Stable Isotope Labeling Methods Research Articles

Rational biodiesel production from oxidation stability perspective: Evaluation of lipid-soluble carbonyl compound formation in the seed oil and biodiesel with long-term storage

The exploration of appropriate feedstock and catalytic transesterification hinders the practical application of biodiesel. Here, we introduced rational biodiesel production from oxidation stability perspective and evaluated the lipid-soluble carbonyl-containing oxidation products in long-term stored biodiesel samples that prepared from different oil source, catalyst for production, and ester bond type. A stable isotope labeling method based on liquid chromatography-high resolution-mass spectrometry (SIL-LC-HRMS) detection is established using a pair of labeling reagents namely1-(2-hydrazinyl-2-oxoethyl)pyridin-1-ium chloride (GP) and [2H5]1-(2-Hydrazinyl-2-oxoethyl)pyridin-1-ium chloride ([2H5]GP) for lipid-soluble carbonyl-containing oxidation products, including aldehydes and ketones. The main benefits of SIL-LC-HRMS are its sensitivity, selectivity, and feature coverage towards carbonyl-containing compounds and realized their non-targeted screening and relative quantification from complex matrices. Using SIL-LC-HRMS analysis, it was estimated the distribution and formation of 131 carbonyl compounds in three different kinds of second-generation feedstock and biodiesels with one-year storage in the dark. Double bond equivalent (DBE)-number of carbon (nC) plots and hierarchical clustering heatmap analysis reveal the effect of the oil source, catalyst type, and alkyl ester bond type on the variance in carbonyl compound formation during long-term biodiesel storage, arguably the most crucial factor oxidation stability in rational biodiesel production. The carbonyl formation and their variations in biodiesel samples are also discussed by theoretically calculating the bond dissociation energy for the primary constituent of seed oil and biodiesel.

Characterizing the remobilization flux of cadmium from pre-anthesis vegetative pools in rice during grain filling using an improved stable isotope labeling method

A clear understanding of the allocation of Cd to grains is essential to manage the level of Cd in cereal diets effectively. Yet, debate remains over whether and how the pre-anthesis pools contribute to grain Cd accumulation, resulting in uncertainty regarding the need to control plant Cd uptake during vegetative growth. To this end, rice seedlings were exposed to 111Cd labeled solution until tillering, transplanted to unlabeled soils, and grown under open-air conditions. The remobilization of Cd derived from pre-anthesis vegetative pools was studied through the fluxes of 111Cd-enriched label among organs during grain filling. The 111Cd label was continuously allocated to the grain after anthesis. The lower leaves remobilized the Cd label during the earlier stage of grain development, which was allocated almost equally to the grains and husks + rachis. During the final stage, the Cd label was strongly remobilized from the roots and, less importantly, the internodes, which was strongly allocated to the nodes and, to a less extent, the grains. The results show that the pre-anthesis vegetative pools are an important source of Cd in rice grains. The lower leaves, internodes, and roots are the source organs, whereas the husks + rachis and nodes are the sinks competing with the grain for the remobilized Cd. This study provides insight into understanding the ecophysiological mechanism of Cd remobilization and setting agronomic measures for lowering grain Cd levels.

Click Isotope Mass Probe for Highly Selective Determination of Trace Steroid Hormones in Food Samples.

Matrix effects are a great challenge for the quantitative analysis of complex food samples by liquid chromatography-tandem mass spectrometry analysis (LC-MS/MS). Stable isotope labeling (SIL) has been widely used as an effective strategy to eliminate matrix effects. Herein, a copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) click-reaction-based SIL method was proposed for a highly sensitive and selective determination of six synthetic steroid hormones in three different food samples (milk, yogurt, and eggs) by high-performance liquid chromatography (HPLC)-MS/MS. A pair of novel SIL agents, N-(2-azidyl ethyl) aniline (d0-AEA) and d5-N-(2-azidyl ethyl) aniline (d5-AEA) were synthesized to label steroid hormones in the samples and standard solution, respectively. The reaction accomplishes in 30 min at 60 °C. The heavy labeled standards were used as internal standards (ISs), which experience the identical ionization processes with light labeled samples to minimize matrix effects. After derivatization, the ionization efficiencies of steroid hormones were greatly improved by 2-54-folds, and the matrix effects ranged from 88.6 to 99.8%. The established method achieved satisfactory detection limits (0.1-2.5 μg L-1) and high recoveries (85-102%). These results demonstrated that the proposed method holds unique advantages for trace steroid hormones analysis in foodstuffs.

