Journal of Proteome Research Statistical analysis was carried out for all three strains grown planktonically and as a bioﬁlm using Principal Component Analysis (PCA), using the XLSTAT software (http://www. xlstat.com/). Each biological sample was analyzed 5 times to assess technical variations. X-ray Photoelectron Spectroscopy (XPS) Analysis XPS analysis was performed as described elsewhere.23 Sulfolobus cells grown planktonically or as a bioﬁlm, were washed twice by centrifugation at 5000 g for 10 min with demineralised water. The pellets were resuspended in water, freeze-dried under sterile conditions and then mounted onto glass coverslips. The samples with the coverslips were mounted on standard sample studs (sample holder) for analysis. The XPS measurements were carried out on a KRATOS AXIS 165 Ultra Photoelectron spectrometer at 10 kV and 20 mA using the Al KR X-ray source (1486.6 eV). The takeoﬀ angle was adjusted at 90 and data was collected for each sample at three random selected locations (technical replicates). The area corresponding to each acquisi- tion was 400 μm in diameter. A survey scan was initially carried out (pass energy 20 eV, 1.0 eV step size) for C, O and N, followed by a high resolution scan (pass energy 20 eV, 0.1 eV step size) for C and O. Deconvolution of the high resolution scan enables the local chemical bond environments between C and O to be investigated. The binding energies of the peaks were determined using the C1s peak at 284.5 eV. CasaXPS 2.3.12 software was used to carry out the spectral integration.23 Elemental composi- tion was expressed as atomic concentration. All measurements were conducted in triplicate. Protein Extraction and iTRAQ Labeling Independent biological duplicate cells from three diﬀerent species harvested as described above were washed twice with cold water before being resuspended in 0.5 M TEAB iTRAQ buﬀer containing 0.1% SDS. Cells were then lysed using ultra- soniﬁcation (Soniﬁer 450, Branson) 4 times (alternatively 1 min on ice) at 70% duty cycle, as described in detail elsewhere24 before centrifugation ﬁrst at 3000 g for 5 min at 4 C and then at 21,000g at 4 C for 15 min. Supernatants were subsequently collected as extracted proteins. Total protein concentrations were determined using the RC-DC protein quantiﬁcation assay (Bio-Rad, U.K.). 100 μg protein (for each phenotype) was used for iTRAQ 4-plex analysis. Proteins were reduced, alkylated, digested and labeled with iTRAQ reagents as described else- where.24 Biological duplicates were used for all three Sulfolobus species, and for each phenotype (bioﬁlm and planktonic). The iTRAQ labeling of all samples is shown in Table S2 (Supporting Information). Labeled peptides (for each species) were subsequently combined before being dried in a vacuum concentrator. Labeled Peptides Separation, Mass Spectrometry and Data Analyses All dried labeled peptide samples were cleaned and fractio- nated using strong cation exchange on a BioLC HPLC system (Dionex, UK) as detailed elsewhere.24 Labeled iTRAQ peptides were collected every minute, and then dried under vacuum. Selected dried labeled peptides were resuspended in 50 μL of buﬀer A containing 0.1% formic acid and 3% acetonitrile before submission to a QStar XL Hybrid ESI Quadrupole time-of-ﬂight tandem mass spectrometer, ESI-qQ-TOF MS/MS (Applied Biosystems/MDS Sciex, Canada), coupled with a nano-LC system (LC Packings Ultimate 3000, Dionex, U.K.). Mass ARTICLE spectrometry was performed as described previously.24 The sample was separated on a PepMap C-18 revered phase capillary column (LC Packings) at a ﬂow rate of 3 μL/min, and a gradient was generated by variation of the percentage of buﬀer B (0.1% formic acid and 97% acetonitrile). The mass detector range was set to 350 to 1800 m/z and operated in the positive ion mode. Peptides with +2, +3, and +4 charge states were selected for fragmentation. All raw mass spectrometry data were converted into MGF format using the mascot.dll script (V1.6) coupled with Analyst QS 2.0 (Applied Biosystems), before submission to an in-house search algorithm Phenyx V 2.6 (Genebio, Geneva) (see Pham et al.,25 for more detail) with the Sulfolobus solfataricus P2, Sulfolobus acidocaldarius, Sulfolobus tokodaii databases down- loaded from NCBI (http://www.ncbi.nlm.nih.gov/Ftp/) in Jan 2009. The search parameters were set as follows: peptide tolerance 0.4 Da, charge +2,+3 and +4, min peptide length, z-score, max p-value and AC score were 5, 5.5, 10 6 and 5.5 respectively, and enzymes used for searching were trypsin, with up to two missed cleavages. Modiﬁcations were analyzed as follows: 4-plex iTRAQ mass shifts (+144 Da, K and N-term), methylthiol (+46 Da) and oxidation of methionine (+16 Da). Data were then exported to Excel (Microsoft 2008, Redmond, WA) for further analysis. Furthermore, the false positive rates (FPR) were also performed by searching data with the reversed databases of these Sulfolobus spp., and calculated as the following equation: FPR = (2 decoy_hits * 100%)/(decoy_hits + true_hits). The quantiﬁcation of identiﬁed proteins was performed based on methods described previously by Pham, et al. (2010), and a rigorous statistical method, including multiple test correction, was also applied to choose signiﬁcantly regulated proteins for biological discussions (see also Pham, et al.25 for full details of methods). First, protein quantiﬁcation was calculated by the geometric means of the ion reporters’ intensities (at least 2 peptides for each protein) before a t test comparison (with R = 0.05) between the reporter ions’ intensities was performed, followed by a Bonferroni correction. Furthermore, proteins were considered as regulated ones when all t tests of these proteins were less than a value of R/P (Onferroni corection) (P: number of quantiﬁed proteins). RNA-Isolation The cell pellet was resuspended in 1 mL ice-cold AE-buﬀer (20 mM sodium-acetate, pH 5.5; 1 mM EDTA) and then centrifuged for 5 min at 4 C. The pellet was resuspended in 600 μL AE-buﬀer. Subsequently, 900 μL of hot phenol-chloro- form-isoamyl alcohol (25:24:1, 60 C) and 10 μL 25% SDS (w/v) were added. The suspension was incubated for 15 min at 60 C and every 2 3 min the tube was inverted. The tube was incubated for 20 30 min on ice and then centrifuged at 15 700 g for 30 min at 4 C. In a new phase lock tube (Phase Lock Gel Light 2 mL, 5 PRIME, Hamburg), one volume of phenol-chloroform-isoamyl alcohol and 62.5 μL 2 M sodium- acetate were added to the supernatant. After centrifugation 15 700 g for 15 min at 4 C the supernatant was transferred in a new 2 mL tube, 2.5 volumes of 96% ethanol was added and incubated for 1 2 days at 80 C. The samples were thawed on ice and centrifuged for 30 min at 4 C. After washing the pellet twice with 500 μL of 70% ethanol, the pellet was air-dried at room temperature. Finally the pellet was resuspended in 100 μL RNase free water (Qiagen, Hilden). The RNA isolation was 4107 dx.doi.org/10.1021/pr2003006 |J. Proteome Res. 2011, 10, 4105–4119