iTRAQ-based Proteomic Technology Research Articles

Proteomics Analysis of Zygosaccharomyces mellis in Response to Sugar Stress

The high-osmotic-pressure environment of honey is not suitable for the survival of microorganisms, except for osmotic-tolerant fungal and bacterial spores. In this study, shotgun metagenomic sequencing technology was used to identify yeast species present in honey samples. As a result, Zygosaccharomyces spp. yeast, including Zygosaccharomyces rouxii, Z. mellis and Z. siamensis, were isolated. The intracellular trehalose and glycerin concentrations of yeast, as well as the antioxidant-related CAT, SOD and POD enzyme activities, increased under a high-glucose environment (60%, w/v). To learn more about the osmotic resistance of Z. mellis, iTRAQ-based proteomic technology was used to investigate the related molecular mechanism at the protein level, yielding 522 differentially expressed proteins, of which 303 (58.05%) were upregulated and 219 (41.95%) were downregulated. The iTRAQ data showed that the proteins involved in the pathway of the cell membrane and cell-wall synthesis, as well as those related to trehalose and glycerin degradation, were all downregulated, while the proteins in the respiratory chain and TCA cycle were upregulated. In addition, formate dehydrogenase 1 (FDH1), which is involved in NADH generation, displayed a great difference in response to a high-sugar environment. Furthermore, the engineered Saccharomyces cerevisiae strains BY4741△scFDH1 with a knocked-out FDH1 gene were constructed using the CRISPR/Cas9 method. In addition, the FDH1 from Z. mellis was expressed in BY4741△scFDH1 to construct the mutant strain BY4717zmFDH1. The CAT, SOD and POD enzyme activities, as well as the content of trehalose, glycerin, ATP and NADH, were decreased in BY4741△scFDH1. However, those were all increased in BY4717zmFDH1. This study revealed that Z. mellis could increase the contents of trehalose and glycerin and promote energy metabolism to improve hypertonic tolerance. In addition, FDH1 had a significant effect on yeast hypertonic tolerance.

Membrane proteomic analysis reveals the intestinal development is deteriorated by intrauterine growth restriction in piglets.

The alterations of the intestinal proteome were observed in intrauterine growth restriction (IUGR) piglets during early life by gel-based approaches. Nevertheless, how IUGR affects the intestinal membrane proteome during neonatal development remains unclear. Here, we applied the iTRAQ-based proteomics technology and biochemical analysis to investigate the impact of IUGR on the membrane proteome of the jejunal mucosa in the piglets. Three hundred sixty-one membrane proteins were screened by functional prediction. Among them, eight, five, and one differentially expressed membrane proteins were identified between IUGR and NBW piglets at day 0, day 7, and day 21 after birth, respectively. Differentially expressed membrane proteins (DEMPs) including F1SBL3, F1RRW8, F1S539, F1S2Z2, F1RIR2, F1RUF2 I3LP60, Q2EN79, and F1SIH8 were reduced while the relative abundance of I3L6A2, F1SCJ1, F1RI18, I3LRJ7, and F1RNN0 were increased in IUGR piglets than NBW piglets. From the aspects of function, F1RRW8, F1S539, F1S2Z2, and F1RIR2 are mainly associated with D2 dopamine receptor binding, transmembrane transport of small molecules, signal transduction, and translocation of GLUT4, respectively, and F1SIH8, I3LRJ7, and F1RNN0 are related to autophagy, metabolism of vitamins, and intracellular protein transport. Additionally, IUGR decreased the level of proteins (F1RRW8, Q2EN79, and F1RI18) that are involved in response to oxidative stress.