Stable isotope labeling of nanomaterials for biosafety evaluation and drug development

Quantitative information, such as environmental migration, absorption, biodistribution, biotransformation, and elimination, is fundamental and essential for the nanosafety evaluations of nanomaterials. Due to the complexity of biological and environmental systems, it is challenging to develop quantitative approaches and tools that could characterize intrinsic behaviors of nanomaterials in the organisms. The isotopic tracers are ideal candidates to tune the physical properties of nanomaterials while preserving their chemical properties. In this review article, we summarized the stable isotope labeling methods of nanomaterials for evaluating their environmental and biological effects. The skeleton labeling protocols of carbon nanomaterials and metal/metal oxide nanoparticles were introduced. The advantages and disadvantages of stable isotope labeling were discussed in comparison with other quantitative methods for nanomaterials. The quantitative information of nanomaterials in environmental and biological systems was summarized along with the biosafety data. The benefits for drug development of nanomedicine were analyzed based on the targeting effects, persistent accumulation, and safety. Finally, the challenges and future perspectives of stable isotope labeling in nanoscience and nanotechnology were discussed.

An In Vivo Stable Isotope Labeling Method to Investigate Individual Matrix Protein Synthesis, Ribosomal Biogenesis, and Cellular Proliferation in Murine Articular Cartilage.

Targeting chondrocyte dynamics is a strategy for slowing osteoarthritis progression during aging. We describe a stable-isotope method using in vivo deuterium oxide labeling and mass spectrometry to measure protein concentration, protein half-life, cell proliferation, and ribosomal biogenesis in a single sample of murine articular cartilage. We hypothesized that a 60-d labeling period would capture age-related declines in cartilage matrix protein content, protein synthesis rates, and cellular proliferation. Knee cartilage was harvested to the subchondral bone from 25- to 90-wk-old female C57BL/6J mice treated with deuterium oxide for 15, 30, 45, and 60 d. We measured protein concentration and half-lives using targeted high resolution accurate mass spectrometry and d2ome data processing software. Deuterium enrichment was quantified in isolated DNA and RNA to measure cell proliferation and ribosomal biogenesis, respectively. Most collagen isoforms were less abundant in aged animals, with negligible collagen synthesis at either age. In contrast, age altered the concentration and half-lives of many proteoglycans and other matrix proteins, including several with greater concentration and half-lives in older mice such as proteoglycan 4, clusterin, and fibronectin-1. Cellular proteins were less abundant in older animals, consistent with reduced cellularity. Nevertheless, deuterium was maximally incorporated into 60% of DNA and RNA by 15 d of labeling in both age groups, suggesting the presence of two large pools of either rapidly (<15 d) or slowly (>60 d) proliferating cells. Our findings indicate that age-associated changes in cartilage matrix protein content and synthesis occur without detectable changes in the relative number of proliferating cells.

Stable isotope labeling method with sensitive identification and accurate quantitation function for aldehydes in fried foods

Deuterated dansylhydrazine (d6-DH) was prepared by one step simple reaction, and applied to the stable isotope labeling (SIL) of aldehydes with the aid of commercially available dansylhydrazine (DH). Common multiple reaction monitoring (MRM) parameters was obtained by a few standard samples and then applied to other aldehydes. Hence levels of unknown aldehydes in real samples was identified using the sensitive MRM mode but not the traditional scan mode. The SIL strategy also enhanced the quantitation accuracy because samples and standards were d6-DH and DH labeled respectively, and then mass spectrum (MS) analyzed simultaneously. Analytes and standards were analyzed in identical MS conditions, and thus matrix effect which caused by different ionization efficiency between samples and standards was greatly minimized. The proposed method was applied to the SIL analysis of 21 aldehydes with limits of detection (LODs) ranged from 8.0 − 14.2 ng/kg. The recoveries were in the range of 85.3–96.4%, and the matrix effect was in the range of 90.1–102.6%.

