The 15N isotope effect in Escherichia coli: A neutron can make the difference

Several techniques based on stable isotope labeling are used for quantitative MS. These include stable isotope metabolic labeling methods for cells in culture as well as live organisms with the assumption that the stable isotope has no effect on the proteome. Here, we investigate the ¹⁵N isotope effect on Escherichia coli cultures that were grown in either unlabeled (¹⁴N) or ¹⁵N‐labeled media by LC‐ESI‐MS/MS‐based relative protein quantification. Consistent protein expression level differences and altered growth rates were observed between ¹⁴N and ¹⁵N‐labeled cultures. Furthermore, targeted metabolite analyses revealed altered metabolite levels between ¹⁴N and ¹⁵N‐labeled bacteria. Our data demonstrate for the first time that the introduction of the ¹⁵N isotope affects protein and metabolite levels in E. coli and underline the importance of implementing controls for unbiased protein quantification using stable isotope labeling techniques.     

A practical method for cell-free protein synthesis to avoid stable isotope scrambling and dilution

During recent years, the targets of protein structure analysis using nuclear magnetic resonance spectroscopy have become larger and more complicated. As a result, a complete and precise stable isotope labeling technique has been desired. A cell-free protein synthesis system is appropriate for this purpose. In the current study, we achieved precise and complete ¹⁵N and ²H labeling using an Escherichia coli cell extract-based cell-free protein synthesis system by controlling the metabolic reactions in the system with their chemical inhibitors. The addition of aminooxyacetate, d-malate, l-methionine sulfoximine, S-methyl-l-cysteine sulfoximine, 6-diazo-5-oxo-l-norleucine, and 5-diazo-4-oxo-l-norvaline was quite effective for precise amino acid-selective ¹⁵N labeling even for aspartic acid, asparagine, glutamic acid, and glutamine, which generally suffer from severe isotope scrambling and dilution when using the conventional cell-free system. For ²H labeling, the back-protonation of the H^α and H^β positions, which commonly occurred in the conventional system, was dramatically suppressed by simply adding aminooxyacetate and d-malate to the cell-free system except for the H^α positions in methionine and cysteine.     

Relative and accurate measurement of protein abundance using 15N stable isotope labeling in Arabidopsis (SILIA)

In the quantitative proteomic studies, numerous in vitro and in vivo peptide labeling strategies have been successfully applied to measure differentially regulated protein and peptide abundance. These approaches have been proven to be versatile and repeatable in biological discoveries. ¹⁵N metabolic labeling is one of these widely adopted and economical methods. However, due to the differential incorporation rates of ¹⁵N or ¹⁴N, the labeling results produce imperfectly matched isotopic envelopes between the heavy and light nitrogen-labeled peptides. In the present study, we have modified the solid Arabidopsis growth medium to standardize the ¹⁵N supply, which led to a uniform incorporation of ¹⁵N into the whole plant protein complement. The incorporation rate (97.43 ± 0.11%) of ¹⁵N into ¹⁵N-coded peptides was determined by correlating the intensities of peptide ions with the labeling efficiencies according to Gaussian distribution. The resulting actual incorporation rate (97.44%) and natural abundance of ¹⁵N/¹⁴N-coded peptides are used to re-calculate the intensities of isotopic envelopes of differentially labeled peptides, respectively. A modified ¹⁵N/¹⁴N stable isotope labeling strategy, SILIA, is assessed and the results demonstrate that this approach is able to differentiate the fold change in protein abundance down to 10%. The machine dynamic range limitation and purification step will make the precursor ion ratio deriving from the actual ratio fold change. It is suggested that the differentially mixed ¹⁵N-coded and ¹⁴N-coded plant protein samples that are used to establish the protein abundance standard curve should be prepared following a similar protein isolation protocol used to isolate the proteins to be quantitated.     

