同位素富集-稀释法研究食性转变对鱼类不同组织N同位素转化率的影响

稳定同位素技术广泛地用于描绘生态系统中食物网的食物来源和营养级关系,但是消费者不同组织转化率的研究相对较少。通过锦鲤摄食人工添加15N蓝藻的食性转化实验,研究不同组织N同位素转化率的差异,探讨组织生长和代谢对同位素转化的相对贡献,为不同时间尺度的稳定同位素研究取样奠定基础。结果表明,通过42d的加富蓝藻饲喂,各组织的N稳定同位素发生显著变化。肝的δ15N为(19.3±1.4)‰,显著高于其它组织,其次为鱼鳍((15.6±1.0)‰)和血液((12.6±0.4)‰),肌肉的δ15N‰最低,为(9.9±0.7)‰。在随后的同位素稀释实验中,锦鲤的体重增加,相对生长速率为0.011d-1,鳍肉的转化率最快,达到11.4%/d,半衰期仅为6.1d,其次是血液和肝,肌肉的转化率最低,仅有3.8%/d,半衰期最长,为18.4d。代谢衰减指数c和-1不存在显著差异,表明锦鲤各组织的N同位素转化主要由组织生长引起。结论显示,同位素富集-稀释法可以有效评价鱼类食性转变对不同组织同位素转化的差异,鳍肉和血液同位素分析可以作为锦鲤食性转变快速追踪的手段。     

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 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.     

15N-enrichment of plant tissue to mark phytophagous insects, associated parasitoids, and flower-visiting entomophaga

New techniques are presented on the use of ¹⁵N to mark insects. ¹⁵N, a stable isotope of nitrogen, was enriched above natural abundance in plant and insect tissues. Two laboratory studies demonstrated that enriched ¹⁵N-concentrations could be tracked from plant to insect using mass spectrometry. In the first study, adult Cotesia plutellae (Kurdjimov) (Hymenoptera: Braconidae) and Hippodamia convergens Guérin-Méneville (Coleoptera: Coccinellidae) were allowed to feed at the flowers of rapid-cycling Chinese cabbage plants that had been fertilized with ¹⁵N-enriched potassium nitrate (KNO₃-¹⁵NO₃). Both insect groups were found to have significantly elevated ¹⁵N levels after visiting the flowers of the ¹⁵N-enriched plants for 48 hours. In the second study, ¹⁵N-enriched bean plant (Phaseolus vulgaris L.) tissue was incorporated into an insect diet and fed to navel orangeworms, Amyelois transitella (Walker) (Lepidoptera: Pyralidae). When the navel orangeworm larvae were 4th instars, they were removed from the diet and exposed to the parasitoid, Goniozus legneri Gordh (Hymenoptera: Bethylidae). Results indicated that the enriched ¹⁵N-concentration of the bean plants was transferred to the navel orangeworms and, subsequently, to the parasitoids. This work may provide useful techniques to help establish whether agriculturally important entomophaga visiting ¹⁵N-enriched flowers or parasitizing enriched sentinel larvae in the field can be effectively marked with ¹⁵N.     

Carbon isotopic composition of legumes with photosynthetic stems from mediterranean and desert habitats

The carbon isotopic compositions of leaves and stems of woody legumes growing in coastal mediterranean and inland desert sites in California were compared. The overall goal was to determine what factors were most associated with the carbon isotope composition of photosynthetic stems in these habitats. The carbon isotope signature (d13C) of photosynthetic stems was less negative than that of leaves on the same plants by an average of 1.51 ± 0.42 ;pp. The d13C of bark (cortical chlorenchyma and epidermis) was more negative than that of wood (vascular tissue and pith) from the same plant for all species studied on all dates. Desert woody legumes had a higher d13C (less negative) and a lower intercellular CO2 concentration (Ci ) (for both photosynthetic tissues) than that of woody legumes from mediterranean climate sites. Differences in the d13C of stems among sites could be entirely accounted for by differences among site air temperatures. Thus, the d13C composition of stems did not indicate a difference in whole-plant integrated water use efficiency (WUE) among sites. In contrast, stems on all plants had a lower stem Ci and a higher d13C than leaves on the same plant, indicating that photosynthetic stems improve long-term, whole-plant water use efficiency in a diversity of species.     

