the functionalized MNPs were demonstrated to be promising probes for the specific separation of targets from proteins to cells and proteomics.

An informatics-assisted label-free approach for personalized tissue membrane proteomics: case study on colorectal cancer.

We developed a multiplexed label-free quantification strategy, which integrates an efficient gel-assisted digestion protocol, high-performance liquid chromatography tandem MS analysis, and a bioinformatics alignment method to determine personalized proteomic profiles for membrane proteins in human tissues. This strategy provided accurate (6% error) and reproducible (34% relative S.D.) quantification of three independently purified membrane fractions from the same human colorectal cancer (CRC) tissue. Using CRC as a model, we constructed the personalized membrane protein atlas of paired tumor and adjacent normal tissues from 28 patients with different stages of CRC. Without fractionation, this strategy confidently quantified 856 proteins (≥2 unique peptides) across different patients, including the first and robust detection (Mascot score: 22,074) of the well-documented CRC marker, carcinoembryonic antigen 5 by a discovery-type proteomics approach. Further validation of a panel of proteins, annexin A4, neutrophils defensin A1, and claudin 3, confirmed differential expression levels and high occurrences (48-70%) in 60 CRC patients. The most significant discovery is the overexpression of stomatin-like 2 (STOML2) for early diagnostic and prognostic potential. Increased expression of STOML2 was associated with decreased CRC-related survival; the mean survival period was 34.77 ± 2.03 months in patients with high STOML2 expression, whereas 53.67 ± 3.46 months was obtained for patients with low STOML2 expression. Further analysis by ELISA verified that plasma concentrations of STOML2 in early-stage CRC patients were elevated as compared with those of healthy individuals (p < 0.001), suggesting that STOML2 may be a noninvasive serological biomarker for early CRC diagnosis. The overall sensitivity of STOML2 for CRC detection was 71%, which increased to 87% when combined with CEA measurements. This study demonstrated a sensitive, label-free strategy for differential analysis of tissue membrane proteome, which may provide a roadmap for the subsequent identification of molecular target candidates of multiple cancer types.

Differential cellular responses to FDA-approved nanomedicines: an exploration of albumin-based nanocarriers and liposomes in protein corona formation.

Albumin nanoparticles (NPs) and PEGylated liposomes have garnered tremendous interest as therapeutic drug carriers due to their unique physicochemical properties. These unique properties also have significant effects on the composition and structure of the protein corona formed around these NPs in a biological environment. Herein, protein corona formation on albumin NPs and liposomes was simultaneously evaluated through in vitro and simulation studies. The sizes of both types of NPs increased with more negatively charged interfaces upon being introduced into fetal bovine serum. Gel electrophoresis and label-free quantitative proteomics were performed to identify proteins recruited to the hard corona, and fewer proteins were found in albumin NPs than in liposomes, which is in accordance with isothermal titration calorimetry. The cellular uptake efficiency of the two NPs significantly differed in different serum concentrations, which was further scrutinized by loading an anticancer compound into albumin NPs. The presence of the hard protein corona increased the cellular uptake of albumin NPs in comparison with liposomes. In our simulation study, a specific receptor present in the membrane was greatly attracted to the albumin-apolipoprotein E complex. Overall, this study not only evaluated protein corona formation on albumin NPs, but also made promising advancements toward albumin- and liposome-based therapeutic systems.

Importance of surface charge of soot nanoparticles in determining inhalation toxicity in mice.

Physicochemical properties of nanoparticles are important in regulating nanoparticle toxicity; however, the contribution of nanoparticle charge remains unclear. The objective of this study was to investigate the pulmonary effects of inhalation of charged soot nanoparticles. We established a stably charged nanoparticle generation system for whole-body exposure in BALB/c mice, which produced positively charged, negatively charged, and neutral soot nanoparticles in a wide range of concentrations. After a 7-day exposure, pulmonary toxicity was assessed, together with proteomics analysis. The charged soot nanoparticles on average carried 1.17-1.35 electric charges, and the sizes for nanoparticles under different charging conditions were all fixed at 69 ~ 72 nm. We observed that charged soot nanoparticles induced cytotoxic LDH and increased lung permeability, with the release of 8-isoprostane and caspase-3 and systemic IL-6 in mice, especially for positively charged soot nanoparticles. Next, we observed that positive-charged soot nanoparticles upregulated Eif2, Eif4, sirtuin, mammalian target of rapamycin (mTOR), peroxisome proliferator-activated receptors (PPAR), and HIPPO-related signaling pathways in the lungs compared with negatively charged soot nanoparticles. HIF1α, sirt1, E-cadherin, and Yap were increased in mice's lungs by positively charged soot nanoparticle exposure. In conclusion, carbonaceous nanoparticles carrying electric ions, especially positive-charged, are particularly toxic when inhaled and should be of concern in terms of pulmonary health protection.