Toxicoproteomic analysis of pulmonary carbon nanotube exposure using LC-MS/MS

Abstract

Toxicoproteomics is a developing field that utilizes global proteomic methodologies to investigate the physiological response as a result of adverse toxicant exposure. The aim of this study was to compare the protein secretion profile in lung bronchoalveolar lavage fluid (BALF) from mice exposed to non-functionalized multi-walled carbon nanotubes (U-MWCNTs) or MWCNTs functionalized by nanoscale Al2O3 coatings (A-MWCNT) formed using atomic layer deposition (ALD). Proteins were identified using liquid chromatography tandem mass spectrometry (LC-MS/MS), and quantified using a combination of two label-free proteomic methods: spectral counting and MS1 peak area analysis. On average 465 protein groups were identified per sample and proteins were first screened using spectral counting and the Fisher's exact test to determine differentially regulated species. Significant proteins by Fisher's exact test (p<0.05) were then verified by integrating the intensity under the extracted ion chromatogram from a single unique peptide for each protein across all runs. A two sample t-test based on integrated peak intensities discovered differences in 27 proteins for control versus U-MWCNT, 13 proteins for control versus A-MWCNT, and 2 proteins for U-MWCNT versus A-MWCNT. Finally, an in-vitro binding experiment was performed yielding 4 common proteins statistically different (p<0.05) for both the in-vitro and in-vivo study. Several of the proteins found to be significantly different between exposed and control groups are known to play a key role in inflammatory and immune response. A comparison between the in-vitro and in-vivo CNT exposure emphasized a true biological response to CNT exposure.

Keywords: Carbon nanotube; Label-free proteomics; Pulmonary fibrosis; Toxicoproteomics.

Fig. 1

Transmission electron photomicrographs of (A) non-functionalized MWCNTs and (B) ALD Al₂O₃-functionalized MWCNTs.

Fig. 2

General overview of sample preparation and data analysis. (A) Mouse exposure toMWCNTs, (B) lung lavage extraction, (C) tryptic digestion of lung lavage and SPE sample clean up, (D) LC-MS/MS, and (E) Label-free protein quantification by: (a) spectral counting, and (b) verification by MS1 peak area analysis of unique peptides.

Fig. 3

Volcano plots of log₂ spectral count fold change versus-log₁₀ p-value calculated by Fisher’s exact test for: (A) U-MWCNT/control, (B) A-MWCNT/control, and (C) U-MWCNT/A-MWCNT. Proteins plotted as a log2 fold change of 3 and -3 represent protein detection in exposed groups, but not control, and vice versa (respectively). Positive fold change in spectral count data represent up-regulation in exposed groups versus the control. Shaded areas highlight significance of p<0.05. Abundant proteins (i.e., human serum albumin) were included in calculations of the Fisher’s exact test, but not shown in plots. α = Pulmonary surfactant-associated protein-B. β =Myeloperoxidase. γ = Lactotransferrin.

Fig. 4

Peak area analysiswas conducted on proteins that were discovered to be significant using the Fisher’s Exact test on the spectral count data. (A) Chromatogram overlay used to assess changes in peak area by group. (B) Peak area was tested for significance across every group using the two-sample t-test: Control versus U-MWCNT (p = 7.02E- 08), Control versus A-MWCNT (p = 3.96E-05), and U-MWCNT versus A-MWCNT = 0.679). *Control peak area = 0, but imputed as a value of 10 to allow for log adjustment. Peaks were identified using a combination of MS2 identification (blue lines), mass measurement accuracy (<3 ppm), dot products of theoretical isotope abundance, and retention time reproducibility (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).

Fig. 5

GO analysis terms for (A) Biological Process, and (B) Molecular Function for significant proteins by peak area analysis both of the exposed groups versus the control. Significance (p<0.05) was calculated by a modified Fisher’s exact test (EASE score) and adjusted by the Benjamini correction and reported as the −log₁₀ Benjamini p-value. Significant GO terms are illustrated to the right of the dashed line (Huang da et al., 2009).