The in vivo nitrogen isotope discrimination among organic plant compounds

The bulk delta 15 N-value of plant (leaf) biomass is determined by that of the inorganic primary nitrogen sources NO(3)(-), NH(4)(+) and N(2), and by isotope discriminations on their uptake or assimilation. NH(4)(+) from these is transferred into "organic N" mainly by the glutamine synthetase reaction. The involved kinetic nitrogen isotope effect does not become manifest, because the turnover is quantitative. From the product glutamine any further conversion proceeds in a "closed system", where kinetic isotope effects become only efficient in connection with metabolic branching. The central and most important corresponding process is the GOGAT-reaction, involved in the de novo nitrogen binding and in recycling processes like the phenylpropanoid biosynthesis and photorespiration. The reaction yields relatively 15N-depleted glutamate and remaining glutamine, source of 15N-enriched amide-N in heteroaromatic compounds. Glutamate provides nitrogen for all amino acids and some other compounds with different 15N-abundances. An isotope equilibration is not connected to transamination; the relative delta 15 N-value of individual amino acids is determined by their metabolic tasks. Relative to the bulk delta 15 N-value of the plant cell, proteins are generally 15N-enriched, secondary products like chlorophyll, lipids, amino sugars and alkaloids are depleted in 15N. Global delta 15 N-values and 15N-patterns of compounds with several N-atoms can be calculated from those of their precursors and isotope discriminations in their biosyntheses.     

Study of early leaf senescence in Arabidopsis thaliana by quantitative proteomics using reciprocal 14N/15N labeling and difference gel electrophoresis

Leaf senescence represents the final stage of leaf development and is associated with fundamental changes on the level of the proteome. For the quantitative analysis of changes in protein abundance related to early leaf senescence, we designed an elaborate double and reverse labeling strategy simultaneously employing fluorescent two-dimensional DIGE as well as metabolic (15)N labeling followed by MS. Reciprocal (14)N/(15)N labeling of entire Arabidopsis thaliana plants showed that full incorporation of (15)N into the proteins of the plant did not cause any adverse effects on development and protein expression. A direct comparison of DIGE and (15)N labeling combined with MS showed that results obtained by both quantification methods correlated well for proteins showing low to moderate regulation factors. Nano HPLC/ESI-MS/MS analysis of 21 protein spots that consistently exhibited abundance differences in nine biological replicates based on both DIGE and MS resulted in the identification of 13 distinct proteins and protein subunits that showed significant regulation in Arabidopsis mutant plants displaying advanced leaf senescence. Ribulose 1,5-bisphosphate carboxylase/oxygenase large and three of its four small subunits were found to be down-regulated, which reflects the degradation of the photosynthetic machinery during leaf senescence. Among the proteins showing higher abundance in mutant plants were several members of the glutathione S-transferase family class phi and quinone reductase. Up-regulation of these proteins fits well into the context of leaf senescence since they are generally involved in the protection of plant cells against reactive oxygen species which are increasingly generated by lipid degradation during leaf senescence. With the exception of one glutathione S-transferase isoform, none of these proteins has been linked to leaf senescence before.     

Uptake, metabolism and distribution of nitrogen in crop plants traced by enriched and natural 15N: Progress over the last 30 years

The major findings of many years of research into plant N cycling are summarised in this review, firstly as revealed by ¹⁵N-enriched methods and secondly, in relation to natural ¹⁵N abundance (δ¹⁵N) in plants and their metabolites. This work has mainly been done in an agricultural context. As many groups especially attempt to relate δ¹⁵N to N cycling, atmospheric N deposition and the interactions of N with carbon budgets, we deem it useful to synthesize these major findings. Primary assimilation and distribution of N within plants were investigated from the ¹⁵N enrichment in individual plant organs and in individual amino acids after feeding them ¹⁵N-labelled compounds. In both roots and leaves, NH₄ ⁺ and NO₃ ^? were assimilated into amino acids, largely by a combination of glutamine synthetase (GS) and glutamate synthase (GOGAT). In the leaves, the transfer of glutamine (amide) N to glutamic acid was accelerated in the light, and amino N in some amino acids was deaminated to ammonia in the dark, followed by its incorporation into glutamine. The N in the growing parts such as growing leaves, filling grains and growing root parts were from two sources: re-allocation (phloem supply) of reserved N (amino acids), and currently-absorbed N. The metabolites from the mature parts may perform the roles of substrates for plant growth and signals for gene expression. δ¹⁵N values, measured for plants/soils and plant metabolites (inorganic N, amino acids, polyamines) were related with the acquisition, metabolism and distribution of N in plants. Small ¹⁵N/¹⁴N fractionation in the acquisition of N₂ and NO₃ ^? and large ¹⁵N/¹⁴N fractionation in NH₄ ⁺ uptake were found. The δ¹⁵N values of whole shoots or grains from field-grown crops were largely reflected major sources of N. In some legumes, ¹⁵N was enriched in their nodules and an extremely ¹⁵N-enriched compound was homospermidine. Nitrate reduction to ammonia (NR) and ammonia assimilation to glutamine (GS) showed large ¹⁵N/¹⁴N fractionations. Specific attention was paid to the δ¹⁵N values in xylem and phloem exudates compared to those of plant organs.     