Stable Isotope Metabolic Labeling with a Novel 15N-Enriched Bacteria Diet for Improved Proteomic Analyses of Mouse Models for Psychopathologies

The identification of differentially regulated proteins in animal models of psychiatric diseases is essential for a comprehensive analysis of associated psychopathological processes. Mass spectrometry is the most relevant method for analyzing differences in protein expression of tissue and body fluid proteomes. However, standardization of sample handling and sample-to-sample variability are problematic. Stable isotope metabolic labeling of a proteome represents the gold standard for quantitative mass spectrometry analysis. The simultaneous processing of a mixture of labeled and unlabeled samples allows a sensitive and accurate comparative analysis between the respective proteomes. Here, we describe a cost-effective feeding protocol based on a newly developed ¹⁵N bacteria diet based on Ralstonia eutropha protein, which was applied to a mouse model for trait anxiety. Tissue from ¹⁵N-labeled vs. ¹⁴N-unlabeled mice was examined by mass spectrometry and differences in the expression of glyoxalase-1 (GLO1) and histidine triad nucleotide binding protein 2 (Hint2) proteins were correlated with the animals'' psychopathological behaviors for methodological validation and proof of concept, respectively. Additionally, phenotyping unraveled an antidepressant-like effect of the incorporation of the stable isotope ¹⁵N into the proteome of highly anxious mice. This novel phenomenon is of considerable relevance to the metabolic labeling method and could provide an opportunity for the discovery of candidate proteins involved in depression-like behavior. The newly developed ¹⁵N bacteria diet provides researchers a novel tool to discover disease-relevant protein expression differences in mouse models using quantitative mass spectrometry.     

Topograph,a Software Platform for Precursor Enrichment Corrected Global Protein Turnover Measurements

Defects in protein turnover have been implicated in a broad range of diseases, but current proteomics methods of measuring protein turnover are limited by the software tools available. Conventional methods require indirect approaches to differentiate newly synthesized protein when synthesized from partially labeled precursor pools. To address this, we have developed Topograph, a software platform which calculates the fraction of peptides that are from newly synthesized proteins and their turnover rates. A unique feature of Topograph is the ability to calculate amino acid precursor pool enrichment levels which allows for accurate calculations when the precursor pool is not fully labeled, and the approach used by Topograph is applicable regardless of the stable isotope label used. We validate the Topograph algorithms using data acquired from a mouse labeling experiment and demonstrate the influence that precursor pool corrections can have on protein turnover measurements.Methods of measuring protein synthesis and degradation using stable or radioactive isotope labels have existed for decades. The isotope label is introduced in the form of a labeled amino acid or amino acid precursor, and the incorporation or removal of that label from protein is used to estimate average protein turnover rates (1, 2). Historically, the amount of stable isotope label incorporated into a protein is measured by enriching for the protein (e.g. affinity chromatography, gel electrophoresis, and other biochemical methods), hydrolyzing the protein to amino acids, derivatizing the amino acids, and measuring the labeled amino acid by gas chromatography-mass spectrometry or gas chromatography-combustion-isotope ratio mass spectrometry (3, 4). More recently, proteomics methods have been developed that measure the labeled amino acid on the peptide level, eliminating the need for a protein enrichment step and enabling the monitoring of many proteins in a single experiment (5).Proteomics approaches to measuring protein turnover rates in mice have been accomplished by the introduction of a ¹⁵N stable isotope label. The labeled diets were created by supplementing a protein-free diet with a ¹⁵N enriched protein source. Price et al. (6) generated ¹⁵N-labeled protein from the alga, Spirulina platensis and Zhang et al. (7) introduced ¹⁵N-label in the form of lysate from the bacterium, Ralstonia eutropha. An advantage of using complete ¹⁵N labeling is the rapid incorporation of ¹⁵N and separation of isotope distributions between labeled and natural isotope abundance peptides, which reduces the need to deconvolute the two distributions. However, current methods require that the dietary protein content be derived from bacterial or alga lysate, a diet that is not normally fed to laboratory mice. As a result, measurements of protein turnover may not reflect conventional mouse model systems because of effects of diet on protein and amino acid metabolism. A more recent work by Claydon et al. (8) demonstrated a stable isotope labeling method by supplementing labeled valine into a standard mouse diet.The complex data generated from these analyses creates a data processing and analysis challenge; exemplified by recent software platforms that have been developed. Guan et al. (9) and Hoopmann et al. (10) demonstrated data analysis pipelines for ¹⁵N labeled SILAM and SILAC experiments. Here we describe the software platform, Topograph, we have developed for the analysis of liquid chromatography-tandem MS (LC-MS/MS) data from samples with isotopic labels. Topograph is able to deconvolute the complex spectra that may result from overlapping isotope distributions, regardless of the isotope label used. More uniquely, Topograph is able to calculate the relative isotope abundance (RIA)¹ of the amino acid precursor pool, which is necessary to correctly determine the amount of newly synthesized peptide and to subsequently calculate peptide and protein turnover rates.     

