We describe a synthesis strategy for the preparation of lysine isotopologues that differ in mass by as little as 6 mDa. We demonstrate that incorporation of these molecules into the proteomes of actively growing cells does not affect cellular proliferation, and we discuss how to use the embedded mass signatures (neutron encoding (NeuCode)) for multiplexed proteome quantification by means of high-resolution mass spectrometry. NeuCode SILAC amalgamates the quantitative accuracy of SILAC with the multiplexing of isobaric tags and, in doing so, offers up new opportunities for biological investigation. Large-scale technologies for the comparative analysis of proteomes have become essential for modern biology and medicine (1, 2). To satisfy this increasing demand and boost statistical power, parallel processing of proteomes (i.e. multiplexing) is key. The ground was broken in this field about two decades ago by advances in both MS and stable isotope labeling (3-5). Since then, two distinct strategies have emerged, each with its own strengths and weaknesses. The first approach, stable isotope labeling by amino acids in cell culture (SILAC), 1 metabolically incorporates labeled amino acids into proteins and is considered the gold standard (6 -8). SILAC quantification provides unmatched accuracy, but simultaneous comparison of more than three proteomes, although possible, is not practical for most global studies (9, 10). A second, and increasingly popular, method is to chemically modify peptides originating from up to 10 different sources (a 3-to 5-fold boost in throughput over SILAC) with isobaric reagents (e.g. TMT or iTRAQ) (11)(12)(13)(14). The escalated throughput afforded by this strategy is, for many applications, essential; however, multiplexing via isobaric tagging comes at the cost of quantitative accuracy (15-17). Furthermore, because each sample is handled independently prior to labeling, systematic and random variation that occurs during sample processing cannot be accounted for as it is with metabolic labeling. Thus, experimenters designing a quantitative proteomics experiment must choose between accuracy and throughput.Recently we described a new approach that blends the SILAC and isobaric tagging methods (18). The strategy, neutron encoding (NeuCode), relies on the mass defects of atoms and their isotopes (19). In studies using two isotopologues of lysine, differing by 36 mDa, NeuCode SILAC quantified proteins as well as traditional SILAC, but it allowed deeper proteome coverage. NeuCode harnesses the exceptional resolving power of modern FT-MS systems so that quantitative information is only revealed by high-resolution scanning when desired, in either MS or tandem MS scans (20,21).Owing to the lack of suitable lysine isotopologues, our initial work with NeuCode SILAC offered only duplex quantification; consequently, we could only predict the utility of NeuCode SILAC (18). To test our supposition that NeuCode SILAC has the potential to combine the benefits of traditional SILAC and
One of the most important steps in the numerical simulation of a hydrogeological system is the precise definition of initial and boundary conditions. The better these are characterized, the more efficient the calculation and the more accurate are the simulation result. In case of simulating processes in the unsaturated soil zone, the water retention curve, the relationship between volumetric water content and matric potential, is of great importance. However, the retention parameters determined locally by different standard methods often do not represent the whole soil system under consideration due to heterogeneities in the soil body caused by variability or different compaction of the soil. Resulting over- or underestimation of the parameters is leading to a worse performance of simulations of the water balance including to a higher calibration effort. Therefore, it is more favorable to identify these soil parameters by a method representing the whole soil system to avoid uncertainties. For this reason, a dike experiment was performed to investigate how soil parameters determined locally and globally can represent the properties of the whole soil system. When comparing the simulation results of the numerical models, a better agreement of measured and simulated water contents as well as a lower effort for calibration is observed by using the soil parameters determined globally.
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