Identification of Novel Biomarker and Therapeutic Target Candidates for Diagnosis and Treatment of Follicular Adenoma

ollicular adenoma is a type of benign and encapsulated nodule in the thyroid gland, but some adenomas have the potential to progress to follicular carcinoma. Therefore, it is important to monitor the state and progress of follicular adenoma in the clinic and discover drug development targets for the treatment of follicular adenoma to prevent its worsening to follicular carcinoma. Currently, the study of biomarkers and therapeutic targets lacks applications of up-to-date technologies, including proteomics and bioinformatics. To discover novel protein biomarker and therapeutic target candidates, a liquid chromatography-tandem mass spectrometry approach was applied to directly compare follicular adenoma with normal thyroid tissue samples. The proteomics analysis revealed 114 protein biomarker candidates out of 1,780 identified and quantified proteins. A comprehensive approach to prioritize the biomarker candidates by category and rank revealed CD63, DDB1, TYMP, VDAC2, and DCXR as the top five biomarker candidates. Upstream regulator analysis using the Ingenuity Pathway Analysis (IPA) software discovered four therapeutic target candidates for follicular adenoma, including TGFB1, MYC, ANGPT2, and NFE2L2. This study provided biomarker and therapeutic target candidates for a follow-up study, which will facilitate monitoring and treatment of follicular adenoma

Identification of novel biomarker candidates for immunohistochemical diagnosis to distinguish low-grade chondrosarcoma from enchondroma

Chondrosarcoma is the third most common primary bone cancer, requiring surgical resection. However, differentiation of low-grade chondrosarcoma (grade 1) from enchondroma that is benign and only requires regular follow-up is one of the most frequent diagnostic dilemmas facing orthopedic oncologists in clinical management. Although multiple techniques are applied to make the distinction, immunohistochemistry is an important ancillary technique, especially when a histopathological stain of specimen must be obtained in order to guarantee an accurate confirmation. Currently, no adequate immunohistochemical diagnostic protein biomarkers are available to distinguish low-grade chondrosarcoma from enchondroma. To discover novel protein biomarker candidates, an LC-MS/MS approach was applied to directly compare formalin-fixed, paraffin-embedded low-grade chondrosarcoma with enchondroma tissue samples. The proteomics analysis revealed 17 protein biomarker candidates. A principle was developed to prioritize the candidates using category and ranking. An algorithm, prioritization index of biomarker candidates for immunohistochemistry on tissue specimens, was developed to rank the candidates inside each category. Using the proteomics data and bioinformatics results, the prioritization index of biomarker candidates for immunohistochemistry on tissue revealed periostin as a top candidate. Immunohistochemical staining of periostin in 23 low-grade chondrosarcoma and 31 enchondroma tissue specimens disclosed 87% specificity and 70% sensitivity

Proteomic Techniques in the Physiological Proteomics Core Facility

poster abstractA new software package, IdentiQuantXL, has been developed in the Physiological Proteomics Core Facility to provide large-scale protein identification and label-free quantification using either low or high resolution LC-MS/MS data. Though many software packages have been developed to perform label-free quantification of proteins in complex biological samples using peptide intensities generated by liquid chromatography - tandem mass spectrometry (LC-MS/MS), two important issues hinder the use of peptide intensity measurements: (i) It is difficult to accurately determine the retention time of each peptide peak, especially for low resolution data, and (ii) many peptides cannot be used for protein quantification. To address these two key issues, we have developed a new method to enable accurate peptide peak retention time determination and multiple filters to eliminate unqualified peptides for protein quantification. Repeatability and linearity have been tested using ion trap-derived low resolution data from six very different samples, i.e., standard peptides, kidney tissue lysates, HT29-MTX cell lysates, depleted human serum, human serum albumin-bound proteins, and standard proteins spiked in kidney tissue lysates. In all these unique experiments, at least 90.8% of proteins (up to 1,390) had CVs ≤ 3 0% across 10 technique replicates, and at least 92.1% of proteins (up to 2,013) had R2 ≥ 0.9500 across 7 concentrations. The performance of our strategy was verified using identical amounts of standard protein (lysozyme) spiked in complex biological samples (cell culture media containing secreted proteins) with a CV of 8.6% across eight injections. The excellent performance was further confirmed by comparing label-free mass spectrometry to Western blot detection of prolactin, which was decreased 17.1fold in dwarfed mice compared to wild-type using the label-free quantification strategy and very low or undetectable using Western blot. The results indicate that our new platform, named IdentiQuantXL, accurately quantifies thousands of peptides and proteins in complex samples. It has been applied in the aqueous humor proteome in patients with Fuchs endothelial corneal dystrophy. While many software packages focus only on high resolution data, our strategy is designed for both high and low resolution data. Consequently, it is very useful for data generated by low resolution mass spectrometers such as the LTQ, especially when the dynamic exclusion of ions in data acquisition is enabled to obtain more MS/MS fragments of low-abundance peptides to maximally identify proteins in a complex biological sample. Supported by NIEHS RC2ES018810 and NIGMS R01GM08521