Tissue Turnover Rates and Isotopic Trophic Discrimination Factors in the Endothermic Teleost,Pacific Bluefin Tuna (Thunnus orientalis)

Stable isotope analysis (SIA) of highly migratory marine pelagic animals can improve understanding of their migratory patterns and trophic ecology. However, accurate interpretation of isotopic analyses relies on knowledge of isotope turnover rates and tissue-diet isotope discrimination factors. Laboratory-derived turnover rates and discrimination factors have been difficult to obtain due to the challenges of maintaining these species in captivity. We conducted a study to determine tissue- (white muscle and liver) and isotope- (nitrogen and carbon) specific turnover rates and trophic discrimination factors (TDFs) using archived tissues from captive Pacific bluefin tuna (PBFT), Thunnus orientalis, 1–2914 days after a diet shift in captivity. Half-life values for ¹⁵N turnover in white muscle and liver were 167 and 86 days, and for ¹³C were 255 and 162 days, respectively. TDFs for white muscle and liver were 1.9 and 1.1‰ for δ ¹⁵N and 1.8 and 1.2‰ for δ ¹³C, respectively. Our results demonstrate that turnover of ¹⁵N and ¹³C in bluefin tuna tissues is well described by a single compartment first-order kinetics model. We report variability in turnover rates between tissue types and their isotope dynamics, and hypothesize that metabolic processes play a large role in turnover of nitrogen and carbon in PBFT white muscle and liver tissues. ¹⁵N in white muscle tissue showed the most predictable change with diet over time, suggesting that white muscle δ ¹⁵N data may provide the most reliable inferences for diet and migration studies using stable isotopes in wild fish. These results allow more accurate interpretation of field data and dramatically improve our ability to use stable isotope data from wild tunas to better understand their migration patterns and trophic ecology.     

Effect of nutritional condition on variation in δ13C and δ15N stable isotope values in Pumpkinseed sunfish (Lepomis gibbosus) fed different diets

Stable isotope analysis is frequently used to infer resource use in natural populations of fishes. Studies have examined factors, other than diet, that influence δ¹⁵N and δ¹³C including tissue-specific rates of equilibration and starvation. Most such studies completed under laboratory conditions tightly control food quantity and its isotopic composition, but it is also necessary to evaluate the influence of these factors under more natural conditions. Using pumpkinseed sunfish (Lepomis gibbosus) we evaluated whether restricted rations below minimum daily requirements affects tissue equilibration to a change in diet by holding fish on two treatments that often reflect divergent resource use in natural populations (pelagic zooplankton or littoral macroinvertebrates). Over 42 days, δ¹⁵N values increased while δ¹³C values did not change, additionally neither were related to diet treatment. Increased δ¹⁵N values were negatively related to body condition while δ¹³C values were not, indicating that stable isotope values were more affected by decreasing body condition than by diet. Additionally, δ¹⁵N values changed more in the blood and liver tissues than in white muscle tissue, indicating that restricting food availability had greater effects on tissues with greater metabolic activity. We hypothesize that stable isotope values of consumers are subject to a tissue-specific trade-off between sensitivity to changes in resource use and resistance to the effects of low resource availability. This trade-off may require consideration in stable isotope studies of wild populations facing periodic limitations of food availability.     

Efficient uniform isotope labeling of Abl kinase expressed in Baculovirus-infected insect cells

This report shows for the first time the efficient uniform isotope labeling of a recombinant protein expressed using Baculovirus-infected insect cells. The recent availability of suitable media for ¹⁵N- and ¹³C/¹⁵N-labeling in insect cells, the high expression of Abl kinase in these labeling media and a suitable labeling protocol made it possible to obtain a ¹H–¹⁵N-HSQC spectrum for the catalytic domain of Abl kinase of good quality and with label incorporation rates > 90%. The presented isotope labeling method should be applicable also to further proteins where successful expression is restricted to the Baculovirus expression system.     

