, 2007; Pandhal et al., 2007). iTRAQ was chosen as this technique has a clear advantage over more conventional proteomic methods through conferring reproducibility and statistical confidence to the measurements Oligomycin A in vivo of protein abundance
within a cell at a fixed point in time (Ross et al., 2004; Gan et al., 2007). For a complete description of the Materials and methods refer to the Supporting Information Appendix S1; in brief, however, P. marinus strain MED4 was grown in biological triplicate under two separate conditions: P-deplete and P-replete PCR-S11 media. The cells were harvested at the same time in the late exponential phase (after 10 days), and proteins were extracted (Meijer & Wijffels, 1998). Approximately 100 μg of protein from each replicate was then reduced, alkylated, digested and labelled with 8-plex iTRAQ reagents according to the manufacturer’s (Applied Biosystems, Framingham, MA) protocol. The replicates were then pooled before primary strong cation exchange (SCX)
fractionation (Pandhal et al., 2007). Mass spectrometeric analysis of the SCX fractions was performed using both a HCTUltra ESI TRAP MS/MS (Bruker Daltonics GmbH, Bremen, Germany) and a QStar XL Hybrid ESI Quadrupole time-of-flight tandem mass spectrometer, ESI-qQ-TOF-MS/MS (Applied Biosystems; MDS-Sciex, Concord, Ontario, Canada), coupled with an online capillary liquid chromatography system (Ultimate 3000, Dionex/LC Packings, the Netherlands) (Pandhal et al., 2007). Preliminary data analysis, peptide identification and quantification were carried out using the phenyx [Geneva Bioinformatics (GeneBio), Geneva, Switzerland] software. Ninety-eight proteins PI3K Inhibitor Library datasheet were identified by ≥1 peptides [coefficient of variation (CV)=1.07] and eight false positives were identified [false positive rate (FPR)=0.016]. However, for accurate determination of protein identification, ≥2 peptides are required. With this restriction, 68 proteins were identified (CV=1.05), with three false positives (FPR=0.05), with quantification only possible for 62 of the identified proteins. For a full list
of identified proteins, see Supporting Information, Table S1. This figure, while lower than other iTRAQ experimental Etofibrate data of other cyanobacteria, such as Synechocystis sp. PCC6803 (Gan et al., 2007) and Nostoc sp. (Ow et al., 2009a), shows a broad coverage across the chromosome for MED4 (Fig. 1). It is also similar to the only other iTRAQ shotgun proteomic experiment conducted on MED4, where 70 proteins were identified by ≥2 peptides (Pandhal et al., 2007). Also, there was a significant bias towards identification of particular proteins within the results, where 75% of the peptides identified only mapped to 19% of the identified proteins (Table S1). This strongly suggests that the cell’s proteome, particularly under P-stressed conditions, is dominated by a small number of these particular proteins.