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.     

Experimental evidence for diel δ15N‐patterns in different tissues,xylem and phloem saps of castor bean (Ricinus communis L.)

Nitrogen isotope signatures in plants might give insights in the metabolism and allocation of nitrogen. To obtain a deeper understanding of the modifications of the nitrogen isotope signatures, we determined δ¹⁵N in transport saps and in different fractions of leaves, axes and roots during a diel course along the plant axis. The most significant diel variations were observed in xylem and phloem saps where δ¹⁵N was significantly higher during the day compared with during the night. However in xylem saps, this was observed only in the canopy, but not at the hypocotyl positions. In the canopy, δ¹⁵N was correlated fairly well between phloem and xylem saps. These variations in δ¹⁵N in transport saps can be attributed to nitrate reduction in leaves during the photoperiod as well as to ¹⁵N‐enriched glutamine acting as transport form of N. δ¹⁵N of the water soluble fraction of roots and leaves partially affected δ¹⁵N of phloem and xylems saps. δ¹⁵N patterns are likely the result of a complex set of interactions and N‐fluxes between plant organs. Furthermore, the natural nitrogen isotope abundance in plant tissue is not constant during the diel course – a fact that needs to be taken into account when sampling for isotopic studies.     

Preparation of amino-acid-type selective isotope labeling of protein expressed in Pichia pastoris

We report the culture conditions for successful amino-acid-type selective (AATS) isotope labeling of protein expressed in Pichia pastoris (P. pastoris). Rhodostomin (Rho), a six disulfide-bonded protein expressed in P. pastoris with the correct fold, was used to optimize the culture conditions. The concentrations of [alpha-15N] selective amino acid, nonlabeled amino acids, and ammonium chloride, as well as induction time, were optimized to avoid scrambling and to increase the incorporation rate and protein yield. The optimized protocol was successfully applied to produce AATS isotope-labeled Rho. The labeling of [alpha-15N]Cys has a 50% incorporation rate, and all 12 cysteine resonances were observed in HSQC spectrum. The labeling of [alpha-15N]Leu, -Lys, and -Met amino acids has an incorporation rate greater than 65%, and the expected number of resonances in the HSQC spectra were observed. In contrast, the labeling of [alpha-15N]Asp and -Gly amino acids has a low incorporation rate and the scrambling problem. In addition, the culture condition was successfully applied to label dendroaspin (Den), a four disulfide-bonded protein expressed in P. pastoris. Therefore, the described condition should be generally applicable to other proteins produced in the P. pastoris expression system. This is the first report to present a protocol for AATS isotope labeling of protein expressed in P. pastoris for NMR study.     