Stable isotope labeling of glycoprotein expressed in silkworms using immunoglobulin G as a test molecule

Silkworms serve as promising bioreactors for the production of recombinant proteins, including glycoproteins and membrane proteins, for structural and functional protein analyses. However, lack of methodology for stable isotope labeling has been a major deterrent to using this expression system for nuclear magnetic resonance (NMR) structural biology. Here we developed a metabolic isotope labeling technique using commercially available silkworm larvae. The fifth instar larvae were infected with baculoviruses for co-expression of recombinant human immunoglobulin G (IgG) as a test molecule, with calnexin as a chaperone. They were subsequently reared on an artificial diet containing ¹⁵N-labeled yeast crude protein extract. We harvested 0.1 mg of IgG from larva with a ¹⁵N-enrichment ratio of approximately 80 %. This allowed us to compare NMR spectral data of the Fc fragment cleaved from the silkworm-produced IgG with those of an authentic Fc glycoprotein derived from mammalian cells. Therefore, we successfully demonstrated that our method enables production of isotopically labeled glycoproteins for NMR studies.     

Combination of online enzyme digestion with stable isotope labeling for high‐throughput quantitative proteome analysis

Various enzyme reactors and online enzyme digestion strategies have been developed in recent years. These reactors greatly enhanced the detection sensitivity and proteome coverage in qualitative proteomics. However, these devices have higher rates of miscleavage in protein digestion. Therefore, we investigated the effect of online enzyme digestion on the quantification accuracy of quantitative proteomics using chemical or metabolic isotope labeling approaches. The incomplete digestion would introduce some unexpected variations in comparative quantification when the samples are digested and then chemically isotope labeled in different aliquots. Even when identical protein aliquots are processed on these devices using post‐digestion chemical isotope labeling and the CVs of the ratios controlled to less than 50% in replicate analyses, about 10% of the quantified proteins have a ratio greater than two‐fold, whereas in theory the ratio is 1:1. Interestingly, the incomplete digestion with enzyme reactor is not a problem when metabolic isotope labeling samples were processed because the proteins are isotopically labeled in vivo prior to their simultaneous digestion within the reactor. Our results also demonstrated that both high quantification accuracy and high proteome coverage can be achieved in comparative proteome quantification using online enzyme digestion even when a limited amount of metabolic isotope labeling samples is used (1683 proteins comparatively quantified from 10⁵ Hela cells).     

Production of stable isotope-labeled acyl-coenzyme A thioesters by yeast stable isotope labeling by essential nutrients in cell culture

Acyl-coenzyme A (CoA) thioesters are key metabolites in numerous anabolic and catabolic pathways, including fatty acid biosynthesis and β-oxidation, the Krebs cycle, and cholesterol and isoprenoid biosynthesis. Stable isotope dilution-based methodology is the “gold standard” for quantitative analyses by mass spectrometry. However, chemical synthesis of families of stable isotope-labeled metabolites such as acyl-CoA thioesters is impractical. Previously, we biosynthetically generated a library of stable isotope internal standard analogs of acyl-CoA thioesters by exploiting the essential requirement in mammals and insects for pantothenic acid (vitamin B5) as a metabolic precursor for the CoA backbone. By replacing pantothenic acid in the cell medium with commercially available [¹³C₃¹⁵N₁]-pantothenic acid, mammalian cells exclusively incorporated [¹³C₃¹⁵N₁]-pantothenate into the biosynthesis of acyl-CoA and acyl-CoA thioesters. We have now developed a much more efficient method for generating stable isotope-labeled CoA and acyl-CoAs from [¹³C₃¹⁵N₁]-pantothenate using stable isotope labeling by essential nutrients in cell culture (SILEC) in Pan6-deficient yeast cells. Efficiency and consistency of labeling were also increased, likely due to the stringently defined and reproducible conditions used for yeast culture. The yeast SILEC method greatly enhances the ease of use and accessibility of labeled CoA thioesters and also provides proof of concept for generating other labeled metabolites in yeast mutants.     

