Uniform stable-isotope labeling in mammalian cells: formulation of a cost-effective culture medium

Uniform stable-isotope labeling of mammalian cells is achieved via a novel formulation of a serum-free cell culture medium that is based on stable-isotope-labeled autolysates and lipid extracts of various microbiological origin. Yeast autolysates allow complete replacement of individual amino acids and organic acids in a chemically defined medium (DMEM/F12), enabling a cost-effective formulation of a stable-isotope-labeled culture medium for mammalian cells. In addition, biomass-derived hydrolysates, autolysates, and lipid extracts of various classes of algae were explored as cell culture components, both separately and in combination with yeast autolysates. Optimal autolysate concentrations were established. Such novel medium formulations were tested on mammalian cell lines, often used for recombinant protein production, i.e., Chinese hamster ovary (CHO) and human embryonic kidney (HEK 293). Special attention was paid to the adaptation of these mammalian cell lines to serum-free media. Formulation of the novel proprietary cell culture medium PLIm, based on yeastolates instead of individual amino acids and organic acids, allows a four- to eightfold cost reduction for ¹⁵N and ¹³C,¹⁵N stable-isotope-labeling, respectively, in CHO cells and a three- to sixfold cost reduction in HEK 293 cells. A high level of stable-isotope enrichment of mammalian cells (>90%) was achieved within four passages by complete replacement of carbon and nitrogen sources in the medium with their stable-isotope-labeled analogs. These conditions can be used to more cost-effectively produce labeled recombinant proteins in mammalian cells.     

Abstracts: Albany 2005, The 14th Conversation

Abstract

We report a cost efficient approach for amino-acid-type selective isotope labeling of proteins expressed in Leishmania tarentolae. The method provides an economically advantageous alternative to recently established protocol for isotopic labeling using expensive synthetic media. The method is based on cultivation of the L. tarentolae expression strain in a cheap complex medium supplemented with labeled amino acid(s). In this protocol, a labeled amino acid is deliberately diluted in the medium of undefined composition, which leads to a low-level isotope enrichment upon protein over-expression. The economic advantage of the protocol is achieved by avoiding large volumes of expensive synthetic medium. Decreased sensitivity of a NMR experiment due to low-level isotope enrichment is compensated by a five- to seven-fold increase of the yield of the recombinant protein in complex medium as compared to that in the synthetic medium. In addition, the decreased sensitivity can be compensated by using a higher magnetic field, cryo-detection system or higher number of transients during the NMR data acquisition. We show that enrichment as low as 5% does not compromise a NMR experiment and makes preparation of the recombinant proteins over- expressed in L. tarentolae economically viable. The method is demonstrated by selective labeling of the ～27 kDa enhanced green fluorescent protein (EGFP) with ¹⁵N-labeled valine.     

An economic approach to isotopic enrichment of glycoproteins expressed from Sf9 insect cells

It is estimated that over half of all proteins are glycosylated, yet only a small number of the structures in the protein data bank are of intact glycoproteins. One of the reasons for the lack of structural information on glycoproteins is the high cost of isotopically labeling proteins expressed from eukaryotic cells such as in insect and mammalian cells. In this paper we describe modifications to commercial insect cell growth medium that reduce the cost for isotopically labeling recombinant proteins expressed from Sf9 cells. A key aspect of this work was to reduce the amount of glutamine in the cell culture medium while maintaining sufficient energy yielding metabolites for vigorous growth by supplementing with glucose and algae-derived amino acids. We present an analysis of cell growth and protein production in Sf9 insect cells expressing secreted Thy1-GFP fusion construct. We also demonstrate isotopic enrichment of the Thy-1 protein backbone with ¹⁵N and carbohydrates with ¹³C by NMR spectroscopy.Electronic supplementary material is available in the online version of this article at and is accessible for authorized users.     

