Isotope Labeling In Combination Research Articles

Approaches for the quantification of protein concentration ratios.

The function of a protein is modulated by its abundance and its degree of specific post-translational modifications such as phosphorylation, glycosylation or truncation. Consequently, changes of protein concentration and the extent of their post-translational modifications has a great influence on the activity of intracellular substrate degradation processes, on the activity of intracellular biosynthetic pathways, on the cell cycle or on the function of a single cell in a whole organism. Defects in this area lead to diseases like cancer or neurodegeneration. Therefore, it is a challenge to quantify changes within the proteome in the diseased state or between developmental stages and to use the results obtained for the maximization of product yields in biotechnology or for the development of new drug targets to fight against diseases. In order to determine the intracellular concentration of a protein it is necessary to spike the cell sample with the same protein in a pure form. If the concentration changes of many proteins have to be determined, it takes a long time to obtain all these proteins in a pure form. Therefore, most approaches in this field are restricted to the determination of protein abundance ratios between two states such as diseased or healthy tissues. In this case the proteins in the sample of state A function as an internal standard for the proteins in the sample of state B and vice versa. The most common techniques in this field are the comparison of two-dimensional gel spot intensities after staining or the integration of mass spectrometric peak intensities after stable isotope labelling with (13)C, (15)N, (18)O or deuterium. The results, advantages and drawbacks of these approaches are discussed. Stable isotope labelling in combination with mass spectrometry is more accurate than the comparison of spot intensities and has the potential for the investigation of highly complex tissue samples.

Incorporation of 15N from ammonium into the N-linked oligosaccharides of an immunoadhesin glycoprotein expressed in Chinese hamster ovary cells

Elevated ammonium concentrations in the medium of cultivated cells have been shown to increase the intracellular levels of uridine-5'-diphospho-N-acetylglucosamine (UDP-GlcNAc) and uridine-5'-diphospho-N-acetylgalactosamine (UDP-GalNAc; Ryll et al., 1994). These sugar nucleotides are substrates for glycosyltransferases in the glycosylation pathway. In our experiments, recombinant Chinese hamster ovary cells producing an immunoadhesin glycoprotein (GP1-IgG) have been cultivated under controlled cell culture conditions in the presence of different ammonium concentrations.15N-Labeled ammonium chloride (15NH4Cl) was added exogenously to the cell culture media to determine if ammonium was incorporated into UDP-GlcNAc and cytidine-5'-monophospho-N-acetylneuraminic acid (CMP-NANA) pools, and subsequently incorporated into GP1-IgG as N-linked glycans. The intracellular pools of UDP-activated hexosamines (UDP-GNAc) were followed during the time course of the experiment. To assess the extent of15NH4+incorporation into the glycans of GP1-IgG, the glycoprotein was first purified to homogeneity by protein A chromatography. Enzymatically released N-glycans were then analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. N-Glycans synthesized in the presence of15NH4Cl revealed an N-glycan-dependent increase in mass-to-charge of 2.5-4.8 Da. These results indicate that 60-70% of the total nitrogen containing monosaccharides had incorporated15N. Presumably,15NH4+was incorporated into GlcNAc and N-acetylneuraminic acid as proposed earlier (Ryll et al., 1994). This might be a universal and previously not described reaction in mammalian cells when exposed to nonphysiological but in cell culture commonly found concentrations of ammonium. The data presented here are of significance for glycoprotein production in mammalian cell culture, since it has been shown previously that elevated levels of UDP-activated hexosamines affect N-glycan characteristics such as branching and degree of amino sugar incorporation. In addition, our results demonstrate that isotope labeling in combination with MALDI-TOF-MS can be used as an alternate tool to radioactive labeling of sugar substrates in metabolic studies.