Evaluation of the anti-hypertensive effect of Tengfu Jiangya tablet by combination of UPLC-Q-exactive-MS-based metabolomics and iTRAQ-based proteomics technology

ObjectiveTengfu Jiangya tablet (TJT) is a traditional Chinese medicine formulation composed of Uncaria rhynchophylla and Semen raphani. It is a hospital preparation that is widely used in clinics for treating hypertension. A previous metabolomics study reported that TJT exerted a protective effect on hypertension by restoring impaired NO production, ameliorating the inflammatory state, and vascular remodeling. A clinical proteomics study also revealed five key target proteins during TJT intervention. This study aimed to integrate proteome and metabolome data sets for a holistic view of the molecular mechanisms of TJT in treating hypertension. MethodsSerum samples from spontaneously hypertensive rats and Wistar Kyoto rats were analyzed using ultra-high performance liquid chromatography coupled to Q Exactive hybrid quadrupole-Orbitrap mass spectrometry (UPLC-Q-Exactive-MS)-based metabolomics technology and isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomics technology. Moreover, we selected two candidate proteins and determined their expression levels in rat serum using an enzyme-linked immunosorbent assay (ELISA). ResultsA total of 20 potential biomarkers and 14 differential proteins in rat serum were identified. These substances were mainly involved in three biological pathways: the kallikrein–kinin pathway, the lipid metabolism pathway, and the PPARγ signaling pathway. ConclusionsThe results suggested that TJT could effectively treat hypertension, partially by regulating the above three metabolic pathways. The combination of proteomics and metabolomics provided a feasible method to uncover the underlying interventional effect and therapeutic mechanism of TJT on spontaneously hypertensive rats.

Establishment of a CRISPR/Cas9-Mediated Cysltr1 Knockout Mouse Model and iTRAQ-Based Proteomic Analysis.

To clarify the role of Cysteinyl leukotrienes receptor type 1 (CYSLTR1) and find the potential predictors of CYSLTR1 antagonists (leukotriene receptor antagonists [LTRAs]) responsiveness in vivo. Cysltr1 knockout (KO) mouse model is established by the CRISPR/Cas9 system. The phenotype of Cysltr1 KO mice are tested by western blotting (WB), histological examinations, and experiment of zymosan A-induced peritoneal inflammation. The differentially expressed proteins (DEPs) between the Cysltr1 KO and the wild type (WT) mice lung tissues are analyzed by the iTRAQ-based proteomic technology. WB is used to validate a subset of DEPs. The total nitric oxide (NO) concentration in lung tissues are measured. The Cysltr1 KO mice show the decrease of vascular permeability in comparison with the WT mice. Our quantitative proteomic analysis identified 239 DEPs in total. WB confirms an increased expression of protein kinase C-δ (PKC-δ), while N(G),N(G)-dimethylarginine dimethylaminohydrolase 1 (DDAH1) and β-Catenin expression are reduced. The total NO concentrations are significantly reduced in lungs from Cysltr1 KO mice. This study not only provides a comprehensive dataset on overall protein changes in Cysltr1 KO mice lung tissues, but also sheds light on interpreting the description of lower vascular permeability in Cysltr1 KO mice.

The Role of FABP5 in Radiation-Induced Human Skin Fibrosis.

Radiation-induced skin fibrosis is a detrimental and chronic disorder that occurs after radiation exposure. The molecular changes underlying the pathogenesis of radiation-induced fibrosis of human skin have not been extensively reported. Technical advances in proteomics have enabled exploration of the biomarkers and molecular pathogenesis of radiation-induced skin fibrosis, with the potential to broaden our understanding of this disease. In this study, we compared protein expression in radiation-induced fibrotic human skin and adjacent normal tissues using iTRAQ-based proteomics technology. We identified 186 preferentially expressed proteins (53 upregulated and 133 downregulated) between radiogenic fibrotic and normal skin tissues. The differentially expressed proteins included keratins (KRT5, KRT6A, KRT16 and KRT17), caspase-14, fatty acid-binding protein 5 (FABP5), SLC2A14 and resistin. Through bioinformatic analysis of the proximal promoters, common motifs and corresponding transcriptional factors were identified that associate with the dysregulated proteins, including PAX5, TBX1, CLOCK and AP2D. In particular, FABP5 (2.15-fold increase in fibrotic skin tissues), a transporter of hydrophobic fatty acids, was investigated in greater detail. Immunohistochemistry confirmed that the protein level of FABP5 was increased in fibrotic human skin tissues, especially in the epidermis. Overexpression of FABP5 resulted in nuclear translocation of SMAD2 and significant activation of the profibrotic TGF-β signaling pathway in human fibroblast WS1 cells. Moreover, exogenous FABP5 (FABP5-EGFP) could be incorporated by skin cells and intensify TGF-β signaling, indicating a communication between the microenvironment and skin fibrosis. Taken together, our findings illustrate the molecular changes during radiation-induced human skin fibrosis and the critical role of FABP5 in activating the TGF-β signaling pathway.