Enhancing the sensitivity of multidimensional NMR experiments by using triply-compensated π pulses

An improved expression protocol is proposed for amino acid type-specific [¹³C], [¹⁵N]-isotope labeling of proteins in baculovirus-infected (BV) insect cell cultures. This new protocol modifies the methods published by Gossert et al. (J Biomol NMR 51(4):449–456, 2011) and provides efficient incorporation of isotopically labeled amino acids, with similar yields per L versus unlabeled expression in rich media. Gossert et al. identified the presence of unlabeled amino acids in the yeastolate of the growth medium as a major limitation in isotope labeling using BV-infected insect cells. By reducing the amount of yeastolate in the growth medium ten-fold, a significant improvement in labeling efficiency was demonstrated, while maintaining good protein expression yield. We report an alternate approach to improve isotope labeling efficiency using BV-infected insect cells namely by replacing the yeast extracts in the medium with dialyzed yeast extracts to reduce the amount of low molecular weight peptides and amino acids. We report the residual levels of amino acids in various media formulations and the amino acid consumption during fermentation, as determined by NMR. While direct replacement of yeastolate with dialyzed yeastolate delivered moderately lower isotope labeling efficiencies compared to the use of ten-fold diluted undialized yeastolate, we show that the use of dialyzed yeastolate combined with a ten-fold dilution delivered enhanced isotope labeling efficiency and at least a comparable level of protein expression yield, all at a scale which economizes use of these costly reagents.     

Mammalian production of an isotopically enriched outer domain of the HIV-1 gp120 glycoprotein for NMR spectroscopy

NMR spectroscopic characterization of the structure or the dynamics of proteins generally requires the production of samples isotopically enriched in ¹⁵N, ¹³C, or ²H. The bacterial expression systems currently in use to obtain isotopic enrichment, however, cannot produce a number of eukaryotic proteins, especially those that require post-translational modifications such as N-linked glycosylation for proper folding or activity. Here, we report the use of an adenovirus vector-based mammalian expression system to produce isotopically enriched ¹⁵N or ¹⁵N/¹³C samples of an outer domain variant of the HIV-1 gp120 envelope glycoprotein with 15 sites of N-linked glycosylation. Yields for the ¹⁵N- and ¹⁵N/¹³C-labeled gp120s after affinity chromatography were 45 and 44 mg/l, respectively, with an average of over 80% isotope incorporation. Recognition of the labeled gp120 by cognate antibodies that recognize complex epitopes showed affinities comparable to the unlabeled protein. NMR spectra, including ¹H-¹⁵N and ¹H-¹³C HSQCs, ¹⁵N-edited NOESY-HSQC, and 3D HNCO, were of high quality, with signal-to-noise consistent with an efficient level of isotope incorporation, and with chemical shift dispersion indicative of a well-folded protein. The exceptional protein yields, good isotope incorporation, and ability to obtain well-folded post-translationally modified proteins make this mammalian system attractive for the production of isotopically enriched eukaryotic proteins for NMR spectroscopy.     

Analysis of 13C labeling enrichment in microbial culture applying metabolic tracer experiments using gas chromatography-combustion-isotope ratio mass spectrometry

The applicability of gas chromatography–combustion–isotope ratio mass spectrometry (GC–C–IRMS) for the quantification of ¹³C enrichment of proteinogenic amino acids in metabolic tracer experiments was evaluated. Measurement of the ¹³C enrichment of proteinogenic amino acids from cell hydrolyzates of Corynebacterium glutamicum growing on different mixtures containing between 0.5 and 10% [1-¹³C]glucose shows the significance of kinetic isotope effects in metabolic flux studies at low degree of labeling. We developed a method to calculate the ¹³C enrichment. The approach to correct for these effects in metabolic flux studies using δ¹³C measurement by GC–C–IRMS uses two parallel experiments applying substrate with natural abundance and ¹³C-enriched tracer substrate, respectively. The fractional enrichment obtained in natural substrate is subtracted from that of the enriched one. Tracer studies with C. glutamicum resulted in a statistically identical relative fractional enrichment of ¹³C in proteinogenic amino acids over the whole range of applied concentrations of [1-¹³C]glucose. The current findings indicate a great potential of GC–C–IRMS for labeling quantification in ¹³C metabolic flux analysis with low labeling degree of tracer substrate directly in larger scale bioreactors.     