Development, validation, and comparison of four methods to simultaneously quantify L-arginine, citrulline, and ornithine in human plasma using hydrophilic interaction liquid chromatography and electrospray tandem mass spectrometry

To understand the role of l-arginine depletion in impaired nitric oxide synthesis in disease, it is important to simultaneously quantify arginine, citrulline, and ornithine in the plasma. Because the three amino acids are endogenous analytes, true blank matrix for them is not available. It is necessary and valuable to compare the performance of different approaches due to lack of regulatory clarity for validation. A two-step sample preparation method using methanol as protein precipitation reagent was developed in this study is used for sample preparation. Because true blank matrix for endogenous analytes is not available, water as blank matrix, 1% BSA in PBS as blank matrix, surrogate analyte, and background subtraction were designed to establish successful quantification methods. Four methods to simultaneously quantify arginine, citrulline, and ornithine in human plasma using hydrophilic interaction liquid chromatography and electrospray tandem mass spectrometry were developed, validated, and compared. The developed two-step sample preparation method using methanol as protein precipitation reagent in this study needs less time and provides higher recovery comparing with other approaches. Three of the four methods, water as blank matrix, 1% BSA in PBS as blank matrix, and surrogate analyte, have been successful in fulfilling all the criteria, while background subtraction has failed. Results of the measured concentrations in 97 human plasma samples using the three methods show that the difference between any two methods or among the three methods presents 100% of samples with less than 20% for all the three amino acids and majority of them are under 10%. The developed two-step sample preparation method using methanol as protein precipitation reagent is simple and convenient. Three of the four methods are fully validated and the validation is successful. The BSA functioned effectively as a blank matrix for these three amino acids, considering cost, data quality, matrix similarity, and practicality

Metabolic and Molecular Regulation of Dietary Polyunsaturated Fatty Acids on Prostate Cancer

Purpose The aim of this study is to investigate the role of n-3 and n-9 fatty acids in crucial processes involved in prostate cancer cell growth through a large-scale proteomic analysis. Experimental design We used a label-free protein quantification method to profile global protein expression of fish oil and oleic acid treated PCa cells and validated a panel of differentially expressed proteins by either Western blot or multiple reaction monitoring. Bioinformatic analysis was also performed to uncover the pathways involved in fatty acid metabolism. Results Fish oil, not oleic acid, suppresses prostate cancer cell viability. Assessment of fatty acid synthesis pathway activity also shows that oleic acid is a more potent inhibitor than fish oil on de novo fatty acid synthesis. Although fatty acid synthase activity decreases with fish oil treatment, the inhibition of its activity occurs over time while reduction in viability occurs within 24 h. Bioinformatic analysis revealed the pathways altered by these fatty acid treatments. Conclusions and clinical relevance This study suggests that suppression of cell viability by fish oil is independent of fatty acid synthase and fish oil regulates prostate cancer cells through activation of other pathways depending upon length of exposure to fish oil

