Statistically enhanced spectral counting approach to TCDD cardiac toxicity in the adult zebrafish heart

Abstract

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a persistent environmental pollutant and teratogen that produces cardiac toxicity in the developing zebrafish. Here we adopted a label free quantitative proteomic approach based on normalized spectral abundance factor (NSAF) to investigate the disturbance of the cardiac proteome induced by TCDD in the adult zebrafish heart. The protein expression level changes between heart samples from TCDD-treated and control zebrafish were systematically evaluated by a large scale MudPIT analysis, which incorporated triplicate analyses for both control and TCDD-exposed heart proteomic samples to overcome the data-dependent variation in shotgun proteomic experiments and obtain a statistically significant protein data set with improved quantification confidence. A total of 519 and 443 proteins were identified in hearts collected from control and TCDD-treated zebrafish, respectively, among which 106 proteins showed statistically significant expression changes. After correcting for the experimental variation between replicate analyses by statistical evaluation, 55 proteins exhibited NSAF ratios above 2 and 43 proteins displayed NSAF ratios smaller than 0.5, with statistical significance by t test (p < 0.05). The proteins identified as altered by TCDD encompass a wide range of biological functions including calcium handling, myocardium cell architecture, energy production and metabolism, mitochondrial homeostasis, and stress response. Collectively, our results indicate that TCDD exposure alters the adult zebrafish heart in a way that could result in cardiac hypertrophy and heart failure and suggests a potential mechanism for the diastolic dysfunction observed in TCDD-exposed embryos.

Figure 1

Workflow of label free quantitative proteomic approach to TCDD cardiac toxicity in adult zebrafish heart. Adult zebrafish (3 month old) were treated in triplicates each by DMSO and TCDD, respectively. Proteins were extracted and digested following the same sample preparation protocol. The peptide samples were analyzed by parallel shotgun proteomic approach. The protein expression was analyzed by spectral count method supplemented by statistical test for significant protein expression change.

Figure 2

Overall protein expression results by spectral count combined with statistical evaluation. 106 proteins were identified to be significantly changed by TCDD treatment.

Figure 3

Experimental reproducibility and biological variability by quantitative proteomic spectral count method. (a) Spectral counts from two replicate analyses of TCDD treated heart samples show good experimental reproducibility of SpC method; (b) Spectral counts from parallel DMSO and TCDD samples revealed biological variability between two distinct samples, corresponding to potential protein differential expression induced by TCDD treatment.

Figure 4

Significantly changed protein expressions by spectral count and statistical analysis. The two blue lines indicated 2 fold up- and down-regulation of proteins in TCDD treated samples. Dark dots represent proteins with statistically significant expression changes. Proteins are labeled by their encoding gene names.

Figure 5

Representative proteins with significant SpC changes. (a)–(d) Representative proteins from significantly changed functional categories.

Figure 6

Gene ontology annotation of significantly changed proteins detected by spectral count method. Significantly changed proteins were analyzed for their (a) biological function and (b) cellular localization by the program GoMiner. The significantly enriched GO categories were listed to show comparison between TCDD and DMSO samples.