Probing in vivo metabolism by stable isotope labeling of storage lipids and proteins in developing Brassica napus embryos

Developing embryos of Brassica napus accumulate both triacylglycerols and proteins as major storage reserves. To evaluate metabolic fluxes during embryo development, we have established conditions for stable isotope labeling of cultured embryos under steady-state conditions. Sucrose supplied via the endosperm is considered to be the main carbon and energy source for seed metabolism. However, in addition to 220 to 270 mM carbohydrates (sucrose, glucose, and fructose), analysis of endosperm liquid revealed up to 70 mM amino acids as well as 6 to 15 mM malic acid. Therefore, a labeling approach with multiple carbon sources is a precondition to quantitatively reflect fluxes of central carbon metabolism in developing embryos. Mid-cotyledon stage B. napus embryos were dissected from plants and cultured for 15 d on a complex liquid medium containing (13)C-labeled carbohydrates. The (13)C enrichment of fatty acids and amino acids (after hydrolysis of the seed proteins) was determined by gas chromatography/mass spectrometry. Analysis of (13)C isotope isomers of labeled fatty acids and plastid-derived amino acids indicated that direct glycolysis provides at least 90% of precursors of plastid acetyl-coenzyme A (CoA). Unlabeled amino acids, when added to the growth medium, did not reduce incorporation of (13)C label into plastid-formed fatty acids, but substantially diluted (13)C label in seed protein. Approximately 30% of carbon in seed protein was derived from exogenous amino acids and as a consequence, the use of amino acids as a carbon source may have significant influence on the total carbon and energy balance in seed metabolism. (13)C label in the terminal acetate units of C(20) and C(22) fatty acids that derive from cytosolic acetyl-CoA was also significantly diluted by unlabeled amino acids. We conclude that cytosolic acetyl-CoA has a more complex biogenetic origin than plastidic acetyl-CoA. Malic acid in the growth medium did not dilute (13)C label incorporation into fatty acids or proteins and can be ruled out as a source of carbon for the major storage components of B. napus embryos.     

Amino acid-selective isotope labeling of proteins for nuclear magnetic resonance study: Proteins secreted by Brevibacillus choshinensis

Here we report the first application of amino acid-type selective (AATS) isotope labeling of a recombinant protein secreted by Brevibacillus choshinensis for a nuclear magnetic resonance (NMR) study. To prepare the ¹⁵N-AATS-labeled protein, the transformed B. choshinensis was cultured in ¹⁵N-labeled amino acid-containing C.H.L. medium, which is commonly used in the Escherichia coli expression system. The analyses of the ¹H-¹⁵N heteronuclear single quantum coherence (HSQC) spectra of the secreted proteins with a ¹⁵N-labeled amino acid demonstrated that alanine, arginine, asparagine, cysteine, glutamine, histidine, lysine, methionine, and valine are suitable for selective labeling, although acidic and aromatic amino acids are not suitable. The ¹⁵N labeling for glycine, isoleucine, leucine, serine, and threonine resulted in scrambling to specific amino acids. These results indicate that the B. choshinensis expression system is an alternative tool for AATS labeling of recombinant proteins, especially secretory proteins, for NMR analyses.     

Glutamine and glutamate nitrogen exchangeable pools in cultured fibroblasts: a stable isotope study

Glutamine's role as an energetic fuel has been extensively studied in the past using ¹⁴C- and ³H-labeled tracers in cultured human cells. Yet another prominent role of glutamine, that of a nitrogen shuttle, cannot be approached without an N-tracer. We therefore used ¹⁵N-labeled glutamine and glutamate to address the following questions: (1) is it possible to study the exchangeable pools of intracellular free glutamine and glutamate nitrogen with stable isotope methods? and (2) to what extent is intracellular glutamine pool regulated by extracellular glutamine? We observed that: (1) intracellular [¹⁵N]-glutamine enrichment reached a plateau at 80% within 20 min of incubation in a buffer containing 0.7 mM pure ¹⁵N-glutamine and no glutamate; in contrast, intracellular ¹⁵N-glutamate enrichment rose only to 40% after 4 hours of incubation in a buffer containing 0.5 mM pure ¹⁵N-glutamate and no glutamine; (2) the cell-free glutamine content was tightly dependent on extracellular glutamine level, while the cell-free glutamate remained steady irrespective of the extracellular glutamate level; (3) the cells took up glutamine and glutamate against a concentration gradient; the rate of glutamine uptake accounted for 90% of the cell glutamine turnover rate; and (4) when cells were confronted with a glutamine-free medium, only one fourth of intracellular glutamine was derived from the exchangeable glutamate. We conclude that: (1) The size and turnover rate of the intracellular pool of free glutamine nitrogen are measurable using stable isotope methodology; (2) glutamine uptake from the extracellular medium accounts for most of glutamine turnover rate in cultured fibroblasts; and (3) intracellular free glutamate is divided up between several pools in cultured human fibroblasts.     

