A MS data search method for improved 15N‐labeled protein identification

Quantitative proteomics using stable isotope labeling strategies combined with MS is an important tool for biomarker discovery. Methods involving stable isotope metabolic labeling result in optimal quantitative accuracy, since they allow the immediate combination of two or more samples. Unfortunately, stable isotope incorporation rates in metabolic labeling experiments using mammalian organisms usually do not reach 100%. As a consequence, protein identifications in ¹⁵N database searches have poor success rates. We report on a strategy that significantly improves the number of ¹⁵N‐labeled protein identifications and results in a more comprehensive and accurate relative peptide quantification workflow.     

A method for efficient isotopic labeling of recombinant proteins

A rapid and efficient approach for preparing isotopically labeled recombinant proteins is presented. The method is demonstrated for ¹³C labeling of the C-terminal domain of angiopoietin-2, ¹⁵N labeling of ubiquitin and for ²H/¹³C/¹⁵N labeling of the Escherichia coli outer-membrane lipoprotein Lpp-56. The production method generates cell mass using unlabeled rich media followed by exchange into a small volume of labeled media at high cell density. Following a short period for growth recovery and unlabeled metabolite clearance, the cells are induced. The expression yields obtained provide a fourfold to eightfold reduction in isotope costs using simple shake flask growths.     

Optimized procedure to generate heavy isotope and selenomethionine-labeled proteins for structure determination using <Emphasis Type="Italic">Escherichia coli</Emphasis>-based expression systems

Generating sufficient quantities of labeled proteins represents a bottleneck in protein structure determination. A simple protocol for producing heavy isotope as well as selenomethionine (Se-Met)-labeled proteins was developed using T7-based Escherichia coli expression systems. The protocol is applicable for generation of single-, double-, and triple-labeled proteins (¹⁵N, ¹³C, and ²H) in shaker flask cultures. Label incorporation into the target protein reached 99% and 97% for ¹⁵N and ¹³C, respectively, and 75% of (non-exchangeable) hydrogen for ²H labeling. The expression yields and final cell densities (OD600 ∼16) were the same as for the production of non-labeled protein. This protocol is also applicable for Se-Met labeling, leading to Se-Met incorporation into the target protein of 70% or 90% using prototrophic or methionine auxotrophic E. coli strains, respectively.     

Stable isotopic labeling‐based quantitative targeted glycomics (i‐QTaG)

Mass spectrometry (MS) analysis combined with stable isotopic labeling is a promising method for the relative quantification of aberrant glycosylation in diseases and disorders. We developed a stable isotopic labeling‐based quantitative targeted glycomics (i‐QTaG) technique for the comparative and quantitative analysis of total N‐glycans using matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS). We established the analytical procedure with the chemical derivatizations (i.e., sialic acid neutralization and stable isotopic labeling) of N‐glycans using a model glycoprotein (bovine fetuin). Moreover, the i‐QTaG using MALDI‐TOF MS was evaluated with various molar ratios (1:1, 1:2, 1:5) of ¹³C₆/¹²C₆‐2‐aminobenzoic acid‐labeled glycans from normal human serum. Finally, this method was applied to direct comparison of the total N‐glycan profiles between normal human sera (n = 8) and prostate cancer patient sera (n = 17). The intensities of the N‐glycan peaks from i‐QTaG method showed a good linearity (R² > 0.99) with the amount of the bovine fetuin glycoproteins. The ratios of relative intensity between the isotopically 2‐AA labeled N‐glycans were close to the theoretical molar ratios (1:1, 1:2, 1:5). We also demonstrated that the up‐regulation of the Lewis antigen (～82%) in sera from prostate cancer patients. In this proof‐of‐concept study, we demonstrated that the i‐QTaG method, which enables to achieve a reliable comparative quantitation of total N‐glycans via MALDI‐TOF MS analysis, has the potential to diagnose and monitor alterations in glycosylation associated with disease states or biotherapeutics. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:840–848, 2015     

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.     

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.     

