Journal of Proteome Research Figure 1. Overlay of normalized spectra FTIR data of (A) S. acidocal- darius, (B) S. solfataricus and (C) S. tokodaii grown either as bioﬁlm (solid line) or planktonically (dotted line). Spectra are baseline cor- rected and normalized to 2930 cm 1. (spectral region: 900 to 1200 cm 1) in bioﬁlm cells of both S. acidocaldarius (Figure 1A) and S. solfataricus (Figure 1B) strains in comparison to their planktonic counterparts. However, despite the same normalization across all strains, the signiﬁcant increase in absorption band intensity in carbohydrate region of S. tokodaii bioﬁlm cells compared to planktonic cells was not found (Figure 1C). Moreover, additional speciﬁc banding assign- ment for chemical function groups was diﬃcult in this region (1200 and 900 cm 1), as it is made up of vibrations correspond- ing to the stretching of diverse polysaccharides groups.23 Additionally, XPS analysis was performed to further describe the cell surface chemical composition in all three Sulfolobus spp. ARTICLE XPS analysis allows for the quantiﬁcation of the elemental surface composition and to assess the local chemical environment of carbon, oxygen and nitrogen atoms on the cell surface. XPS wide scan data (see Figure S2 and Table S1 in the Supporting Information) showed that the cell surface (approximately 1 10 nm in depth) was mainly comprised of C, N, O. The abundance of C, N, O was therefore estimated for each Sulfolobus strains and in comparison between bioﬁlm-associated cells and planktonic cells samples. N/C and O/C atomic con- centration ratios indicate the fraction of carbon linked to either nitrogen or oxygen atoms on the cell surface, respectively (Table 1). The results indicated an excess of O/C linkages on the cell surface of all three strains (Table 1). Furthermore, when compared to planktonic cell samples, an increase in the O/C ratios was determined for bioﬁlm-associated cells of S. acidocaldarius and S. solfataricus. The opposite was found in the S. tokodaii bioﬁlm cell surface (Table 1). Since polysaccharides predominantly contain O/C linkages in their structures, this ratio might be attributed to an increase in polysaccharide moieties. Morever, O/C ratios were found to be higher than N/C ratios, indicating that O/C ratios might arise from polysaccharide moieties rather than from the amide linkages (C-NHCO-C) on the protein moieties (Table 1), as a 1:1 ratio (approx.) between O/C and N/C values is expected for a proteinaceous cell surface. Thus, the XPS analysis correlated with the FT-IR spectra in that a statistically signiﬁcant increase was determined in the polysaccharide moieties on the S. acidocaldarius and S. solfataricus bioﬁlm cell surfaces. The opposite trend was determined in the S. tokodaii bioﬁlm cell surface (Table 1). Using both FTIR and XPS, diﬀerences between bioﬁlm and planktonic modes of growth in all three Sulfolobus species were noted, which is most likely due to changes in the carbohydrate composition. Comparative Proteomic Analysis of Biofilm versus Plank- tonic Cells Taking the spectroscopic evidence that Sulfolobus bioﬁlm population shows distinctive features in comparison to the free-living cells, we further assessed the impact of this mode of growth on the proteome of Sulfolobus species. Total protein extracts of S. acidocaldarius, S. solfataricus and S. tokodaii from bioﬁlm-associated and planktonic populations were compara- tively analyzed using iTRAQ. Planktonic and bioﬁlm cell samples of each strain at the same time (2 days of growth) were used in the proteomic experiments. Using the Phenyx program for searching within correlated databases, 11063, 10122, and 11419 peptides corresponding to 481, 463, and 542 quantiﬁed proteins (g2 peptides) were identiﬁed for S. solfataricus, S. acidocaldarius, and S. tokodaii, respectively (see sheet 1 for details of peptides lists and sheet 2 for details of quantiﬁed proteins lists in pro- teomics Supporting Information section, Excel ﬁles 1, 2, and 3). Furthermore, false positive rates of 0.25%, 0.18% and 0.31% were also estimated for S. solfataricus, S. acidocaldarius, and S. tokodaii, respectively, as described in Experimental procedures section. The numbers of all signiﬁcantly regulated proteins are summar- ized in Table S3 (Supporting Information section). Since two biological replicates for each condition (bioﬁlm (iTRAQ labels 116 and 117) and planktonic (iTRAQ labels 114 and 115) for each Sulfolobus strain) were carried out, four t tests were calculated for each Sulfolobus species. To pick up regulated proteins, we required all t test values of these proteins to be less than a value of R/P (Bonferroni correction) (where R = 0.05 and 4109 dx.doi.org/10.1021/pr2003006 |J. Proteome Res. 2011, 10, 4105–4119