Cost-effective and uniform (13)C- and (15)N-labeling of the 24-kDa N-terminal domain of the Escherichia coli gyrase B by overexpression in the photoautotrophic cyanobacterium Anabaena sp. PCC 7120

Structural studies of biomolecules using nuclear magnetic resonance (NMR) rely on the availability of samples enriched in (13)C and (15)N isotopes. While (13)C/(15)N-labeled proteins are generally obtained by overexpression in transformed Escherichia coli cells cultured in the presence of an expensive mixture of labeled precursors, those of the photoautotrophic cyanobacterium Anabaena sp. PCC 7120 can be uniformly labeled by growing them in medium containing Na(15)NO(3) and NaH(13)CO(3) as the sole nitrogen and carbon sources. We report here a novel vector-host system suitable for the efficient preparation of uniformly (13)C/(15)N-labeled proteins in Anabaena sp. PCC 7120. The 24-kDa N-terminal domain of the E. coli gyrase B subunit, used as a test protein, was cloned into the pRL25C shuttle vector under the control of the tac promoter. The transformed Anabaena cells were grown in the presence of the labeled mineral salts and culture conditions were optimized to obtain over 90% of (13)C and (15)N enrichment in the constitutively expressed 24-kDa polypeptide. The yield of purified 24-kDa protein after dual isotope labeling under anaerobic conditions was similar to that obtained with E. coli cells bearing a comparable expression vector and cultured in parallel in a commercially available labeling medium. Furthermore, as probed by NMR spectroscopy and mass spectrometry, the 24-kDa N-terminal domain expressed in Anabaena was identical to the E. coli sample, demonstrating that it was of sufficient quality for 3D-structure determination. Because the Anabaena system was far more advantageous taking into consideration the expense for the labels that were necessary, these results indicate that Anabaena sp. PCC 7120 is an economic alternative for the (13)C/(15)N-labeling of soluble recombinant proteins destined for structural studies.     

Evaluation of isotope discrimination in C-based metabolic flux analysis

In a ¹³C experiment for metabolic flux analysis (¹³C MFA), we examined isotope discrimination by measuring the labeling of glucose, amino acids, and hexose monophosphates via mass spectrometry. When Escherichia coli grew in a mix of 20% fully labeled and 80% naturally labeled glucose medium, the cell metabolism favored light isotopes and the measured isotopic ratios (δ¹³C) were in the range of −35 to −92. Glucose transporters might play an important role in such isotopic fractionation. Flux analysis showed that both isotopic discrimination and isotopic impurities in labeled substrates could affect the solution of ¹³C MFA.     