Abstract 4410: LSR promotes ovarian cancer cell growth following the activation of β-oxidation and its antibody inhibits lipid catabolism

Abstract Ovarian cancer is the most lethal gynecologic malignancy; thus developing new treatment options is urgently required. Molecular targeted therapies for cancers, which are generally more tolerable than widely used cytotoxic agents, have shown highly specific inhibition of target molecules. In this study, we aimed to identify a new ovarian cancer antigen and to develop a novel monoclonal antibody (mAb). Furthermore we evaluated its preclinical efficacy and analyzed the function of its antigen in ovarian cancer.To identify a new ovarian cancer antigen, cell surface membrane proteins of normal ovarian epithelial and ovarian cancer cell lines were analyzed by iTRAQ-based proteomic technology. We identified lipolysis-stimulated lipoprotein receptor (LSR) as the new therapeutic target for ovarian cancer. By the immunohistochemical analysis, significant poor prognosis was observed in high-LSR expression patients with ovarian cancer compared to patients with low-LSR expression by survival assay (p < 0.05). Our newly developed anti-LSR mAb showed significant inhibition of tumor growth in vivo against xenograft model of LSR-positive ovarian cancer cell line and patient derived LSR-positive ovarian cancer tissue (p < 0.05). In LSR-positive ovarian cancer cells, high number and large lipid droplets were observed compared to LSR-negative cells and anti-LSR mAb decreased these droplets. Moreover addition of VLDL to LSR-positive ovarian cancer cells significantly promoted the cell proliferation (p < 0.05) and anti-LSR mAb inhibited that in vitro (p < 0.05). Supporting these data, addition of VLDL to LSR-positive ovarian cancer cells significantly promoted β-oxidation-mediated lipid catabolism (p < 0.05) and anti-LSR mAb also inhibited that (p < 0.05). In addition, this anti-LSR mAb which cross-reacted with mouse LSR did not show any cytotoxicity on normal organs and lipid metabolism in mice. In summary, high expression of LSR in ovarian cancer was the poor prognostic factor. Our newly developed anti-LSR mAb showed significant tumor growth inhibition against not only LSR-positive ovarian cancer cell line but also patient derived LSR-positive ovarian cancer tissue. In LSR-positive ovarian cancer cells, high number and large lipid droplets were observed and LSR promoted cell proliferation following β-oxidation-mediated lipid catabolism. Anti-LSR mAb inhibited these processes. Our preclinical data demonstrated that targeting LSR by mAb is a promising therapy for patients with LSR-positive ovarian cancer. Citation Format: Kosuke Hiramatsu, Satoshi Serada, Takayuki Enomoto, Takuhei Yokoyama, Yusuke Takahashi, Minoru Fujimoto, Kiyoshi Yoshino, Tadashi Kimura, Tetsuji Naka. LSR promotes ovarian cancer cell growth following the activation of β-oxidation and its antibody inhibits lipid catabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4410. doi:10.1158/1538-7445.AM2017-4410

Abstract 4743: Identification of metastasis-associated biomarker PLEC1 by iTRAQ-based proteomic approach and prognostic role of PLEC1 in patients with hepatocellular carcinoma