Isotopic fractionation with morphological change and sexual specificity in the lappet moth Euthrix potatoria

The δ¹⁵N values of adult holometabolous insects exceed those of larvae, but otherwise little information on terrestrial invertebrates has been obtained in food‐web analyses using stable isotope ratios (δ¹⁵N, δ¹³C). Changes in δ¹³C during metamorphosis and differences between males and females have not been examined. We collected the larvae and cocoons of Euthrix potatoria (L.) (Lepidoptera: Lasiocampidae) in the field and used them to assess the species’ isotopic fractionation. Each emerged moth was divided into five body parts. We conducted stable N and C isotope analyses for each body part, as well as for cocoons and exuviae, and also compared stable isotope ratios between sexes. We confirmed δ¹⁵N enrichment through metamorphosis and estimated that δ¹⁵N enrichment is accomplished by the relative concentration of ¹⁵N due to the excretion of copious meconium, which contains abundant ¹⁴N. We also observed changes in δ¹³C values through metamorphosis. Both isotope values tended to change more in males than in females. The proportion of the whole‐adult weight represented by meconium was higher in males than in females, suggesting that high meconium secretion in males contributes to the sexual difference in δ¹⁵N. These phenomena may be common in Holometabola, which require a pupal stage. For more accurate food‐web assessments, it is important to consider stable isotope changes during different life cycles, as well as sexual differences.     

Dual‐tracer‐based isotope turnover rates in a highly invasive mysid Limnomysis benedeni from Lake Constance

Understanding the ecological patterns of invasive species and their habitats require an understanding of the species’ foraging ecology. Stable carbon (δ¹³C) and nitrogen (δ¹⁵N) isotope values provide useful information into the study of animal ecology and evolution, since the isotope ratios of consumers reflect consumer's dietary patterns. Nevertheless, the lack of species‐ and element‐specific laboratory‐derived turnover rates could limit their application. Using a laboratory‐based dual stable isotope tracer approach (Na¹⁵NO₃ and NaH¹³CO₃), we evaluated the δ¹⁵N and δ¹³C isotope turnover rates in full‐grown adult invasive Limnomysis benedeni from Lake Constance. We provide δ¹⁵N and δ¹³C turnover rates based on nonlinear least‐squares regression and posterior linear regression models. Model precisions and fit were evaluated using Akaike's information criterion. Within a couple of days, the δ¹⁵N and δ¹³C of mysids began to change. Nevertheless, after about 14 days, L. benedeni did not reach equilibrium with their new isotope values. Since the experiment was conducted on adult subjects, it is evident that turnover was mainly influenced by metabolism (in contrast to growth). Unlike traditional dietary shifts, our laboratory‐based dual stable isotope tracer approach does not shift the experimental organisms into a new diet and avoids dietary effects on isotope values. Results confirm the application of isotopic tracers to label mysid subpopulations and could be used to reflect assimilation and turnover from the labeled dietary sources. Field‐based stable isotope studies often use isotopic mixing models commonly assuming diet‐tissue steady state. Unfortunately, in cases where the isotopic composition of the animal is not in equilibrium with its diet, this can lead to highly misleading conclusions. Thus, our laboratory‐based isotopic incorporation rates assist interpretation of the isotopic values from the field and provide a foundation for future research into using isotopic tracers to investigate invasion ecology.     

Natural Isotopic Signatures of Variations in Body Nitrogen Fluxes: A Compartmental Model Analysis

Body tissues are generally ¹⁵N-enriched over the diet, with a discrimination factor (Δ¹⁵N) that varies among tissues and individuals as a function of their nutritional and physiopathological condition. However, both ¹⁵N bioaccumulation and intra- and inter-individual Δ¹⁵N variations are still poorly understood, so that theoretical models are required to understand their underlying mechanisms. Using experimental Δ¹⁵N measurements in rats, we developed a multi-compartmental model that provides the first detailed representation of the complex functioning of the body''s Δ¹⁵N system, by explicitly linking the sizes and Δ¹⁵N values of 21 nitrogen pools to the rates and isotope effects of 49 nitrogen metabolic fluxes. We have shown that (i) besides urea production, several metabolic pathways (e.g., protein synthesis, amino acid intracellular metabolism, urea recycling and intestinal absorption or secretion) are most probably associated with isotope fractionation and together contribute to ¹⁵N accumulation in tissues, (ii) the Δ¹⁵N of a tissue at steady-state is not affected by variations of its P turnover rate, but can vary according to the relative orientation of tissue free amino acids towards oxidation vs. protein synthesis, (iii) at the whole-body level, Δ¹⁵N variations result from variations in the body partitioning of nitrogen fluxes (e.g., urea production, urea recycling and amino acid exchanges), with or without changes in nitrogen balance, (iv) any deviation from the optimal amino acid intake, in terms of both quality and quantity, causes a global rise in tissue Δ¹⁵N, and (v) Δ¹⁵N variations differ between tissues depending on the metabolic changes involved, which can therefore be identified using simultaneous multi-tissue Δ¹⁵N measurements. This work provides proof of concept that Δ¹⁵N measurements constitute a new promising tool to investigate how metabolic fluxes are nutritionally or physiopathologically reorganized or altered. The existence of such natural and interpretable isotopic biomarkers promises interesting applications in nutrition and health.     