AMINO ACID METABOLISM IN GLIAL CELLS: HOMEOSTATIC REGULATION OF INTRA- and EXTRACELLULAR MILIEU BY C-6 GLIOMA CELLS

Abstract— The amino acid and carbohydrate metabolism of confluent cultures of C-6 glioma cells has been investigated. It was observed that the presence of glutamine in the incubation fluid was essential to maintain high glutamine levels in the cells during a 2 h incubation. When cells were incubated in a cerebrospinal fluid-like medium glutamate, glutamine, aspartate and γ-aminobutyrate (GABA) levels were comparable to those occurring in whole forebrain of adult rat in vivo. Glucose uptake was high, approx 1 μmol/mg protein/2 h, 50% of which was accounted for by lactate production. Of the remaining glucose uptake a substantial proportion was unaccounted for by known oxygen-coupled citric acid cycle flux, or glycogen or amino acid synthesis. Interestingly, the cells released into the medium significant amounts of the neuroinhibitory amino acids, GABA and glycine, and rapidly cleared the medium of the neuroexcitatory amino acids glutamate and aspartate. Metabolism of [2-¹⁴C]glucose and [³H]acetate by the cells indicated rapid labelling of the glutamate and aspartate pools of the cells by glucose in 1 h, but the relative specific activities of glutamine and GABA were much lower. The metabolism of tracer concentrations of [³H]acetate to glutamate by the cells indicated greater dilution of this isotope compared to that of labelled glucose. However, the ratio of ³H to ¹⁴C radioactivity in glutamate and other amino acids was similar to that in the mixture of glucose and acetate added to the medium. Therefore, some active route of acetate metabolism which communicates metabolically with the route of glucose metabolism to glutamate appears to exist in the cells. Significant acetate activation and fatty acid turnover would explain the present results. Some of the amino acid labelling patterns observed in these studies are not consistent with these glial-like cells behaving as models for the small compartment of amino acid metabolism in brain. Enzyme measurements corroborated the metabolic studies. Glutamate decarboxylase activity was 3–10% of the level found in whole brain. GABA transaminase was also low compared to brain as was glutamine synthetase. Glutamate dehydrogenase was present at levels equal to or higher than those of whole brain.     

Interrelationships of Leucine and Glutamate Metabolism in Cultured Astrocytes

Abstract: The aim was to study the extent to which leu-cine furnishes α-NH₂ groups for glutamate synthesis via branched-chain amino acid aminotransferase. The transfer of N from leucine to glutamate was determined by incubating astrocytes in a medium containing [¹⁵N]leucine and 15 unlabeled amino acids; isotopic abundance was measured with gas chromatography-mass spectrometry. The ratio of labeling in both [¹⁵N]glutamate/[¹⁵N]leucine and [2-¹⁵N]glutamine/[¹⁵N]leucine suggested that at least one-fifth of all glutamate N had been derived from leucine nitrogen. At the same time, enrichment in [¹⁵N]leucine declined, reflecting dilution of the ¹⁶N label by the unlabeled amino acids that were in the medium. Isotopic abundance in [¹⁶N]-isoleucine increased very quickly, suggesting the rapidity of transamination between these amino acids. The appearance of ¹⁵N in valine was more gradual. Measurement of branched-chain amino acid transaminase showed that the reaction from leucine to glutamate was approximately six times more active than from glutamate to leucine (8.72 vs. 1.46 nmol/min/mg of protein). However, when the medium was supplemented with α-ketoisocaproate (1 mM), the ketoacid of leucine, the reaction readily ran in the “reverse” direction and intraastrocytic [glutamate] was reduced by ～50% in only 5 min. Extracellular concentrations of α-ketoisocaproate as low as 0.05 mM significantly lowered intracellular [glutamate]. The relative efficiency of branched-chain amino acid transamination was studied by incubating astrocytes with 15 unlabeled amino acids (0.1 mM each) and [¹⁵N]glutamate. After 45 min, the most highly labeled amino acid was [¹⁵N]alanine, which was closely followed by [¹⁵N]leucine and [¹⁵N]isoleucine. Relatively little ¹⁵N was detected in any other amino acids, except for [¹⁵N]serine. The transamination of leucine was ～17 times greater than the rate of [1-¹⁴C]leucine oxidation. These data indicate that leucine is a major source of glutamate nitrogen. Conversely, reamination of a-ketoisocaproate, the ketoacid of leucine, affords a mechanism for the temporary “buffering” of intracellular glutamate.     

2H- and13C-labeled amino acids generated by obligate methylotrophs Biosynthesis and MS monitoring

Summary Biosynthetic preparation of²H- and¹³C- labeled amino acids was studied using a leucine-producing mutant of the obligate methylotroph,Methylobacillus flagellatum. The strain was cultivated in various media containing¹³C- or²H-analogs of methanol. The total protein from each experiment was subjected to acid hydrolysis and converted into a mixture of dansyl amino acid methyl esters. The samples of excreted leucine were converted into methyl esters of dansyl and benzyloxycarbonyl derivatives. Electron impact mass spectrometry was performed to detect stable isotope enrichment of the amino acids. According to the mass spectrometric analysis it is feasible to use methylotrophic microorganisms for the preparation of²H- and¹³C- analogs of amino acids by labeled methanol bioconversion; the excreted amino acids can be convenient for express analysis as an indicator of isotopic enrichment of the total protein. The data obtained testified to a high efficiency of dansyl derivatization for mass spectrometric analysis of complex amino acid mixtures.     