Abstract Purpose: Hepatocellular Carcinoma(HCC)is the fifth most prevalent cancer and the second most frequent cause of cancer-related death in men worldwide because of high incidence of recurrence and widespread metastasis. However, to date, there have been no direct metastesis-associated biomarker which is critical for the effective treatment of patients with HCC, we thus aimed to identify novel HCC metastasis-associated biomarkers with prognostic significance.E xperimental Design: Two human HCC cell lines, MHCC97-L and MHCC97-H with similar genetic background and stepwise increasing metastatic potentials, were used to explore potential metastasis-associated biomarkers for HCC by isobaric tag for relative and absolute quantitation (iTRAQ) combination with 2D-LC-MS/MS. Ninety-five HCC cancerous and paracancerous tissues were further analyzed by immunohistochemistry to validate the correlations between the expression levels of PLEC1 with various clinicopathologic factors and patient survival. Results: Using iTRAQ combination with 2D-LC-MS/MS, a total of 47 differentially expressed proteins were found in MHCC97-L and MHCC97-H cells, of which 26 proteins were up regulated and 21 proteins were down regulated. Plectin-1 (PLEC1) was identified and re-confirmed as a potential biomarker for HCC that was highly up-regulated in tumor tissues, in comparison with that of para-cancerous tissues. Over-expression of PLEC1 was also strongly correlated with tumor size (P < 0.05) and TMN stages (P < 0.05). In addition, both univariate and multivariate analyses demonstrate that PLEC1 expression is an independent prognostic factor for patients with HCC(p<0.05). Conclusions: In this study, iTRAQ-based proteomic technology was explored to uncover novel metastasis-associated biomarkers in HCC. We identified and validated metastasis-associated protein PLEC1 as a potential prognostic marker in patients with HCC. Note: This abstract was not presented at the meeting. Citation Format: Lin-lan Song, Yu Chen, Di Mu, Xiao-Yan Jiang, Yu-Nong Li, Ling-Yu Jiang, Zhen-Fang Zhang, Liang Yan, Min Yang, Yi-xuan Yang, Hong Ren, Yong Liao. Identification of metastasis-associated biomarker PLEC1 by iTRAQ-based proteomic approach and prognostic role of PLEC1 in patients with hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4743. doi:10.1158/1538-7445.AM2017-4743

Determination of the protein expression profiles of Propsilocerus akamusi (Tokunaga) Malpighian tubules response to cadmium stress by iTRAQ coupled LC–MS/MS

Propsilocerus akamusi (Tokunaga) is an important prey species for fish and aquatic birds, which represent an important link in the aquatic food chain as other species of Chironomidae. Malpighian tubules (MT) play an important role in metabolism and detoxification of harmful compounds and metal accumulation. Our previous studies have reported that P. akamusi have an extraordinary resistance to Cd. In this paper, iTRAQ-based proteomics technology was first used to study the differential proteomics of MT of Propsilocerus akamusi under sublethal Cd stress for different time courses. 118 differential expressed proteins (DEPs) were identified through LC–MS/MS and further classified into 26 GO functional groups. Our results showed that metabolic process was the main biological functional categories. Cluster of orthologous groups of proteins (COG) analysis was also performed based on the number of unique proteins identified in each functional category, 21 metabolism-related DEPs were identified. P. akamusi adapted to Cd stress mainly by inducing the processes of lipid metabolism of MT and lipid may play a possible protection role in cadmium resistance in MT. The results provide important information for research on molecular mechanisms of Cd-stress adaption in Malpighian tubules of P. akamusi. Biological significancePropsilocerus akamusi (old species name: Tokunagayusurika akamusi) is an important prey species for fish and aquatic birds, which represent an important link in the aquatic food web as other species of Chironomidae. Malpighian tubules constitute a versatile organ which is able to exert excretory of chemical compounds, acting in the insect metabolism and detoxification of harmful compounds and metal accumulation. Our previous studies have reported that P. akamusi have an extraordinary resistance to Cd. However, up to now, there has been a lack of research on the molecular mechanisms of adaption to Cd stress in the Malpighian tubules of this species. Study on the adaptive mechanisms to Cd stress in Malpighian tubules of Propsilocerus akamusi through comparative proteome analysis is one important way to solve this problem. The aim of the present study was to understand the molecular mechanisms of adaption of the Malpighian tubules of P. akamusi exposed to Cd stress and assess the high expression of proteins involved in cellular adaptation to Cd stress. This will provide information about how this organ is responding to disturbances of contaminated ecosystems.