A Review of Stable Isotope Labeling and Mass Spectrometry Methods to Distinguish Exogenous from Endogenous DNA Adducts and Improve Dose-Response Assessments.

Cancer remains the second most frequent cause of death in human populations worldwide, which has been reflected in the emphasis placed on management of risk from environmental chemicals considered to be potential human carcinogens. The formation of DNA adducts has been considered as one of the key events of cancer, and persistence and/or failure of repair of these adducts may lead to mutation, thus initiating cancer. Some chemical carcinogens can produce DNA adducts, and DNA adducts have been used as biomarkers of exposure. However, DNA adducts of various types are also produced endogenously in the course of normal metabolism. Since both endogenous physiological processes and exogenous exposure to xenobiotics can cause DNA adducts, the differentiation of the sources of DNA adducts can be highly informative for cancer risk assessment. This review summarizes a highly applicable methodology, termed stable isotope labeling and mass spectrometry (SILMS), that is superior to previous methods, as it not only provides absolute quantitation of DNA adducts but also differentiates the exogenous and endogenous origins of DNA adducts. SILMS uses stable isotope-labeled substances for exposure, followed by DNA adduct measurement with highly sensitive mass spectrometry. Herein, the utilities and advantage of SILMS have been demonstrated by the rich data sets generated over the last two decades in improving the risk assessment of chemicals with DNA adducts being induced by both endogenous and exogenous sources, such as formaldehyde, vinyl acetate, vinyl chloride, and ethylene oxide.

Noncovalently Tagged Gas Phase Complex Ions for Screening Unknown Contaminant Metabolites in Plants.

Screening the metabolites of emerging organic contaminants (EOCs) from complicated biological matrices is an important but challenging task. Although stable isotope labeling (SIL) is frequently used to facilitate the identification of contaminant metabolites from redundant interfering components, the isotopically labeled reagents are expensive and difficult to synthesize, which greatly constrains the application of the SIL method. Herein, a new online noncovalent tagging method was developed for screening the metabolites of 1H-benzotriazol (BT) based on the characteristic structural moieties reserved in the metabolites. By selecting β-cyclodextrin (β-CD) as a macrocyclic tagging reagent, metabolites with the reserved moiety were expected to exhibit a characteristic shift of the mass-to-charge ratio (Δm/z = 1134.3698) after being noncovalently tagged by β-CD. Based on the characteristic mass shift, the suspected features were reduced by 1 order of magnitude, as numerous interfering species that could not be effectively tagged by β-CD were excluded. From these suspected features, two metabolites of BT that have not been reported before were successfully screened out. The significant characteristic mass shift caused by the noncovalent tagging method is easier to identify with more confidence than the previously reported SIL method. Besides, noncovalent tagging reagents can be much more accessible and less expensive than isotopically labeled reagents. Hence, this online noncovalent tagging method can be an intriguing alternative to the conventional SIL method.

Complement and Coagulation Cascades are Potentially Involved in Dopaminergic Neurodegeneration in α-Synuclein-Based Mouse Models of Parkinson's Disease.