Using the dual isotope method to assess cecal amino acid absorption of goat whey protein in rats,a pilot study

Measurement of ileal amino acids (AA) bioavailability is recommended to evaluate protein quality. A dual isotope tracer method, based on plasma isotopic enrichment ratios, has been proposed to determine true digestibility in humans. In a pilot study, we aimed to evaluate whether this method could be implemented in rats to determine AA bioavailability based on isotopic enrichment ratios measured in cecal digesta or plasma samples. Goat milk proteins were intrinsically labeled with ¹⁵N and ²H. Wistar rats were fed a meal containing the doubly labeled goat whey proteins and a tracer dose of ¹³C-spirulina. Blood samples were collected 0, 1 h and 3 h after meal ingestion from the tail vein. The rats were euthanized 4 h (n?=?6) or 6 h (n?=?6) after meal to collect plasma and intestinal contents. True orocecal protein digestibility and AA bioavailability were assessed by means of ¹⁵N and ²H enrichment in cecum content and compared with absorption indexes determined at the plasma or cecum level using isotopic ratios. Plasma kinetics of isotopic enrichment could not be completed due to the limited quantity of plasma obtained with sequential blood collection. However, the absorption indexes determined from cecal ¹⁵N or ²H/¹³C ratios gave coherent values with true orocecal AA bioavailability. This dual isotope approach with measurements of isotopic ratios in digestive content could be an interesting strategy to determine true AA bioavailability in ileal digesta of rats.

     

NMR resonance assignments for sparsely <Superscript>15</Superscript>N labeled proteins

For larger proteins, and proteins not amenable to expression in bacterial hosts, it is difficult to deduce structures using NMR methods based on uniform ¹³C, ¹⁵N isotopic labeling and observation of just nuclear Overhauser effects (NOEs). In these cases, sparse labeling with selected ¹⁵N enriched amino acids and extraction of a wider variety of backbone-centered structural constraints is providing an alternate approach. A limitation, however, is the absence of resonance assignment strategies that work without uniform ¹⁵N, ¹³C labeling or preparation of numerous samples labeled with pairs of isotopically labeled amino acids. In this paper an approach applicable to a single sample prepared with sparse ¹⁵N labeling in selected amino acids is presented. It relies on correlation of amide proton exchange rates, measured from data on the intact protein and on digested and sequenced peptides. Application is illustrated using the carbohydrate binding protein, Galectin-3. Limitations and future applications are discussed.     

Production of yeastolates for uniform stable isotope labelling in eukaryotic cell culture

Preparation of stable isotope-labelled yeastolates opens up ways to establish more cost-effective stable isotope labelling of biomolecules in insect and mammalian cell lines and hence to employ higher eukaryotic cell lines for stable isotope labelling of complex recombinant proteins. Therefore, we evaluated several common yeast strains of the Saccharomycetoideae family as a source of high-quality, non-toxic yeastolates with the major aim to find a primary amino acid source for insect and mammalian cell culture that would allow cost-effective uniform stable isotope labelling (¹³C, ¹⁵N). Strains of the facultative methylotrophic yeasts Pichia pastoris and Hansenula polymorpha (Pichia angusta) as well as a strain of the baker’s yeast Saccharomyces cerevisiae were compared as a source of yeastolate with respect to processing, recovery and ability to sustain growth of insect and mammalian cell lines. The best growth-supporting yeastolates were prepared via autolysis from yeast obtained from fed-batch cultures that were terminated at the end of the logarithmic growth phase. Yeastolates obtained from H. polymorpha performed well as a component of insect cell cultures, while yeastolates from S. cerevisiae and H. polymorpha both yielded good results in mammalian cell cultures. Growth of yeasts in Heine’s medium without lactic acid allows relatively low concentrations of ¹³C and ¹⁵N sources, and this medium can be reused several times with supplementation of the ¹³C source only.     