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.     

Comparison of full versus partial metabolic labeling for quantitative proteomics analysis in Arabidopsis thaliana

In recent years a variety of quantitative proteomics techniques have been developed, allowing characterization of changes in protein abundance in a variety of organisms under various biological conditions. Because it allows excellent control for error at all steps in sample preparation and analysis, full metabolic labeling using (15)N has emerged as an important strategy for quantitative proteomics, having been applied in a variety of organisms from yeast to Arabidopsis and even rats. However, challenges associated with complete replacement of (14)N with (15)N can make its application in many complex eukaryotic systems impractical on a routine basis. Extending a concept proposed by Whitelegge et al. (Whitelegge, J. P., Katz, J. E., Pihakari, K. A., Hale, R., Aguilera, R., Gomez, S. M., Faull, K. F., Vavilin, D., and Vermaas, W. (2004) Subtle modification of isotope ratio proteomics; an integrated strategy for expression proteomics. Phytochemistry 65, 1507-1515), we investigate an alternative strategy for quantitative proteomics that relies upon the subtle changes in isotopic envelope shape that result from partial metabolic labeling to compare relative abundances of labeled and unlabeled peptides in complex mixtures. We present a novel algorithm for the automated quantitative analysis of partial incorporation samples via LC-MS. We then compare the performance of partial metabolic labeling with traditional full metabolic labeling for quantification of controlled mixtures of labeled and unlabeled Arabidopsis peptides. Finally we evaluate the performance of each technique for comparison of light- versus dark-grown Arabidopsis with respect to reproducibility and numbers of peptide and protein identifications under more realistic experimental conditions. Overall full metabolic labeling and partial metabolic labeling prove to be comparable with respect to dynamic range, accuracy, and reproducibility, although partial metabolic labeling consistently allows quantification of a higher percentage of peptide observations across the dynamic range. This difference is especially pronounced at extreme ratios. Ultimately both full metabolic labeling and partial metabolic labeling prove to be well suited for quantitative proteomics characterization.     

Cost-effective large-scale expression of proteins for NMR studies

We present protocols for high-level expression of isotope-labelled proteins in E. coli in cost-effective ways. This includes production of large amounts of unlabeled proteins and ¹³C-methyl methionine labeling in rich media, where yields of up to a gram of soluble protein per liter of culture are reached. Procedures for uniform isotope labeling of ²H, ¹³C and ¹⁵N using auto-induction or isopropyl-β-d-1-thiogalactopyranoside-induction are described, with primary focus on minimal isotope consumption and high reproducibility of protein expression. These protocols are based on high cell-density fermentation, but the key procedures are easily transferred to shake flask cultures.     

Affordable uniform isotope labeling with <Superscript>2</Superscript>H, <Superscript>13</Superscript>C and <Superscript>15</Superscript>N in insect cells

For a wide range of proteins of high interest, the major obstacle for NMR studies is the lack of an affordable eukaryotic expression system for isotope labeling. Here, a simple and affordable protocol is presented to produce uniform labeled proteins in the most prevalent eukaryotic expression system for structural biology, namely Spodoptera frugiperda insect cells. Incorporation levels of 80 % can be achieved for ¹⁵N and ¹³C with yields comparable to expression in full media. For ²H,¹⁵N and ²H,¹³C,¹⁵N labeling, incorporation is only slightly lower with 75 and 73 %, respectively, and yields are typically twofold reduced. The media were optimized for isotope incorporation, reproducibility, simplicity and cost. High isotope incorporation levels for all labeling patterns are achieved by using labeled algal amino acid extracts and exploiting well-known biochemical pathways. The final formulation consists of just five commercially available components, at costs 12-fold lower than labeling media from vendors. The approach was applied to several cytosolic and secreted target proteins.