The (15) N isotope effect as a means for correlating phenotypic alterations and affected pathways in a trait anxiety mouse model

Stable isotope labeling techniques hold great potential for accurate quantitative proteomics comparisons by MS. To investigate the effect of stable isotopes in vivo, we metabolically labeled high anxiety-related behavior (HAB) mice with the heavy nitrogen isotope (15) N. (15) N-labeled HAB mice exhibited behavioral alterations compared to unlabeled ((14) N) HAB mice in their depression-like phenotype. To correlate behavioral alterations with changes on the molecular level, we explored the (15) N isotope effect on the brain proteome by comparing protein expression levels between (15) N-labeled and (14) N HAB mouse brains using quantitative MS. By implementing two complementary in silico pathway analysis approaches, we were able to identify altered networks in (15) N-labeled HAB mice, including major metabolic pathways such as the tricarboxylic acid (TCA) cycle and oxidative phosphorylation. Here, we discuss the affected pathways with regard to their relevance for the behavioral phenotype and critically assess the utility of exploiting the (15) N isotope effect for correlating phenotypic and molecular alterations.     

QuantiSpec — Quantitative mass spectrometry data analysis of N-metabolically labeled proteins

For relative protein quantitation by mass spectrometry we metabolically labeled E. coli bacteria with ¹⁵N-enriched diets. Proteins extracted from ¹⁵N-labeled and unlabeled E. coli bacteria were mixed, separated by two-dimensional gel electrophoresis and enzymatically digested. The resulting tryptic peptides were analyzed by MALDI mass spectrometry. For the relative protein quantitation we developed fully automated software, QuantiSpec (Quantitative Mass Spectrometry Analysis Software), which uses data from MALDI TOF mass spectrometry and the Mascot database search engine. QuantiSpec detects natural as well as partially or fully labeled peptide isotope distributions. For each identified peptide the ¹⁵N incorporation rate is determined by comparing the experimental to a set of theoretical isotope patterns based on the peptide sequence. Relative quantitation is accomplished by calculating the signal intensity ratios for each ¹⁴N/¹⁵N peptide pair.     

Effects of decomposition and storage conditions on the δ13C and δ15N isotope values of killer whale (Orcinus orca) skin and blubber tissues

Several different factors in the collection and preservation of whale skin and blubber samples were examined to determine their effect on the results obtained by stable nitrogen and carbon isotope (δ¹⁵N and δ¹³C) analysis. Samples of wet killer whale skin retained their original stable isotope values for up to 14 d at 4°C or lower. However, decomposition significantly changed the δ¹⁵N value within 3 d at 20°C. Storage at ?20°C was as effective as ?80°C for the preservation of skin and blubber samples for stable isotope analysis for at least a year. By contrast, once a skin sample had been freeze‐dried and lipid extracted, the stable isotope values did not change significantly when it was stored dry at room temperature for at least 12 mo. Preservation of whale skin samples for a month in DMSO‐salt solution, frozen or at room temperature, did not significantly change the δ¹⁵N and δ¹³C values of lipid extracted tissues, although the slight changes seen could influence results of a study if only small changes are expected.     

Ratio‐dependent significance thresholds in reciprocal 15N‐labeling experiments as a robust tool in detection of candidate proteins responding to biological treatment

Metabolic labeling of plant tissues with ¹⁵N has become widely used in plant proteomics. Here, we describe a robust experimental design and data analysis workflow implementing two parallel biological replicate experiments with reciprocal labeling and series of 1:1 control mixtures. Thereby, we are able to unambiguously distinguish (i) inherent biological variation between cultures and (ii) specific responses to a biological treatment. The data analysis workflow is based on first determining the variation between cultures based on ¹⁵N/¹⁴N ratios in independent 1:1 mixtures before biological treatment is applied. In a second step, ratio‐dependent SD is used to define p‐values for significant deviation of protein ratios in the biological experiment from the distribution of protein ratios in the 1:1 mixture. This approach allows defining those proteins showing significant biological response superimposed on the biological variation before treatment. The proposed workflow was applied to a series of experiments, in which changes in composition of detergent resistant membrane domains was analyzed in response to sucrose resupply after carbon starvation. Especially in experiments involving cell culture treatment (starvation) prior to the actual biological stimulus of interest (resupply), a clear distinction between culture to culture variations and biological response is of utmost importance.     

15N-metabolic labeling for comparative plasma membrane proteomics in Arabidopsis cells

An important goal for proteomic studies is the global comparison of proteomes from different genotypes, tissues, or physiological conditions. This has so far been mostly achieved by densitometric comparison of spot intensities after protein separation by 2-DE. However, the physicochemical properties of membrane proteins preclude the use of 2-DE. Here, we describe the use of in vivo labeling by the stable isotope 15N as an alternative approach for comparative membrane proteomic studies in plant cells. We confirm that 15N-metabolic labeling of proteins is possible and efficient in Arabidopsis suspension cells. Quantification of 14N versus 15N MS signals reflects the relative abundance of 14N and 15N proteins in the sample analyzed. We describe the use of 15N-metabolic labeling to perform a partial comparative analysis of Arabidopsis cells following cadmium exposure. By focusing our attention on plasma membrane proteins, we were able to confidently identify proteins showing up to 5-fold regulation compared to unexposed cells. This study provides a proof of principle that 15N-metabolic labeling is a useful technique for comparative membrane proteome studies.     