Microbial synthesis of L-[15N]leucine L-[15N]isoleucine, and L-[3-13C]-and L-[3'-13C]isoleucines studied by nuclear magnetic resonance and gas chromatography-mass spectrometry

The preparation of leucine and isoleucine labeled with 15N and of site-specific 13C-labeled isoleucines is described. This method is based on the induction of the biosynthetic pathways specific for branched chain amino acids in glutamic acid producing bacteria, and controlled provision of stable isotope labeled precursors. Corynebacterium glutamicum (ATCC 13032), a glutamic acid overproducer, was incubated in leucine production medium which consisted of a basal medium supplemented with [15N]ammonium sulfate, glucose, and sodium alpha-ketoisocaproate. production of L-[15N]leucine reached 138 mumol/ml at an isotopic efficiency of 90%. It was purified and checked by proton NMR and GC-MS. The electron impact (EI) spectrum showed 95 atom% enrichment. The cultivation of C. glutamicum in a similar medium containing alpha-ketobutyrate yielded L-[15N]isoleucine at a concentration of 120 mumol/ml. The GC-MS EI and chemical ionization (CI) spectra confirmed enrichment of 96 atom% 15N as that of the labeled precursors. The biosynthesis of L-[13C]isoleucine was carried out by induced cells which were transferred to a similar medium in which [2-13C]- or [3-13C]pyruvic acid replaced glucose. 13C NMR of the product isoleucine revealed single-site enrichment at C-3 or at C-3' respective to the precursor [13C]pyruvate; i.e., C-3 was labeled from [2-13C]pyruvate and C-3' from [3-13C]pyruvate. Mass spectrometric analysis confirmed that all molecules were labeled only in one carbon. This site-specific incorporation of [13C]pyruvate is contrasted with the labeling pattern obtained when producing cells were supplied with [2-13C]acetate, instead of pyruvate, when most label was incorporated into carbons 3 and 3' of the same isoleucine molecule.     

Synthesis of the 15N-Gln11 labeled peptaibol antibiotic zervamicin-IIB

Summary Synthesis of zervamicin IIB, specifically labeled at the α-position of glutamine-11 with¹⁵N, was achieved by the Fmoc/tert.-butyl strategy in solution using a fragment condensation approach. Three fragments of zervamicin IIB were obtained by stepwise elongation with Fmoc amino acids using BOP as a coupling reagent. For the introduction of the highly sterically hindered α-aminoisobutyric acid residues, BOP/DMAP activation was applied. Peptide fragments were coupled by means of the coupling reagent, CF₃-PyBOP. Using the strategy developed, zervamicin IIB specifically¹⁵N labeled has been synthesized in 30% overall yield based on the isotopically labeled amino acid. From 600 MHz NMR spectroscopy the position of the¹⁵N-label was clearly detected. The isotope enrichment (98 ±2%) was determined by FAB-mass spectrometry.     

Harmonizing Labeling and Analytical Strategies to Obtain Protein Turnover Rates in Intact Adult Animals

Changes in the abundance of individual proteins in the proteome can be elicited by modulation of protein synthesis (the rate of input of newly synthesized proteins into the protein pool) or degradation (the rate of removal of protein molecules from the pool). A full understanding of proteome changes therefore requires a definition of the roles of these two processes in proteostasis, collectively known as protein turnover. Because protein turnover occurs even in the absence of overt changes in pool abundance, turnover measurements necessitate monitoring the flux of stable isotope–labeled precursors through the protein pool such as labeled amino acids or metabolic precursors such as ammonium chloride or heavy water. In cells in culture, the ability to manipulate precursor pools by rapid medium changes is simple, but for more complex systems such as intact animals, the approach becomes more convoluted. Individual methods bring specific complications, and the suitability of different methods has not been comprehensively explored. In this study, we compare the turnover rates of proteins across four mouse tissues, obtained from the same inbred mouse strain maintained under identical husbandry conditions, measured using either [¹³C₆]lysine or [²H₂]O as the labeling precursor. We show that for long-lived proteins, the two approaches yield essentially identical measures of the first-order rate constant for degradation. For short-lived proteins, there is a need to compensate for the slower equilibration of lysine through the precursor pools. We evaluate different approaches to provide that compensation. We conclude that both labels are suitable, but careful determination of precursor enrichment kinetics in amino acid labeling is critical and has a considerable influence on the numerical values of the derived protein turnover rates.