Parkinson's disease (PD) is the second most common neurodegenerative disorder that results in motor dysfunction and, eventually, cognitive impairment. α-Synuclein protein is known as a central protein to the pathophysiology of PD, but the underlying pathological mechanism still remains to be elucidated. In an effort to understand how α-synuclein underlies the pathology of PD, various PD mouse models with α-synuclein overexpression have been developed. However, systemic analysis of the brain proteome of those mouse models is lacking. In this study, we established two mouse models of PD by injecting α-synuclein preformed fibrils (PFF) or by inducing overexpression of human A53T α-synuclein to investigate common pathways in the two different types of the PD mouse models. For more accurate quantification of mouse brain proteome, the proteins were quantified using the method of stable isotope labeling with amino acids in mammals . We identified a total of 8355 proteins from the two mouse models; ∼6800 and ∼7200 proteins from α-synuclein PFF-injected mice and human A53T α-synuclein transgenic mice, respectively. Through pathway analysis of the differentially expressed proteins common to both PD mouse models, it was discovered that the complement and coagulation cascade pathways were enriched in the PD mice compared to control animals. Notably, a validation study demonstrated that complement component 3 (C3)-positive astrocytes were increased in the ventral midbrain of the intrastriatal α-synuclein PFF-injected mice and C3 secreted from astrocytes could induce the degeneration of dopaminergic neurons. This is the first study that highlights the significance of the complement and coagulation pathways in the pathogenesis of PD through proteome analyses with two sophisticated mouse models of PD.

Stable Isotope Labeling by Carbon-13 in Bacteria Culture for the Analysis of Residual Avermectin Using Stable Isotope Dilution Liquid Chromatography Tandem Mass Spectrometry.

This study describes the development of a new stable isotope labeling method by carbon-13 in bacteria culture (SILCB) for the analysis of residual antibiotics via liquid chromatography with tandem mass spectrometry (LC-MS/MS). Stable isotope dilution (SID) LC-MS/MS with QuEChERS was employed to determine avermectin, particularly avermectin B1a (AV-a) and B1b (AV-b), based on completely 13C-labeled internal standards (13C-ISs) obtained from the SILCB. Our SILCB was developed from an optimal inorganic medium using 13C6-glucose for Streptomyces avermitilis (14893 strain). A rough extract containing 13C-ISs was purified via high-speed countercurrent chromatography with a volatile two-phase solvent system composed of n-hexane/ethyl acetate/methanol/0.5% formic acid in water (7/3/5/5/, V/V). The purified 13C-ISs were evaluated to confirm the presence of completely 13C-labeled ions with m/z 938 > 326 and m/z 923 > 309 for AV-a and AV-b, respectively. The QuEChERS approach with the 13C-ISs procedure achieved acceptable recovery rates in beef meat samples of 99.5 - 100.0% (RSD < 2.0%, n = 6). For the analysis of residual antibiotics in foodstuffs by SID-LC-MS/MS and QuEChERS, the SILCB represents a significant improvement over previous methods suffering from cumbersome sample preparation and matrix effects.

Lipids at the plant–animal interface: a stable isotope labelling method to evaluate the assimilation of essential fatty acids in the marine copepod Calanus finmarchicus

AbstractLinking production, transfer and subsequent bioavailability of nutritionally significant matter from phytoplankton to higher trophic levels is a fundamental aspect in understanding marine food webs. The plant–animal interface is of interest because of the highly variable transfer between producers and consumers, and the myriad of factors that influence it. Essential fatty acids (EFAs) are dietary nutrients that are necessary for normal function in all consumers, yet it remains unclear how efficiently they are transferred through marine food webs. We introduced a 13C-labelled carbon source to the cryptophyte Rhodomonas salina to quantify primary production of two omega-3 long-chain polyunsaturated fatty acids (PUFAs), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). We investigated transfer and assimilation efficiencies of these EFAs from phytoplankton to the calanoid copepod Calanus finmarchicus in an 8-day feeding experiment. We found low production of both EFAs in R. salina. Assimilation efficiencies of both EFAs ranged from 5 to 15% throughout the experiment, remaining slightly higher on average for DHA. This was mirrored in more efficient trophic transfer of DHA (up to 28%, compared to 13% for EPA). These results add to previously scarce experiments empirically quantifying the assimilation and transfer efficiency of EFAs in a basic marine planktonic food chain.

