assessed by the total immobility time in the TST. Many of the differentially expressed proteins caused by 15N metabolic labeling have been previously reported in the literature to be related to the depression phenotype (Fig. 7.5). However, this finding does not imply that the 15N incorporation has antidepressant, therapeutic properties and one has to be careful to not over- interpret the antidepressant-like effects of the 15N isotope in HAB mice. 15N incorporation to an organism can affect protein architecture (Hartmann et al., 2003) and as a result, enzymatic reactions and pathways can be altered in an unpredictable manner. Yet, the 15N isotope effect provides a means for studying affected pathways involved in depression-like behavior.
On a second level of analysis, the 15N isotope effect was studied in E. coli cultures. E. coli is an organism of lower complexity compared to mice, used both as a model organism for quantitative proteomics experiments and as a stable isotope source to metabolically label higher organisms (Krijgsveld et al., 2003). Strikingly, more than 10% of the quantified proteins were found to be differentially expressed in the 14N and 15N E. coli cytoplasm, demonstrating that the introduction of the 15N isotope on a proteome alters protein expression, a fact that needs to be taken into consideration when planning and executing quantitative proteomics experiments using 15N metabolic labeling. The 18 differentially expressed proteins in 14N and 15N E. coli cytoplasms were primarily involved in pyruvate metabolism, energy production and amino acid biosynthesis. Pyruvate is the endpoint of glycolysis, a central metabolic cascade conserved in all organisms. Via gluconeogenesis, pyruvate can be converted to carbohydrates, whereas through its conversion to oxaloacetate pyruvate can enter the citric acid cycle, thus being a key metabolite for major metabolic cascades. Increased expression of enzymes involved in pyruvate metabolism (PFLB_ECOLI, Q47521_ECOLI, PPSA_ECOLI) as well as elevated pyruvate levels were found in 15
N cytoplasm. In addition, increased expression in 15N cytoplasm of beta galactosidase, an enzyme also involved in energy catabolism was demonstrated in 15N cytoplasm both by MS and Western blot analyses. Lactose catabolism by beta galactosidase leads to the production of galactose and glucose, the starting point of glycolysis. Apart from energy metabolism, alterations were observed in five proteins (AK2H_ECOLI, Q9F6G4_ECOLI, Q6LEL0_ECOLI, Q8RMX0_ECOLI, Q68QZ6_ECOLI, see Table 7.3) involved in aspartate amino acid family biosynthesis. Decreased expression in 15N E. coli cytoplasm was found for MukE (Q6ITT5_ECOLI) and CpsC (Q1PG46_ECOLI), proteins that participate in chromosome
segregation (Yamanaka et al., 1996) and transcription regulation (Bae et al., 2000), respectively. Enrichment analysis revealed that in the group of differentially expressed proteins phosphorylation-related processes were significantly overrepresented compared to all quantified proteins. Intriguingly, six of the differentially expressed proteins exhibit kinase activity (AK2H_ECOLI, PPSA_ECOLI, Q2EVI1_ECOLI, Q6LEL0_ECOLI, Q8RMX0_ECOLI, Q68QZ6_ECOLI, see Table 7.3), whereas PPSA_ECOLI has recently been reported to be phosphorylated (Macek et al., 2008). In conclusion, the introduction of the 15N isotope in E. coli
does not only affect essential metabolic pathways, such as glycolysis but also fundamental functions related to chromosome, transcription and post-translational modification regulation. In the literature, the 15N isotope effect has been considered to be trivial since a substitution of 15N for 14N only results in a relatively small mass difference (Van Langenhove, 1986). To assess the effect of the 15N isotope, Bigeleisen (Bigeleisen, 1949) estimated the maximum ratio of the 14N and 15N specific rate constants (K14N/K15N) and found that it was within the error limits of normal measurements, concluding that the 15N isotope effect on a reaction process is insignificant. On the other hand, it is known that a chemical bond involving a heavy isotope is stronger and therefore more difficult to break than the same bond involving the corresponding light isotope of the same element (Melander and Saunders, 1980). If the cleavage of this bond is the rate-limiting step of a reaction, then the reaction will proceed slower for the molecule with the heavy isotope. We propose that minor synergistic changes introduced by the substitution of 14N for 15N may account for slight structural conformational changes in a protein. When these alterations occur in an enzyme’s active site or in the peptide backbone of a rigid cluster, they could cause alterations in enzyme catalytic activity or distortions in spatial structure, respectively. As shown in our studies of HAB mice and E. coli, such alterations at the molecular level can affect biochemical reactions and pathways in an unpredictable manner. Our hypothesis is further supported by a study that compared 15N- or 13C-labeled to unlabeled Cu-thiolate cluster in yeast and found spectroscopic differences between the clusters, suggesting that stable isotope labeling can indeed affect the molecular architecture of a protein (Hartmann et al., 2003).
We show that the introduction of the 15N isotope affects evolutionary well-conserved pathways present in all organisms such as the glycolysis and the citric acid cycle as well as ubiquitous regulatory mechanisms such as phosphorylation. We have demonstrated the effect of the 15N incorporation at the proteome level both in low complexity organisms and mammals, where