Proteomic analysis of sera of asymptomatic,early‐stage patients with Wilson's disease

Wilson's disease (WD) is characterized by excessive accumulation of intracellular copper in liver and extrahepatic tissues, leading to significant oxidative stress and tissue damage. To date, several diagnostic biomarkers for WD such as serum ceruloplasmin, serum or urine copper levels and copper content in liver have been identified. However, these biomarkers may not be convincing for the diagnosis in some WD patients. To identify additional novel diagnostic biomarkers, we compared the serum protein profiles of asymptomatic childhood WD patients (n=20), without neurologic manifestation or liver cirrhosis, with normal controls (n=13). Fourteen spots, five up‐regulated and nine down‐regulated (>2‐fold), were differentially expressed in WD patients in comparison to normal control on 2‐DE. Among them, three spots were down‐regulated in both male and female WD. MS/MS analysis revealed that the three spots were complement component C3, complement factor B and alpha‐2 macroglobulin. By comparative proteome analysis, complement component C3, complement factor B and alpha‐2 macroglobulin, which are related to oxidative stress and inflammation, turned out to be good candidates for novel diagnostic biomarkers for early stages of WD.     

Protein-induced changes during the maturation process of human dendritic cells: A 2-D DIGE approach

Dendritic cells (DCs) are unique antigen presenting cells, which upon maturation change from a specialized antigen‐capturing cell towards a professional antigen presenting cells. In this study, a 2‐D DIGE analysis of immature and mature DCs was performed, to identify proteins changing in expression upon maturation. The protein expression profile of immature and mature DCs, derived from CD14⁺ peripheral blood monocytes was investigated using two pH ranges (pH 4–7 and 6–9) (n = 4). Ninety one differentially expressed spots (p<0.01) were detected, from which we identified 74 spots (81.32%) corresponding to 41 different proteins. The proteins identified play a role in diverse processes, such as antigen processing/presentation, vesicle transport and cytoskeleton remodeling. In addition, a protein interaction network contained 29 (out of 41) proteins, suggesting that, although they functionally originate from distinct classes, these proteins are acting as a protein‐interactome. In conclusion, the proteins shown here to be altered in expression upon maturation are in line with the morphological and functional changes observed during the maturation process, providing a better understanding of the processes involved. This will open new avenues for investigating treatment regimens for immune‐associated disorders.     

Discovery of putative oocyte quality markers by comparative ExacTag proteomics

Purpose: Identification of the biomarkers of oocyte quality, and developmental and reprogramming potential is of importance to assisted reproductive technology in humans and animals. Experimental design: PerkinElmer ExacTag? Kit was used to label differentially proteins in pig oocyte extracts (oocyte proteome) and pig oocyte‐conditioned in vitro maturation media (oocyte secretome) obtained with high‐ and low‐quality oocytes. Results: We identified 16 major proteins in the oocyte proteome that were expressed differentially in high‐ versus low‐quality oocytes. More abundant proteins in the high‐quality oocyte proteome included kelch‐like ECH‐associated protein 1 (an adaptor for ubiquitin‐ligase CUL3), nuclear export factor CRM1 and ataxia‐telangiectasia mutated protein kinase. Dystrophin (DMD) was more abundant in low‐quality oocytes. In the secretome, we identified 110 proteins, including DMD and cystic fibrosis transmembrane conductance regulator, two proteins implicated in muscular dystrophy and cystic fibrosis, respectively. Monoubiquitin was identified in the low‐quality‐oocyte secretome. Conclusions and clinical implications: A direct, quantitative proteomic analysis of small oocyte protein samples can identify potential markers of oocyte quality without the need for a large amount of total protein. This approach will be applied to discovery of non‐invasive biomarkers of oocyte quality in assisted human reproduction and in large animal embryo transfer programs.     