Relative protein quantification by isobaric SILAC with immonium ion splitting (ISIS)

Metabolic labeling techniques have recently become popular tools for the quantitative profiling of proteomes. Classical stable isotope labeling with amino acids in cell cultures (SILAC) uses pairs of heavy/light isotopic forms of amino acids to introduce predictable mass differences in protein samples to be compared. After proteolysis, pairs of cognate precursor peptides can be correlated, and their intensities can be used for mass spectrometry-based relative protein quantification. We present an alternative SILAC approach by which two cell cultures are grown in media containing isobaric forms of amino acids, labeled either with 13C on the carbonyl (C-1) carbon or 15N on backbone nitrogen. Labeled peptides from both samples have the same nominal mass and nearly identical MS/MS spectra but generate upon fragmentation distinct immonium ions separated by 1 amu. When labeled protein samples are mixed, the intensities of these immonium ions can be used for the relative quantification of the parent proteins. We validated the labeling of cellular proteins with valine, isoleucine, and leucine with coverage of 97% of all tryptic peptides. We improved the sensitivity for the detection of the quantification ions on a pulsing instrument by using a specific fast scan event. The analysis of a protein mixture with a known heavy/light ratio showed reliable quantification. Finally the application of the technique to the analysis of two melanoma cell lines yielded quantitative data consistent with those obtained by a classical two-dimensional DIGE analysis of the same samples. Our method combines the features of the SILAC technique with the advantages of isobaric labeling schemes like iTRAQ. We discuss advantages and disadvantages of isobaric SILAC with immonium ion splitting as well as possible ways to improve it.     

A stable isotopic investigation into the causes of decline in a sub-Antarctic predator, the rockhopper penguin Eudyptes chrysocome

The rockhopper penguin (Eudyptes chrysocome) is a conspicuous apex marine predator that has experienced marked population declines throughout most of its circumpolar breeding distribution. The cause(s) for the declines remain elusive, but the relatively large spatio‐temporal scale over which population decreases have occurred implies that ecosystem‐scale, at‐sea factors are likely to be involved. We employ stable isotope analyses of carbon (¹³C/¹²C, expressed as δ¹³C) and nitrogen (¹⁵N/¹⁴N, δ¹⁵N) in time‐series of rockhopper penguin feather samples, dating back to 1861, in order to reconstruct the species' ecological history. Specifically, we examine whether rockhopper penguin population decline has been associated with a shift towards lower primary productivity in the ecosystem in which they feed, or with a shift to a diet of lower trophic status and lower quality, and we use long‐term temperature records to evaluate whether shifts in isotope ratios are associated with annual variations in sea surface temperature. Having controlled temporally for the Suess Effect and for increases in CO₂ concentrations in seawater, we found that overall, δ¹³C signatures decreased significantly over time in rockhopper penguins from seven breeding sites, supporting the hypothesis that decreases in primary productivity, and hence, carrying capacity, for which δ¹³C signature is a proxy, have been associated with the decline of penguin populations. There was some evidence of a long‐term decline in δ¹⁵N at some sites, and strong evidence that δ¹⁵N signatures were negatively related to sea surface temperatures across sites, indicative of a shift in diet to prey of lower trophic status over time and in warm years. However, a site‐by‐site analysis revealed divergent isotopic trends among sites: five of seven sites exhibited significant temporal or temperature‐related trends in isotope signatures. This study highlights the utility of stable isotope analyses when applied over relatively long timescales to apex predators.     

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.     