ITRAQ-based proteomic technology revealed protein perturbations in intestinal mucosa from manganese exposure in rat models

Manganese (Mn) is an essential metal ion as a biological cofactor, but in excess, it is toxic; however, the homeostatic mechanisms of Mn at the cellular level have not been identified.

ITRAQ-Based Proteomics Screen identifies LIPOCALIN-2 (LCN-2) as a potential biomarker for colonic lateral-spreading tumors.

The improvement and implementation of a colonoscopy technique has led to increased detection of laterally spreading tumors (LSTs), which are presumed to constitute an aggressive type of colonic neoplasm. Early diagnosis and treatment of LSTs is clinically challenging. To overcome this problem, we employed iTRAQ to identify LST-specific protein biomarkers potentially involved in LST progression. In this study, we identified 2,001 differentially expressed proteins in LSTs using iTRAQ-based proteomics technology. Lipocalin-2 (LCN-2) was the most up-regulated protein. LSTs expression levels of LCN-2 and matrix metallopeptidase-9 (MMP-9) showed positive correlation with worse pathological grading, and up-regulation of these proteins in LSTs was also reflected in serum. Furthermore, LCN-2 protein overexpression was positively correlated with MMP-9 protein up-regulation in the tumor tissue and serum of LST patients (former rs = 0.631, P = 0.000; latter rs = 0.815, P = 0.000). Our results suggest that LCN-2 constitutes a potential biomarker for LST disease progression and might be a novel therapeutic target in LSTs.

Protein mechanisms in response to NaCl-stress of salt-tolerant and salt-sensitive industrial hemp based on iTRAQ technology

Industrial hemp is one of the important natural green fiber crops and, up to now, there has been a lack of research on its salt tolerance and the metabolic molecular mechanisms involved. Study on the adaptive mechanisms to salt stress in hemp through comparative proteome analysis is one important way to solve this problem. In this paper, iTRAQ-based proteomics technology was first used to study the differential proteomics of leaves of seedling under salt stress (600mM sodium chloride) for 4 days in salt-resistant industrial hemp and salt-sensitive varieties. There were 403 and 252 differential proteins identified through LC-ESI–MS/MS mass spectrometry, respectively, and these were classified into 14 categories: primary metabolism, energy, protein destination and storage, disease/defense, protein synthesis, photosynthesis, transport, transcription, signal transduction, cell structure, secondary metabolism, intracellular traffic, cell growth/division and unknown. Hemp adapted to stress mainly by improved ATP metabolism, regulating photosynthesis according to light intensity, strengthening chlorophyll synthesis, promoting cell relaxation and enlargement, improving synthesis of osmoregulation substances, enhancing flow of inorganic sulfur in the body, regulating aquaporins, strengthening transmission of ion transport signals, improving signal transmission between proteins and between proteins and cytomembranes, improving selective absorption and transportation speed of organic and inorganic molecular, degrading hemicellulose cell walls, controlling transport of cellular material in and out, and promoting metabolism and cell stability. The results provide important reference information for research on molecular mechanisms of salt-stress adaption in hemp.

Differential expression of serum proteins in rats subchronically exposed to arsenic identified by iTRAQ-based proteomic technology-14-3-3 ζ protein to serve as a potential biomarker.