Membrane Proteome of Invasive Retinoblastoma: Differential Proteins and Biomarkers

Purpose: Retinoblastoma (RB) is a pediatric ocular cancer which is caused due to the aberrations in the RB1 gene. The changes in the membrane proteomics would help in understanding the development of the retinoblastoma and could identify candidates for biomarkers and therapy. Experimental design: Quantitative proteomics is performed on the enriched membrane fractions from pooled normal retina (n = 5) and pooled retinoblastoma tissues (n = 5). The proteins are tryptic‐digested and tagged with iTRAQ labels. Orbitrap mass spectrometry is used to analyze and quantify the deregulated membrane proteins involved in the RB tumor progression. Immunohistochemistry (IHC) is used to further validate few of the differentially expressed proteins. Results: A total of 3122 proteins are identified of which, 663 proteins are found to be deregulated with ≥two fold change in the RB tumor compared to the retina. 282 proteins are upregulated and 381 are downregulated with ≥2 peptide identifications. Bioinformatic analysis revealed that, most of the proteins are involved in the transport, cellular communication, and growth. Overexpression of lamin B1 (LMNB1) and transferrin receptor (TFRC) are observed in RB tumors using IHC. Conclusion and clinical relevance: The present study, is the first comprehensive quantitative membrane proteomic atlas of the differentially regulated proteins in RB compared to the retina. LMNB1 and TFRC could be potential biomarkers for this childhood cancer.     

Identification of circulating endorepellin LG3 fragment: Potential use as a serological biomarker for breast cancer

Comparative proteome analysis was performed on the cultured media of human nontumor and malignant breast cell lines, Hs578Bst and Hs578T, respectively, in search of a serological biomarker(s) for breast cancer. Proteins in the conditioned media were separated by 2‐D PAGE and then visualized by silver‐staining. Eight proteins changed differentially by more than two‐fold were identified by MALDI‐TOF/TOF MS. Among the proteins identified, the terminal laminin‐like globular (LG3) domain of endorepellin, which was recently reported as an antiangiogenesis factor, was decreased in the cancer cell line. We confirmed the bone morphogenic protein‐1 (BMP‐1) mediated cleavage site on the N‐terminus of endorepellin LG3 fragment. This finding suggests that the LG3 fragment is specifically released by a BMP‐1 driven limited proteolytic process. The protein was also detected in plasma by Western blot analysis and selected reaction monitoring (SRM). The plasma level of the endorepellin LG3 fragment was significantly lower in breast cancer patients compared to healthy donors (p = 0.017; n = 12). The LG3 protein concentration in the control plasma was measured at approximately 3.7 pmol/mL compared to 1.8 pmol/mL in plasma from the cancer patients. We suggest that these results support the potential use of the endorepellin LG3 fragment as a new serological biomarker for breast cancer.     

Proteome of human endometrium: Identification of differentially expressed proteins in proliferative and secretory phase endometrium

Purpose: To exploit the potential of proteomics to identify and study additional yet‐unidentified important proteins present in human endometrium. Experimental design: The proteome of human endometrium would be established using 2‐DE and MALDI and the data analyzed to identify differential protein expression in the proliferative and secretory phase of the menstrual cycle using PDQuest software and MALDI. Results: In the present work, 2‐DE of human endometrium protein led to the resolution of over 200 spots. Subsequent MALDI analysis of 215 spots allowed the identification of 194 proteins. A total of 57 out of the 215 spots were found to be differentially expressed, out of which 49 could be identified using MALDI. These differentially expressed proteins included structural proteins, molecular chaperones, signaling proteins, metabolic proteins, proteins related to immunity, RNA biogenesis, protein biosynthesis and others. The differential expressions of seven representative proteins in secretory and proliferative phase endometrium tissue were confirmed by immunoblot analysis. Conclusion and clinical relevance: This study establishes the 2‐D proteome of human endometrium represented by 194 identified protein spots. The present data provides an important clue towards determining the function of these proteins with respect to endometrium related diseases.     

