Quantitative mass spectrometry-based proteomics has become widely used for examining differences

in global expression level of proteins in various cellular states (Bantscheff et al., 2007; Elliott et al., 2009; Walther & Mann, 2010). In this method, proteins from samples obtained from different experimental conditions can be distinguished by incorporation of unique, stable isotopes with disparate masses in one of the samples. In this way, various samples can be combined and analyzed in a single LC-MS/MS analysis allowing estimation of the relative intensities of the peptides of interest from the labeled and unlabelled samples. Metabolic (Ong et al., 2002) and chemical (Boersema et al., 2009) labeling are two common procedures used for introducing heavy isotopes into cellular proteins. A pre-requisite for metabolic labeling of ATM signaling pathway proteins is that the cells efficiently take up a labeled substrate in culture and incorporate

it into proteins. However, this approach does not always result in a sufficient degree of labeling. Alternatively, as used in the present work, isotopic labeling can be performed by chemical labeling of peptides resulting from post-digestion of the cellular protein fractions. The green sulfur bacterium (GSB) Chlorobaculum (Cba.) tepidum is a strictly RO4929097 cost anaerobic, photosynthetic bacterium that lives in anaerobic aquatic environments, where reduced sulfur compounds, predominantly sulfide and light occur at the same time (Wahlund et al., 1991; Overmann, 2008). Chlorobaculum tepidum oxidizes sulfide, elemental sulfur, and thiosulfate for use as electron donor in its photosynthesis. The 2.15-Mbp genome of Cba. tepidum has been sequenced and revealed about 2245 protein-encoding genes (Eisen et al., 2002). Currently, 15 genome sequences of GSB have been determined (Gregersen et al., 2011). This information has allowed a detailed analysis of the sulfur metabolism of GSB, but many processes are still poorly described Bay 11-7085 (Frigaard & Bryant, 2004, 2008; Frigaard & Dahl, 2009; Sakurai

et al., 2010). Table 1 lists 57 enzymes putatively involved in the oxidative sulfur metabolism of Cba. tepidum, some of which have been functionally investigated. Figure 1 shows a simplified scheme of the pathways and enzymes of the oxidative sulfur metabolism of Cba. tepidum. Sulfide is oxidized by sulfide:quinone oxidoreductases (SQR; Chan et al., 2009); additional unknown enzyme activity contributes to sulfide oxidation (Holkenbrink et al., 2011). Thiosulfate is oxidized exclusively by the sulfur oxidation (SOX) enzyme system in the periplasm (Ogawa et al., 2008, 2010; Azai et al., 2009). Both of these processes give rise to a putative oligosulfide pool, which presumably is in equilibrium with an extracellular pool of sulfur globules that sometimes is referred to as ‘elemental sulfur’ (‘S0’). Oxidation of the oligosulfide pool is dependent on the dissimilatory sulfite reductase (DSR) enzyme system (Holkenbrink et al.