不同肥力棕壤全氮和微生物量氮对外源玉米残体氮的响应

以棕壤玉米长期连作定位试验(27a)形成的高低两种肥力水平土壤为研究对象,采用~(15)N标记的玉米植株为试验试材,分别向两种土壤中加入玉米根、茎、叶(共8个处理),采用室内模拟培养与~(15)N同位素示踪技术,旨在弄清玉米根、茎、叶添加后不同肥力土壤全氮含量及微生物量氮的变化规律。结果表明:(1)添加玉米根、茎、叶后低肥力棕壤全氮含量提升幅度分别比高肥力棕壤高5.75%、4.77%和3.75%,外源新氮的贡献率分别比高肥力棕壤高3.54%、3.28%和2.49%,说明不同肥力土壤对玉米残体添加的响应程度不同,低肥力棕壤对外源新氮施入后的响应更敏感,固定能力更强。(2)在添加玉米残体的56d培养时间内,低肥力棕壤中微生物量氮平均增加0.83—0.98倍,高肥力棕壤中微生物量氮平均增加0.87—1.56倍,可以看出不同部位玉米植株添加后均能显著促进土壤微生物量氮的积累,说明外源有机物输入是刺激土壤微生物数量和活性的重要因素,并且在高肥力土壤中刺激作用更加显著。此外,高肥力土壤添加茎和叶处理微生物量氮显著高于根添加处理,但低肥力土壤中根、茎和叶添加处理土壤中微生物量氮之间无显著差异。外源有机氮输入对土壤氮库的贡献与土壤的肥力水平及不同残体部位自身的物质组成特性密切相关。     

15N自然丰度法在陆地生态系统氮循环研究中的应用

随着氮沉降的不断增加以及人们对全球变化问题的日益关注, 稳定同位素技术在全球变化研究中得到广泛的应用。因为植物和土壤的氮同位素组成记录了氮循环影响因子的综合作用, 并且具有测量简单以及不受取样时间和空间限制的优点, 所以氮同位素自然丰度法被用于氮循环的研究中。该文从氮循环过程中植物和土壤的氮分馏入手, 总结国内外相关文献, 阐述了植物和土壤氮自然丰度在预测生态系统氮饱和和氮循环长期变化趋势中的应用; 总结了利用树轮δ ¹⁵N法研究氮循环过程中应该注意的事项以及目前尚未解决的问题。     

Metabolic disturbances innicotiana glauca,a symptomless

Metabolic disturbances inNicotiana glauca, a symptomless carrier of potato witches’ broom were studied. The dry weight content of leaves of diseased plants was slightly decreased, that of the stems increased. The ash content in the dry weight of the whole diseased plant was decreased by 11%. Contrary to tomato plants which had shown considerable disproportions in the distribution of metabolites among the individual organs investigated (ULRYCHOVÁ, LIMBERK 1964), the content of the total nitrogen and of the individual nitrogen fractions of both leaves and stems of diseasedNicotiana glauca were decreased. The content of glutamine was increased in both organs in spite of the decrease of ammonia nitrogen by nearly 30% in both cases. The most striking disturbance found in the diseased plants ofNicotiana glauca was the 67% increase of a low-molecular phosphorus fraction (P extractable with 0-2N HCIO₄) in the leaves. This result agrees with that found in diseased tomato plants and may be primarily associated with the virus infection. The content of all other phosphorus fractions of both leaves and stems was decreased.     

The effect of drought on C and N stable isotopes in different fractions of leaves, stems and roots of sensitive and tolerant beech ecotypes

Beech seedlings from 11 German climatic provenances were exposed to a realistically timed drought treatment in a greenhouse experiment. The stable isotope composition of carbon (C) and nitrogen (N) was analysed in pooled bulk material of roots, stems and leaves, as well as in the aqueous extracts and starch fractions. The delta 13C values increased in bulk samples (BS) of roots, stems and leaves by drought, although no leaf growth occurred during the experimental period. A clear drought effect on delta 13C in aqueous extracts was detected in leaves. In aqueous extracts of stems and roots as well as in starch fractions of all organs, abundance of delta 13C also tended to be increased by drought, but this effect was not statistically significant. For both delta 13C and delta 15N, enrichment was observed from the site of uptake/ source to the site of use/sink. A gradient for delta 13C in all fractions from leaves (-29.49, -28.89 and -27.85 per thousand) to stems (-28.81, -27.48 and -26.98 per thousand) and to roots (-27.60, -26.37 and -26.48 per thousand) was detected in BS, aqueous extracts and starch, respectively. An opposite gradient for delta 15N was found in BS: 1.59 per thousand, 1.84 per thousand and 3.05 per thousand in roots, stems and leaves, respectively. delta 15N was neither affected by drought in the BS nor in aqueous extracts, but an effect of provenance was observed. Particularly in roots and stems, drought-sensitive provenances showed the strongest shifts in delta 13C induced by drought and the lowest delta 15N values. In the present experiment, delta 13C values were more affected by the environmental factor drought, while delta 15N values were more affected by the genetic factor provenance.     