Comparative proteomic analysis of differentially expressed proteins in an in vitro cellular carcinogenesis model of oral squamous cell carcinoma

In vitro cellular model is an important tool to be used to investigate the cellular events related to pathophysiological conditions in humans. We have developed an in vitro cellular carcinogenesis model of oral squamous cell carcinoma (OSCC). In this study, we performed comparative proteomic analysis using 2‐DE and LC‐tandem mass chromatography to separate and identify differentially expressed proteins. Forty‐five proteins were identified, including 24 proteins with decreased expression and 19 proteins with increased expression during carcinogenesis from immortalized oral epithelial cells to squamous cancerous cells. The identified known proteins were classified into three ontologies of cellular component, molecular function, and biological process. Further validation of five identified proteins (ANXA1, ANXA2, CTSB, KRT17, and S100A6) in the cellular carcinogenesis model and cancerous tissues from OSCC patients confirmed the comparative proteomic results. Moreover, Annexin A1 and A2 expression levels correlated with the pathological differentiation grade of cancerous tissues. Thus, this work provides a dynamic protein file of differentially expressed proteins in oral squamous carcinoma cells, which could provide clues to study the mechanisms of OSCC carcinogenesis and possibly be developed as potential biomarkers for clinical diagnosis or prognostic monitoring.     

Altered proteins of the anterior cingulate cortex white matter proteome in schizophrenia

Alterations in the circuitry between and within different brain regions including the anterior cingulate cortex (ACC) is implicated in the neuropathology of schizophrenia. The involvement of white matter in schizophrenia is becoming increasingly apparent with reports of structural, morphological and genetic alterations occurring in the disease. The 2‐DE was employed to reveal significantly altered proteins within the ACC white matter proteome in a schizophrenia cohort (n = 10) relative to controls (n = 10). From the 423 matched spots between the two groups, the levels of 32 protein spots were altered in the schizophrenia ACC white matter. Of these, 30 spots were identified using MS. The majority of the altered proteins in schizophrenia function in metabolism, the cytoskeleton, and the synapse. These proteomic data suggest that the brain circuitry involving the ACC white matter tracts is altered in schizophrenia, possibly caused by depleted glucose metabolism and altered structural components of this region.     

Proteomic analysis of the response of the human neutrophil-like cell line NB-4 after exposure to anthrax lethal toxin

We used 2‐D DIGE to analyze the early response of NB‐4 cells, a human promyelotic leukemia cell line, exposed to lethal toxin from Bacillus anthracis at the proteome level. After a 2 h exposure, cells were still viable and 43% of spots (n = 1042) showed a significant change in protein level. We identified 59 spots whose expression had changed significantly, and these reflected cytoskeleton damage, mitochondrial lysis and endoplasmic reticulum stress. Actin filament assembly was disrupted as evidenced by an increase in both actin subunits and phosphorylated cofilin, whilst levels of tropomyosin, tropomodulin and actin related protein 2/3 complex subunit decreased. Lower levels of ATP synthase subunits and mitochondrial inner membrane protein were identified as markers of mitochondrial lysis. Levels of various stress response proteins rose and, uniquely, levels of Ca²⁺ binding proteins such as translationally controlled tumor protein rose and hippocalcin‐like protein 1 decreased. This response may have mitigated effects brought about by mitochondrial lysis and endoplasmic reticulum stress, and delayed or prevented apoptosis in NB‐4 cells. These results resemble findings of similar proteomics studies in murine macrophages, although quantitative differences were observed.     

Characterization of molecular markers indicative of cervical cancer progression

Cervical cancer originates with human papillomavirus (HPV) infection and progresses via histologically defined premalignant stages. Here we compare normal cervical epithelium and patient‐matched high‐grade squamous intraepithelial lesions (HSIL) with cervical carcinoma tissue from the same patient population (n = 10 per group). Specimens were analyzed by combined laser capture microdissection and 2‐D DIGE. Significant expression changes were seen with 53 spots resulting in identification of 23 unique proteins at the molecular level. These include eight that uniquely distinguish normal epithelium and HSIL and four that uniquely distinguish HSIL and carcinoma. In addition, one protein, cornulin, distinguishes all three states. Other identified proteins included differentiation markers, oncogene DJ‐1, serpins, stress and interferon‐responsive proteins, detoxifying enzymes, and serum transporters. A literature review, performed for all identified proteins, allowed most changes to be assigned to one of three causes: direct or indirect HPV oncoprotein interactions, growth selection during latency, or interactions in the lesion microenvironment. Selected findings were confirmed by immunohistochemistry using either frozen sections from the same cohort or formalin fixed paraffin embedded samples from a tissue microarray. Novel markers described here have potential applications for increasing the predictive value of current screening methods.     