Delineation of Molecular Pathways Involved in Cardiomyopathies Caused by Troponin T Mutations

Familial hypertrophic cardiomyopathy (FHC) is associated with mild to severe cardiac problems and is the leading cause of sudden death in young people and athletes. Although the genetic basis for FHC is well-established, the molecular mechanisms that ultimately lead to cardiac dysfunction are not well understood. To obtain important insights into the molecular mechanism(s) involved in FHC, hearts from two FHC troponin T models (Ile79Asn [I79N] and Arg278Cys [R278C]) were investigated using label-free proteomics and metabolomics. Mutations in troponin T are the third most common cause of FHC, and the I79N mutation is associated with a high risk of sudden cardiac death. Most FHC-causing mutations, including I79N, increase the Ca(2+) sensitivity of the myofilament; however, the R278C mutation does not alter Ca(2+) sensitivity and is associated with a better prognosis than most FHC mutations. Out of more than 1200 identified proteins, 53 and 76 proteins were differentially expressed in I79N and R278C hearts, respectively, when compared with wild-type hearts. Interestingly, more than 400 proteins were differentially expressed when the I79N and R278C hearts were directly compared. The three major pathways affected in I79N hearts relative to R278C and wild-type hearts were the ubiquitin-proteasome system, antioxidant systems, and energy production pathways. Further investigation of the proteasome system using Western blotting and activity assays showed that proteasome dysfunction occurs in I79N hearts. Metabolomic results corroborate the proteomic data and suggest the glycolytic, citric acid, and electron transport chain pathways are important pathways that are altered in I79N hearts relative to R278C or wild-type hearts. Our findings suggest that impaired energy production and protein degradation dysfunction are important mechanisms in FHCs associated with poor prognosis and that cardiac hypertrophy is not likely needed for a switch from fatty acid to glucose metabolism

A microRNA signature in circulating exosomes is superior to exosomal glypican-1 levels for diagnosing pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is a deadly malignancy that often presents clinically at an advanced stage and that may be confused with chronic pancreatitis (CP). Conversely, CP may be misdiagnosed as PDAC leading to unwarranted pancreas resection. Therefore, early PDAC diagnosis and clear differentiation between PDAC and CP are crucial for improved care. Exosomes are circulating microvesicles whose components can serve as cancer biomarkers. We compared exosomal glypican-1 (GPC1) and microRNA levels in normal control subjects and in patients with PDAC and CP. We report that exosomal GPC1 is not diagnostic for PDAC, whereas high exosomal levels of microRNA-10b, (miR-10b), miR-21, miR-30c, and miR-181a and low miR-let7a readily differentiate PDAC from normal control and CP samples. By contrast with GPC1, elevated exosomal miR levels decreased to normal values within 24 h following PDAC resection. All 29 PDAC cases exhibited significantly elevated exosomal miR-10b and miR-30c levels, whereas 8 cases had normal or slightly increased CA 19-9 levels. Thus, our exosomal miR signature is superior to exosomal GPC1 or plasma CA 19-9 levels in establishing a diagnosis of PDAC and differentiating between PDAC and CP

Distinctive and pervasive alterations in aqueous humor protein composition following different types of glaucoma surgery

PURPOSE: To investigate whether specific glaucoma surgeries are associated with differences in aqueous humor protein concentrations compared to eyes without filters. METHODS: In this cross-sectional study, aqueous humor samples were prospectively collected from control subjects who underwent routine cataract surgery (n=14) and from patients who had different glaucoma filters: Baerveldt aqueous shunt (n=6), Ahmed aqueous shunt (n=6), trabeculectomy (n=5), and Ex-Press trabeculectomy (n=3). Total protein concentrations were determined with Bradford assay. Tryptic digests were analyzed with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Proteins were identified with high confidence using stringent criteria and were quantitatively compared with a label-free platform. Relative protein quantities were compared across groups with ANOVA. Post hoc pair-wise comparisons were adjusted for multiple comparisons. RESULTS: Compared to the control eyes, the aqueous humor protein concentration was increased approximately tenfold in the Ahmed and Baerveldt eyes and fivefold in the trabeculectomy and Ex-Press eyes. Overall, 718 unique proteins, splice variants, or isoforms were identified. No differences in the protein concentrations were detected between the Baerveldt and Ahmed groups. Likewise, the trabeculectomy and Ex-Press groups were remarkably similar. Therefore, the aqueous shunt groups were pooled, and the trabeculectomy groups were pooled for a three-way comparison with the controls. More than 500 proteins differed significantly in relative abundance (ANOVA p<0.01) among the control, aqueous shunt, and trabeculectomy groups. Functional analyses suggested these alterations in relative protein abundance affected dozens of signaling pathways. CONCLUSIONS: Different glaucoma surgical procedures were associated with marked increases in the aqueous humor protein concentration and distinctive changes in the relative abundance of numerous proteins involved in multiple signaling pathways

