Follicular 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.
Thyroid follicular adenoma (FA) is a benign tumor of the thyroid gland where cells produce iodine‐containing hormones. FA can precede either follicular carcinoma or papillary carcinoma. To discover the molecular mechanism involved in follicular adenoma, diseased and matched normal adjacent tissue were analyzed by liquid chromatography tandem mass spectrometry (LC‐MS/MS). Using a label‐free quantitative mass spectrometry‐based proteomics approach, IdentiQuantXL, 1,799 unique, non‐redundant proteins were compared between FA and normal tissue samples. Among the 1,799 proteins, 15 proteins had significant changes (p ¡Ü 0.05) between the two groups. Using Ingenuity Pathways Analysis (IPA), their molecular and cellular functions were discovered, including cell‐to‐cell signaling and interaction, cell cycle, cell death and survival, cell morphology, and cellular assembly and organization. Among the 15 proteins, 6 proteins were cancer‐related. In conclusion, differential protein expression was observed, which may reveal the mechanism in the development of follicular adenoma.
<i>Background:</i> Meckel syndrome (MKS) is a fatal autosomal recessive condition with prominent renal cystic pathology. Renal protein misexpression was evaluated in the Wpk rat model of human MKS3 gene disease to identify biomarkers for the staging of renal cystic progression. <i>Methods:</i> Misexpressed proteins were compared between early and late stages of MKS renal cystic disease using proteomic analysis (two-dimensional gel electrophoresis with LC-MS/MS identification) followed by Western blot analysis. <i>Results:</i> A proteomic analysis identified 76 proteins with statistically different, normalized abundance in at least one group. Subsequently, Western blot was used to confirm differential expression in several of these and polycystic kidney disease (PKD)-associated proteins. Galectin-1 and vimentin were identified as overexpressed proteins, which have been previously found in the jck mouse model of nephronophthisis 9. Ciliopathic PKD proteins, polycystins 1 & 2, and fibrocystin were also differentially expressed in Wpk kidney. <i>Conclusion:</i> In the Wpk rat, misexpressed proteins were identified that were also implicated in other forms of cystic disease. Numerous proteins were either over- or underexpressed in late-stage disease. Differences in protein expression may serve as biomarkers of cystic disease and its progression.
The proportionately low abundance of membrane proteins hampers their proteomic analysis, especially for a quantitative LC-MS/MS approach. To overcome this limitation, a method was developed that consists of one cell disruption step in a hypotonic reagent using liquid nitrogen, one isolation step using a low speed centrifugation, and three wash steps using high speed centrifugation. Pellets contained plasma, nuclear, and mitochondrial membranes, including their integral, peripheral, and anchored membrane proteins. The reproducibility of this method was verified by protein assay of four separate experiments with a CV of 7.7%, and by comparative LC-MS/MS label-free quantification of individual proteins between two experiments with 99% of the quantified proteins having a CV ≤30%. Western blot and LC-MS/MS results of markers for cytoplasm, nucleus, mitochondria, and their membranes indicated that the enriched membrane fraction was highly pure by the absence of, or presence of trace amounts of, nonmembrane marker proteins. The average yield of membrane proteins was 237 μg/10 million HT29-MTX cells. LC-MS/MS analysis of the membrane-enriched sample resulted in the identification of 2597 protein groups. In summary, the developed method is reproducible, produces a highly pure membrane fraction, and generates a high yield of membrane proteins.
ADPKD is characterized by localized autonomous cellular proliferation, fluid accumulation within the cysts, and intraparenchymal fibrosis of the kidney. Little is known about the cyst fluid's protein composition. We hypothesized that the complex collection of cystic fluid proteins (cystic proteome) plabys a major role in cyst formation/maintenance and contains yet unknown diagnostic and mechanistic features that are common to all forms of PKD. We analyzed five kidney cyst fluids from four patients with ADPKD. Tryptic peptides from plasma‐protein immunodepleted (ProteoPrep ® ) and undepleted cyst fluid samples were analyzed by LC‐MS/MS. The proteins were identified by SEQUEST and validated via Trans‐Proteomic Pipeline. 391 proteins were identified with >90% confidence; 251 of them in undepleted and 362 in immunodepleted samples. Immunodepletion removed >98% of the cyst fluid protein. A surprisingly large and functionally diverse number of proteins common to all 5 cysts was identified. These proteins may be of mechanistic interest and include Ig γ κ and fragments; complement components; vitronectin; orosomucoid; prostaglandin D2 synthase; and vitamin D‐binding protein. Supported by AFOSR Grant FA9550‐06‐1‐0083.
Although numerous biomarkers or biomarker candidates have been discovered to detect levels of drinking and intervals of time after last drinking episode, only a few biomarkers have been applied to monitor abstinence in a longer interval (≥6 wks) from alcohol abuse. Considering sample sources, sensitivity, and specificity, new biomarkers from blood with better accuracy are needed. To address this, serum proteomic profiles were compared between pre- and post- treatment samples from subjects seeking treatment for alcohol abuse and dependence in an intensive 6 wk daily outpatient program using high-abundance plasma protein immunodepletion and LC-MS/MS techniques. Protein identification, quantification, candidate biomarker selection, and prioritization analyses were carried out. Among the 246 quantified serum proteins, abundance of 13 and 45 proteins in female and male subjects were significantly changed (p ≤ 0.05), respectively. Of these biomarker candidate proteins, 2 (female) and 8 (male) proteins were listed in category 1, with high area under the receiver operating characteristic curve, sensitivity, specificity, and fold change. In summary, several new biomarker candidates have been identified to monitor abstinence from alcohol abuse.
Fuchs endothelial corneal dystrophy (FECD) is a progressive disorder characterized by corneal endothelial decompensation leading to corneal edema, clouding, and vision impairment. Despite improved understanding over the last century since its first description, the exact mechanism(s) behind the pathogenesis of FECD remain unknown, and surgical correction is the only effective treatment available. Previous studies have suggested a role for changes in aqueous humor (AH) composition in FECD pathogenesis, so to explore this possibility, we probed the AH proteome for alterations correlating with end-stage corneal disease. Following albumin depletion we performed label-free quantitative tandem mass spectrometry on proteins isolated from patients with and without FECD who were scheduled to undergo routine cataract extraction. We identified 64 proteins, most of which were identified in previous AH proteomic studies of patients with cataracts, in the albumin-depleted fraction. The levels of five of these were significantly lower (afamin, complement C3, histidine-rich glycoprotein, immunoglobulin heavy [IgH], and protein family with sequence similarity 3, member C [FAM3C]), while the levels of one (suprabasin) was significantly higher in patients with FECD compared to controls (p≤0.01). We also identified 34 proteins in the albumin-bound fraction, four of which were significantly elevated in patients with FECD including a hemoglobin fragment, immunoglobulin kappa (IgK), immunoglobulin lambda (IgL), and uncharacterized protein albumin (ALB), (p≤0.01). Although it has been reported that females have a greater extent of disease than males, we were unable to detect any significant differences in protein levels due to gender. Because FECD is a progressive disorder, regression analyses were performed to determine any significant correlations with age, and of interest retinol-binding protein 3 was significantly correlated with age in patients with FECD (p≤0.01), whereas no proteins in the control group correlated with age. This is the first report indicating alterations in the AH proteome with FECD, and taken together this study suggests several novel hypotheses regarding AH proteins role in FECD pathogenesis.