Discovery of a Novel Connecting Link between Renin–Angiotensin System and Cancer in Barrett's Esophagus by Proteomic Screening

The renin–angiotensin system (RAS) plays a central role in the regulation of homeostasis and blood pressure. This involves an important enzyme called angiotensin‐converting enzyme that leads to the conversion of angiotensin I into angiotensin II. RAS has been reported to show association with inflammation, and in sporadic studies, with cancer. In particular, angiotensin II has been reported to be prevalent in the hypoxic microenvironment and associated with cancer signaling pathways. In a recent study, Bratlie et al. (Proteomics Clin. Appl. 2019 , 4, 1800102) is shown to exploit 2D gel electrophoresis, and mass spectrometry (MS) to identify differentially expressed proteins by comparing low‐grade dysplasia in Barrett's Esophagus (BE) following administration of agents that interfere with RAS, that is, enalapril and candesartan, and identified specific modulation of HSP60, PDIA3, and PPA1. Though 2D gel coupled with MS is a commonly‐used tool for studying proteomes, it still has limitations in terms of a comprehensive analysis due to lack of absolute quantitation in a high‐throughput manner. Despite technical limitations and the small size of the study, preliminary data emerging from the investigation show interference caused by clinically approved RAS inhibitors resulting in alteration of molecular markers associated with tumorigenicity. The authors propose potential factors that may influence the progression of the disease. However, these are conspicuous changes in high‐abundance proteins only. Therefore, there is a need to carry out detailed experimental studies either using an in vitro labeling technique (isobaric labeling for relative and absolute quantitation) for tissues or an in vivo labeling technique (stable isotope labeling in animal cell culture) coupled with LC‐MS/MS to identify differentially‐regulated proteins to delineate the role of RAS in BE.     

Proteomic profile of differentially expressed plasma proteins from dystrophic mice and following suberoylanilide hydroxamic acid treatment

Purpose: Histone Deacetylase Inhibitors (DI) ameliorates dystrophic muscle regeneration restoring muscular strength in the mdx mouse model of Duchenne muscular dystrophy (DMD). The further development of these compounds as drugs for DMD treatment is currently hampered by the lack of knowledge about DIs effect in large dystrophic animal models and that of suitable biomarkers to monitor their efficacy. Experimental design: In this study we applied proteomic analysis to identify differentially expressed proteins present in plasma samples from mdx mice treated with the Suberoylanilide hydroxamic acid (SAHA) and relative normal controls (WT). Results: Several differentially expressed proteins were identified between untreated wild type and mdx mice. Among these, fibrinogen, epidermal growth factor 2 receptor, major urinary protein and glutathione peroxidase 3 (GPX3) were constitutively up‐regulated in mdx, while complement C3, complement C6, gelsolin, leukaemia inhibitory factor receptor (LIFr), and alpha 2 macroglobulin were down‐regulated compared to WT mice. SAHA determined the normalization of LIFr and GPX3 protein level while apoliprotein E was de novo up‐regulated in comparison to vehicle‐treated mdx mice. Conclusions and clinical relevance: Collectively, these data unravel potential serological disease biomarkers of mdx that could be useful to monitor muscular dystrophy response to DI treatment.     

In pursuit of B-cell synovial autoantigens in rheumatoid arthritis: Confirmation of citrullinated fibrinogen, detection of vimentin, and introducing carbonic anhydrase as a possible new synovial autoantigen

We aimed to investigate potential synovial autoantigens in rheumatoid arthritis (RA) that could trigger the induction of B‐cell autoantibodies. Total protein extract of synovial tissue obtained from seven RA patients was pooled and separated by 1‐DE and 2‐DE. The corresponding blots were probed with sera from RA (n = 30) and disease control samples (n = 30). Protein spots showing a sensitivity of >15% were identified by MS. 1‐D immunoblots revealed one protein band with a specificity in RA of 100%, a sensitivity of 43%, which was identified as fibrinogen β chain. 2‐D analysis revealed the subunits of fibrinogen, especially the β and γ chain, as the most prominent synovial autoantigens. We also identified vimentin, the Sa‐antigen and carbonic anhydrase I as a potentially new synovial autoantigen. The protein patterns of these immunoreactive spots were observed as trains. The spots showing the highest autoimmune reactivity occurred at the acidic side of these trains and were recognized by anticitrullinated protein/peptide antibodies positive RA sera. Antimodified citrulline staining of these patterns confirmed protein citrullination. Therefore, PTMs such as citrullination due to alterations of peptidylarginine deiminase activity or generation of RA‐specific epitopes, should be considered as a trigger in tolerance break.     