Comparative Proteomics and Biological Effects of Functionalized Carbon Nanotubes in Intestinal Cell Co-culture

poster abstract“Carbon nanotubes (CNTs) possess unique electrical, mechanical, and thermal properties, with potential applications in the electronics, catalysts, polymer composites, aerospace, and other industries. CNTs are also being developed for a broad range of applications in biomedicine, including oral drug delivery. Functionalized, water dispersible CNTs (fCNTS) can be expected to enter the digestive tract and exert biological effects on its barrier epithelial cells. To characterize these effects, we developed an in vitro model of the large intestinal tract using a coculture of Caco-2 (75%) and HT29-MTX (25%, mucus secreting) cells, and exposed these cells to functionalized single-walled (SWNT) and multi-walled (MWNT) carbon nanotubes at realistic concentrations (500 pg/mL and 10 µg/mL; 48 h). Protein expression was analyzed using our recently developed label-free quantitative mass spectrometry (LFQMS) platform, IdentiQuantXL™, while typical toxicological endpoint assays were used to characterize various cellular responses. LFQMS identified 5,007 unique protein database entries, from which 4,200 proteins were considered qualified for quantitation. These proteins represented 1,978 protein groups (containing isoforms, splice-variants, etc). Differences in expression were calculated by ANOVA (P<0.001) and post hoc Holm Sidak comparisons (P<0.05). fCNT significantly altered protein expression in a moderate number of proteins, the extent and type of which were fCNT specific. Only 13 proteins were universally altered by all exposures (except 500 pg/mL COOHSWNT which had no effect), and these represent a broad range of cellular functions. Bioinformatic analysis using the Gene Ontology Database and Ingenuity Pathway Analysis revealed statistically significant protein associations with a broad range of functional networks and signaling/metabolic pathways. Again, little overlap between fCNT was observed. None of the exposures was associated with overt toxicity or proinflammatory response. The results suggest that significant biological effects result from fCNT exposure, responses that are specific to CNT-type and dose, but occurring in the absence of toxicity or irritation. Supported by NIEHS RC2ES018810.

Proteomic profiling of halloysite clay nanotube exposure in intestinal cell co-culture

Halloysite is aluminosilicate clay with a hollow tubular structure with nanoscale internal and external diameters. Assessment of halloysite biocompatibility has gained importance in view of its potential application in oral drug delivery. To investigate the effect of halloysite nanotubes on an in vitro model of the large intestine, Caco-2/HT29-MTX cells in monolayer co-culture were exposed to nanotubes for toxicity tests and proteomic analysis. Results indicate that halloysite exhibits a high degree of biocompatibility characterized by an absence of cytotoxicity, in spite of elevated pro-inflammatory cytokine release. Exposure-specific changes in expression were observed among 4081 proteins analyzed. Bioinformatic analysis of differentially expressed protein profiles suggest that halloysite stimulates processes related to cell growth and proliferation, subtle responses to cell infection, irritation and injury, enhanced antioxidant capability, and an overall adaptive response to exposure. These potentially relevant functional effects warrant further investigation in in vivo models and suggest that chronic or bolus occupational exposure to halloysite nanotubes may have unintended outcomes