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Open Access Research article

Proteomic analysis of the marine diatom Thalassiosira pseudonana upon exposure to benzo(a)pyrene

Raquel N Carvalho and Teresa Lettieri*

Author Affiliations

European Commission - Joint Research Centre, Institute for Environment and Sustainability, Rural, Water, and Ecosystem Resources Unit, T.P. 270, Via E. Fermi 2749, 21027 Ispra (VA), Italy

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BMC Genomics 2011, 12:159  doi:10.1186/1471-2164-12-159

Published: 24 March 2011

Additional files

Additional file 1:

Workflow for the identification of protein changes induced by BaP treatment in T. pseudonana. Diatom cells were inoculated into fresh medium, cultured for 24 h and exposed to BaP (36 μg/L) or just the solvent (control) for 24 h, in three biological replicates. Following cell lysis, 60 μg of protein were alkylated, reduced and digested with trypsin. Peptides were then labeled with iTRAQ reagents and pooled as shown. Strong cation exchange was then used to remove free iTRAQ reagent and to fractionate peptides for subsequent separation and peptide analysis by LC-MS/MS. MS/MS data was analyzed using Mascot and ProteinPilot software.

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Additional file 2:

Silver staining of protein extract and tryptic digest run on SDS-PAGE. The figure shows the similar amount of proteins run for three biological replicates. Control protein extracts, labeled with iTRAQ 113, 115 and 119 were loaded onto lanes 1, 5 and 9, respectively, while BaP-exposed protein extracts, labeled with iTRAQ 114, 116 and 121 were loaded onto lanes 3, 7 and 11, respectively. Tryptic digested products were loaded on the neighboring lanes to the right of the corresponding protein extracts, and show the protein digestion was complete under the used conditions. Molecular weight markers were loaded onto lanes labeled with M.

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Additional file 3:

Protein identification in T. Pseudonana. The file shows Table S1 listing all the proteins identified by MS/MS from the T. pseudonana extract with a minimum of 2 peptides at >95% confidence.

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Additional file 4:

Peptide fragmentation spectra used for the identification and relative quantification of proteins that have been included in Table 1as potentially regulated. The file contains the fragmentation spectra of the peptide fragment identified with high confidence (> 95%) as well as the fragmentation spectra of other peptides identified with lower confidence, for six different proteins (Supplemental Figures S1-S6). The reporter ion signals shown in the low-mass region of the spectra were used to determine the relative amount of the protein in T. pseudonana exposed to BaP (iTRAQ 114, 116 and 121) when compared to control conditions (iTRAQ 113, 115 and 119).

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Additional file 5:

Principle component analysis for the quantification of the iTRAQ labels in the different experiments. The analysis shows a clustering of the proteome analysis from control diatom cultures, labeled with iTRAQ 113, 115 and 119, as well as a clustering of the proteome analysis from the BaP-exposed conditions, labeled with iTRAQ 114, 116 and 121. An additional experiment has been incorporated in the MS/MS analysis, consisting of control and BaP-exposed conditions, labeled with iTRAQ 117 and 118, respectively, but were not considered for the overall quantification analysis. In this experiment, the same starting density of 0.5 million cells/mL was used, but from a culture in the resting phase. We observed that the growth in the diatom cultures was largely reduced from the expected values, and was also observed after the addition of solvent or BaP. We decided to use the cells arising from this experiment with the remaining iTRAQ labels from the 8-plex kit, to see whether under these abnormal cell culture circumstances, we could still observe significant differences between control and exposure conditions. It is evident that both conditions in this experiment sit as outliers on the graph and in addition, they seem to be clustered together. In fact, from the quantification analysis, it was observed that both 117 and 118 labels showed rather consistent values for most of the peptides analyzed, and not in agreement with the experimental conditions, i.e. control or BaP-exposed. It is interesting to note that the reduced cell growth in this culture, greatly affected the outcome of the quantification. Thus, the apparent sensitivity of these cells seemed to be more relevant than the exposure conditions themselves. These findings, although not unexpected, truly support the importance of reproducibility in biomarker research, since any differences in the initial state of the cells has a substantial effect on the biomolecular pool and thus masks any potential differences of expression, either at gene or protein level.

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