Analysis of protein profiling studies of β‐thalassemia/Hb E disease

A number of studies have used global protein profiling technologies on a range of patient samples to detect proteins that are differentially expressed in β‐thalassemia/Hb E as an aid for understanding the physiopathology of this disease. Seven studies have identified a total of 111 unique, differentially expressed proteins. Seven proteins (prothrombin, alpha‐1‐antichymotrypsin, fibrinogen beta chain, hemoglobin beta, selenium‐binding protein, microtubule‐actin cross‐linking factor and adenomatous polyposis coli protein 2) have been identified in two independent studies, whereas two proteins (carbonic anhydrase 1 and peroxiredoxin‐2) have been identified in three independent studies. Both of these latter two proteins were consistently upregulated in the studies that identified them. Ontological analysis of all differentially regulated proteins identified “response to inorganic substances” as the most significant functional annotation cluster, which is consistent with iron overload being a major pathological consequence of this disease. Despite the range of samples investigated and the relatively small number of studies undertaken, a coherent picture of the mediators of the pathological consequences of β‐thalassemia/Hb E disease is starting to emerge.     

Proteome profiling of vitreoretinal diseases by cluster analysis

Vitreous samples collected in retinopathic surgeries have diverse properties, making proteomics analysis difficult. We report a cluster analysis to evade this difficulty. Vitreous and subretinal fluid samples were collected from 60 patients during surgical operation of non‐proliferative diabetic retinopathy, proliferative diabetic retinopathy, proliferative vitreoretinopathy, and rhegmatogenous retinal detachment. For controls, we collected vitreous fluid from patients of idiopathic macular hole, epiretinal, and from a healthy postmortem donor. Proteins from these samples were subjected to quantitative proteomics using two‐dimensional gel electrophoresis. We selected 105 proteins robustly expressed among ca. 400 protein spots and subjected them to permutation test. By using permutation test analysis we observed unique variations in the expression of some of these proteins in vitreoretinal diseases when compared to the control and to each other: (i) the levels of inflammation‐associated proteins such as alpha1‐antitrypsin, apolipoprotein A4, albumin, and transferrin were significantly higher in all four types of vitreoretinal diseases, and (ii) each vitreoretinal disease elevated a unique set of proteins, which can be interpreted based on the pathology of retinopathy. Our protocol will be effective for the study of protein expression in other types of clinical samples of diverse properties.     

Human duodenal proteome modulations by glutamine and antioxidants

Purpose : Glutamine (Gln) has protective, anti‐inflammatory effects in animal models and humans. Antioxidant nutrients may exert synergistic effects on intestinal functions. Therefore, these combined nutrients may have a therapeutic potential during intestinal inflammation. This study was designed to investigate in humans the effects of a supplement composed of Gln and high‐dosed antioxidant micronutrients compared to isomolar Gln only, on duodenal proteome. Experimental design : Enteral perfusion of Gln (0.8 mmol . kg^?1. h^?1) or supplement was performed in two groups of six healthy volunteers during 5 h before taking endoscopic duodenal biopsies. Protein expression was analyzed by 2‐DE and the relevant proteins identified by MS/MS. Results : About 1500 protein spots were revealed in both supplement and Gln conditions. Comparative proteomics analysis indicated that 11 proteins were differentially and significantly (p≤0.05) expressed in response to the supplement. These proteins were essentially implicated in metabolism pathways, e.g. fatty acid binding protein‐1 and 40S ribosomal protein SA expressions were downregulated while manganese superoxide dismutase and retinal dehydrogenase‐1 expressions were upregulated. Conclusions and clinical relevance : This study provides new information on human duodenal proteome and its nutritional modulation, and supports further clinical investigations designed to evaluate the effects of Gln plus antioxidants during intestinal inflammation and cancer.