Arsenic is a multi-system toxicant. However, the mechanism of arsenic toxicity is not fully clarified and few effective protein biomarkers could be used for arsenic poisoning. This study was to investigate the differentially expressed proteins in the serum of rats subchronically exposed to arsenic. Sixty male rats were randomly divided into four groups, and the dose of sodium arsenite in drinking water for each group was 0, 2, 10, and 50 mg L-1, respectively. The exposure lasted for 12 weeks. An Isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomic approach was used to identify the differentially expressed proteins in serum between control and 50 mg L-1 groups. A total of 201 serum proteins were identified by iTRAQ, of which 12 were significantly changed by arsenic exposure with two up-regulated and ten down-regulated proteins. One down-regulated protein 14-3-3 ζ, an abundant protein expressed in the brain, was verified by ELISA using serum samples and by immunohistochemical, real time PCR, and western blot methods using brain tissues in four groups. Our work provided valuable insight into the serum protein changes in rats exposed to arsenic, and indicated that 14-3-3 ζ may serve as a useful biomarker for nervous damage caused by arsenic poisoning.

Abstract 4382: Anti-human LSR monoclonal antibody inhibits tumor growth of ovarian cancer directly

Abstract Ovarian cancer is the most lethal gynecologic malignancy; thus developing new treatment options is urgently required. Molecular targeted therapies for cancers, which are generally more tolerable than widely used cytotoxic agents, have shown highly specific inhibition of target molecules. We previously identified bone marrow stromal antigen 2 (BST2) as an endometrial cancer antigen using iTRAQ-based quantitative proteomic technology focused on cell surface membrane proteins, and also demonstrated the usefulness of an anti-BST2 monoclonal antibody (mAb) for endometrial cancer. In this study, we aimed to identify a new ovarian cancer antigen. We also aimed to develop a novel monoclonal antibody (mAb) and evaluate its preclinical efficacy against ovarian cancer. To identify a new ovarian cancer antigen, cell surface membrane proteins of normal ovarian epithelial and ovarian cancer cell lines were analyzed by iTRAQ-based proteomic technology. As the new therapeutic target for ovarian cancer, we identified lipolysis-stimulated lipoprotein receptor (LSR) which had one of the largest significant differences in protein level between normal ovarian epithelial and ovarian cancer cell lines. Immunohistochemical analysis showed that the overall survival of ovarian serous carcinoma patients with high LSR expression was significantly shorter than those with low LSR (p = 0.0293). We newly developed anti-LSR mAb and investigated its preclinical efficacy. Anti-LSR mAb showed significant in vivo inhibition of tumor growth against a xenograft model of hLSR-positive ovarian cancer in an antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) independent manner (p = 0.0001). And anti-LSR mAb also induced G0/G1 cell cycle arrest by regulation of MEK and p44/42 MAPK activities and expression levels of cell cycle related proteins in vitro. Furthermore, anti-hLSR mAb, which crossreacts with mouse LSR, had little detectable toxicity in mice. In summary, high expression of LSR in ovarian cancer was the poor prognostic factor. Our newly developed anti-LSR mAb showed significant tumor growth inhibition in ADCC and CDC independent manner in vivo. Anti-human LSR mAb also inhibited LSR function and showed direct tumor growth inhibition inducing G0/G1 cell cycle arrest in vitro. Our preclinical data demonstrated that targeting LSR by mAb is a promising therapy for patients with LSR-positive ovarian cancer. Citation Format: Kosuke Hiramatsu, Satoshi Serada, Takayuki Enomoto, Satoshi Nakagawa, Akiko Morimoto, Minoru Fujimoto, Takuhei Yokoyama, Yusuke Takahashi, Yutaka Ueda, Kiyoshi Yoshino, Eiichi Morii, Tadashi Kimura, Tetsuji Naka. Anti-human LSR monoclonal antibody inhibits tumor growth of ovarian cancer directly. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4382. doi:10.1158/1538-7445.AM2015-4382