The prediction of the retention behavior/time would facilitate the identification and characterization of glycoproteins, particularly the analytical challenges, such as the characterization of low-abundance glycoforms. This task is essential in the biotherapeutics industry, where the type and amount of glycosylation on recombinant IgG alter the efficacy, function, and immunogenicity. Models exist for the prediction of the hydrophilic interaction liquid chromatography retention of peptides and glycans. Here, we have devised a unified model to predict the retention behavior of glycopeptides from human IgGs and applied this to the analysis of glycopeptides from rabbit IgGs. The combined model is capable of accurately predicting the retention of native IgG glycopeptides on 2 completely different liquid chromatography-mass spectrometry systems.

Nitrogen sources are all converted into ammonium/ia as a first step of assimilation. It is reasonable to expect that molecular components involved in the transport of ammonium/ ia across biological membranes connect with the regulation of both nitrogen and central metabolism. We applied both genetic (i.e., Δamt mutation) and environmental treatments to a target biological system, the cyanobacterium Anabaena sp PCC 7120. The aim was to both perturb nitrogen metabolism and induce multiple inner nitrogen states, respectively, followed by targeted quantification of key proteins, metabolites and enzyme activities. The absence of AMT transporters triggered a substantial whole-system response, affecting enzyme activities and quantity of proteins and metabolites, spanning nitrogen and carbon metabolisms. Moreover, the Δamt strain displayed a molecular fingerprint indicating nitrogen deficiency even under nitrogen replete conditions. Contrasting with such dynamic adaptations was the striking near-complete lack of an externally measurable altered phenotype. We conclude that this species evolved a highly robust and adaptable molecular network to maintain homeostasis, resulting in substantial internal but minimal external perturbations. This analysis provides evidence for a potential role of AMT transporters in the regulatory/signalling network of nitrogen metabolism and the existence of a novel fourth regulatory mechanism controlling glutamine synthetase activity.

Nitrogen is a key macro-nutrient required for the metabolism and growth of biological systems. Although multiple nitrogen sources can serve this purpose, they are all converted into ammonium/ammonia as a first step of assimilation. It is thus reasonable to expect that molecular parts involved in the transport of ammonium/ammonia across biological membranes (i.e. catalysed by AMT transporters) connect with the regulation of both nitrogen and central carbon metabolism. In order to test this hypothesis, we applied both (1) genetic (i.e. Δamt mutation) and (2) environmental treatments to a target biological system, the cyanobacterium Anabaena sp. PCC 7120. Cyanobacteria have a key role in the global nitrogen cycle and thus represent a useful model system. The aim was to both (1) perturb sensing and low-affinity uptake of ammonium/ammonia and (2) induce multiple inner N states, followed by targeted quantification of key proteins, metabolites and enzyme activities, with experiments intentionally designed over a longer time-scale than the available studies in literature. We observed that the absence of AMT transporters triggered a substantial response at a whole-system level, affecting enzyme activities and the quantity of both proteins and metabolites, spanning both N and C metabolism. Moreover, the absence of AMT transporters left a molecular fingerprint indicating N-deficiency even under N replete conditions (i.e. greater GS activity, lower 2-OG content and faster nitrogenase activation upon N deprivation). Contrasting with all of the above dynamic adaptations was the striking near-complete lack of any externally measurable phenotype (i.e. growth, photosynthesis, pigments, metabolites). We thus conclude that this species evolved a highly robust and adaptable molecular network to maintain homeostasis, resulting in substantial internal but minimal external perturbations. The analytical data highlights several internal adaptations, including increased N assimilation (i.e. greater GS activity) and nitrogenase activity (i.e. faster activation upon N deprivation) together with altered amino acids metabolism, as indicated by changes in Gln, Glu and 2-OG, indicating an altered C/N balance. The analyses provides evidence for an active role of AMT transporters in the regulatory/signalling network of N metabolism in this biological system, and the existence of a novel fourth IF7A-independent regulatory mechanism controlling GS activity.