Nitrogen storage and remobilization in Brassica napus L. during the growth cycle: nitrogen fluxes within the plant and changes in soluble protein patterns

Oilseed rape (Brassica napus L.) is commonly grown for oil or bio-fuel production, while the seed residues can be used for animal feed. It can also be grown as a catch crop because of its efficiency in extracting mineral N from the soil profile. However, the N harvest index is usually low, due in part to a low ability to remobilize N from leaves and to the fall of N-rich leaves which allows a significant amount of N to return to the environment. In order to understand how N filling of pods occurs, experiments were undertaken to quantify N flows within the plant by (15)N labelling and to follow the changes in soluble protein profiles of tissues presumed to store and subsequently to remobilize N. Whereas N uptake increased as a function of growth, N uptake capacity decreased at flowering to a non-significant level during pod filling. However, large amounts of endogenous N were transferred from the leaves to the stems and to taproots which acted as a buffering storage compartment later used to supply the reproductive tissue. About 15% of the total N cycling through the plant were lost through leaf fall and 48%, nearly all of which had been remobilized from vegetative tissues, were finally recovered in the mature pods. SDS-PAGE analysis revealed that large amounts of a 23 kDa polypeptide accumulated in the taproots during flowering and was later fully hydrolysed. Its putative function of storage protein is further supported by the fact that when plants were grown at lower temperature, both flowering, its accumulation and further mobilization were delayed. The overall results are discussed in relation to plant strategies which optimize N cycling to reproductive sinks by means of buffering vegetative tissues such as stems and taproots.     

Nitrogen fixation in Faidherbia albida,Acacia raddiana,Acacia senegal and Acacia seyal estimated using the 15N isotope dilution technique

A pot experiment was conducted in a greenhouse using the ¹⁵N isotope dilution method and two reference plants, Parkia biglobosa and Tamarindus indica to estimate nitrogen fixed in four Acacia species: A raddiana, A. senegal, A. seyal and Faidherbia albida (synonym Acacia albida). For the reference plants, the ¹⁵N enrichments in leaves, stems and roots were similar. With the fixing plants, leaves and stems had similar ¹⁵N enrichments; they were higher than the ¹⁵N enrichment of roots. The amounts of nitrogen fixed at 5 months after planting were similar using either reference plant. Estimates of the percentage of N derived from fixation (%Ndfa) for the above ground parts, in contrast to %Ndfa in roots, were similar to those for the whole plant. However, none of the individual plant parts estimated accurately total N fixed in the whole plant, and excluding the roots resulted in at least 30% underestimation of the amounts of N fixed. Between species, differences in N₂ fixation were observed, both for %Ndfa and total N fixed. For %Ndfa, the best were A. seyal (average, 63%) and A. raddiana (average, 62%), being at least twice the %Ndfa in A. senegal and F. albida. Because of its very high N content, A. seyal was clearly the best in total N fixed, fixing 1.62 g N plant^–1 compared to an average of 0.48 g N plant^–1 for the other Acacia species. Our results show the wide variability existing between Acacia species in terms of both %Ndfa and total N fixed: A. seyal was classified as having a high N₂ fixing potential (NFP) while the other Acacia species had a low NFP.     

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.     

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.     

Quantitative Proteomics Using Reductive Dimethylation for Stable Isotope Labeling

Stable isotope labeling of peptides by reductive dimethylation (ReDi labeling) is a method to accurately quantify protein expression differences between samples using mass spectrometry. ReDi labeling is performed using either regular (light) or deuterated (heavy) forms of formaldehyde and sodium cyanoborohydride to add two methyl groups to each free amine. Here we demonstrate a robust protocol for ReDi labeling and quantitative comparison of complex protein mixtures. Protein samples for comparison are digested into peptides, labeled to carry either light or heavy methyl tags, mixed, and co-analyzed by LC-MS/MS. Relative protein abundances are quantified by comparing the ion chromatogram peak areas of heavy and light labeled versions of the constituent peptide extracted from the full MS spectra. The method described here includes sample preparation by reversed-phase solid phase extraction, on-column ReDi labeling of peptides, peptide fractionation by basic pH reversed-phase (BPRP) chromatography, and StageTip peptide purification. We discuss advantages and limitations of ReDi labeling with respect to other methods for stable isotope incorporation. We highlight novel applications using ReDi labeling as a fast, inexpensive, and accurate method to compare protein abundances in nearly any type of sample.