Testing the efficiency of three 15N-labeled nitrogen compounds for indirect labeling of grasshoppers via plants in the field

In field studies of plant–insect herbivore interactions it is often difficult to establish which herbivore has fed on a particular plant. We investigated the suitability of three different ¹⁵N‐labeled nitrogen compounds (ammonium, nitrate, and glycine) for indirect marking of three grasshopper species [Omocestus viridulus (L.), Chorthippus parallelus (Zett.), and Chorthippus biguttulus (L.) (Orthoptera: Acrididae)] through labeling their food plants in the field. In two short‐term experiments grassland plots of 1 m² were separately labeled with either one of the different nitrogen compounds. Grasshoppers were caged on three food‐plant species [Dactylis glomerata L., Holcus lanatus L. (Poaceae), and Trifolium repens L. (Fabaceae)] present in these plots for 72 h. Significantly enriched δ¹⁵N values in grasshoppers were found in all plant/grasshopper combinations. Enrichment in grasshoppers was positively correlated with the enrichment of plants and labeling with nitrate resulted in highest ¹⁵N enrichment. In a long‐term experiment, individuals of C. biguttulus were placed in a cage covering an area of 1 m² for 37 days, with sampling of grasshoppers at regular intervals. δ¹⁵N values of the grasshopper and a common food plant, D. glomerata, increased steadily over time, up to 40‐fold by the end of the experiment. Our results demonstrate that ¹⁵N‐labeling of plants is an appropriate tool for the investigation of insect–plant interactions under natural conditions.     

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.     

15N-labeled P22 c2 repressor for nuclear magnetic resonance studies of protein-DNA interactions

The salmonella phage P22 c2 repressor was produced with 90% ¹⁵N isotope labeling of all leucines, using the expression system E. coli W3110 lac I ^Q/pTP 125. The N-terminal DNA-binding domain 1–76 was obtained by chymotrypsin cleavage. Its characterization by biochemical techniques, mass spectrometry, and one- and two-dimensional nuclear magnetic resonance (NMR) showed that highly residue-selective isotope labeling was achieved with the minimal growth medium used. The ability to obtain such isotope labeling opens new avenues for NMR studies of protein-DNA interactions in the P22 operator system.     

A distinctive latitudinal trend of nitrogen isotope signature across urban forests in eastern China

Rapid urbanization has greatly altered nitrogen (N) cycling from regional to global scales. Compared to natural forests, urban forests receive much more external N inputs with distinctive abundances of stable N isotope (δ¹⁵N). However, the large-scale pattern of soil δ¹⁵N and its imprint on plant δ¹⁵N remain less well understood in urban forests. By collecting topsoil (0–20 cm) and leaf samples from urban forest patches in nine large cities across a north–south transect in eastern China, we analyzed the latitudinal trends of topsoil C:N ratio and δ¹⁵N as well as the correlations between tree leaf δ¹⁵N and topsoil δ¹⁵N. We further explored the spatial variation of topsoil δ¹⁵N explained by corresponding climatic, edaphic, vegetation-associated, and anthropogenic drivers. Our results showed a significant increase of topsoil C:N ratio towards higher latitudes, suggesting lower N availability at higher latitudes. Topsoil δ¹⁵N also increased significantly at higher latitudes, being opposite to the latitudinal trend of soil N availability. The latitudinal trend of topsoil δ¹⁵N was mainly explained by mean annual temperature, mean annual precipitation, and atmospheric deposition of both ammonium and nitrate. Consequently, tree leaf δ¹⁵N showed significant positive correlations with topsoil δ¹⁵N across all sampled plant species and functional types. Our findings reveal a distinctive latitudinal trend of δ¹⁵N in urban forests and highlight an important role of anthropogenic N sources in shaping the large-scale pattern of urban forest ¹⁵N signature.     

Evaluation of the accuracy of protein quantification using isotope TMPP‐labeled peptides

N‐succinimidyloxycarbonylmethyl tris(2,4,6‐trimethoxyphenyl) phosphonium bromide (TMPP‐Ac‐OSu) reacts rapidly, mildly, and specifically with the N‐terminals of proteins and peptides. Thus, it can be developed as an ideal isotope‐coded tag to be used in quantitative proteomics. Here, we present a strategy for light and heavy TMPP‐based quantitative proteomic analysis, in which peptides in a mixture can be quantified using an on‐tip TMPP derivatization approach. To demonstrate the accuracy of this strategy, light and heavy TMPP‐labeled peptides were combined at different ratios and subsequently analyzed by LC‐MS/MS. The MS spectra and scatter plots show that peptide and protein ratios were both consistent with the mixed ratios. We observed a linear correlation between protein ratios and the predicted ratios. In comparison with SILAC method, the TMPP labeling method produced similarly accurate quantitative results with low CVs. In conclusion, our results suggest that this isotope‐coded TMPP method achieved accurate quantification and compatibility with IEF‐based separation. With the inherent advantages of TMPP derivatization, we believe that it holds great promise for future applications in quantitative proteomics analysis.