Increasing soil age drives shifts in plant-plant interactions from positive to negative and affects primary succession dynamics in a subalpine glacier forefield

The stress gradient hypothesis predicts that plant-plant interactions switch between facilitation (positive) and competition (negative) along environmental gradients, with facilitation being more common under high abiotic stress conditions relative to more moderate abiotic stress conditions. Our aim was to reveal, whether the interactions between Populus purdomii Rehder and Salix rehderiana Schneider switch from positive to negative during the early stages of primary succession in the Gongga Mountain glacier retreat region. We also investigated, whether soil age is a major driving factor for the transformation of interactions between neighboring plants. We analyzed differences between intraspecific interactions and interspecific interactions of Populus and Salix under 20- and 40-year-old soil conditions, including plant biomass accumulation and allocation, nutrient absorption and utilization, relative competition intensity, non-structural carbohydrates, foliar carbon and nitrogen isotope composition, mesophyll cell ultrastructure, soil microbial biomass and community structure, extracellular enzyme activities, and soil organic carbon (SOC), soil total nitrogen (TN), soil ammonium (NH4+-N), and soil nitrate (NO3−-N) contents. We found that P. purdomii and S. rehderiana growing under interspecific interactions had greater contents of aboveground dry matter, belowground dry matter and total dry matter compared to intraspecific interactions in 20-year-old soil. Furthermore, in 40-year-old soil conditions, the phospholipid fatty acid (PLFA) analysis showed that Populus and Salix exposed to interspecific interactions exhibited lower amounts of gram-positive bacteria, fungi (18,1 ω9c) and actinomycetes, and lower levels of total PLFAs than those growing under intraspecific interactions. The redundancy analysis (RDA) results demonstrated that soil N was the most important parameter contributing to the composition of microbial communities. In addition, the 15N stable isotope labeling method showed that Populus and Salix growing under interspecific interactions had higher foliage δ15N derived from NO3− (δ15N-NO3−) than those growing under intraspecific interactions in 20-year-old soil. In summary, our results demonstrated that Populus-Salix interactions exhibited positive effects on survival in 20-year-old soil. Conversely, under 40-year-old soil conditions, Populus-Salix interactions presented negative effects in relation to nutrients and elimination by neighboring plants. Moreover, soil age is a major driving factor for plant-plant interactions that shift from positive to negative with an increasing soil age in the Gongga Mountain glacier forefield. In all, our results support the stress gradient hypothesis. Our findings improve understanding of plant-plant interactions and plant-soil feedbacks during the early stages of soil development, and of the construction of vegetation communities.

Study on the cyanobacterial toxin metabolism of Microcystis aeruginosa in nitrogen-starved conditions by a stable isotope labelling method.

In this study, the biosynthesis of microcystins (MCs) was investigated after long-term nitrogen-starved conditions in cyanobacterium Microcystis aeruginosa. The results demonstrated that the algal cells were able to survive in a non-growing state with nitrogen starvation for more than one month. The physiological properties of the algal cells were studied to elucidate the mechanisms of viability under nitrogen-deprivation conditions. After the state of nitrogen chlorosis, new toxins could be resynthesized and tracked using 15N-stable isotope-labelled nitrogen. Nitrogen starvation of nutritionally replete cells resulted in a significant increase of microcystin-LY (MC-LY), thereby suggesting that MC-LY may undergo catabolism to provide nitrogen or that MC-LY may be produced to play an important role in the cell in response to nitrogen deprivation. The rank order of different types of nitrogen in algal cells assimilation was N-ammonium > N-urea > N-nitrate > N-alanine. The relationship between the production of toxin variants and various environmental conditions is an interesting issue for future research and may help improve the understanding of the ecological role of cyanobacterial toxins.

Identification of naringin metabolites mediated by human intestinal microbes with stable isotope-labeling method and UFLC-Q-TOF-MS/MS

Widely presented in medicinal plants, naringin is one of the major flavanones with various pharmaceutical bioactivities. After oral administration, naringin predominantly undergoes metabolisms mediated by liver cytochrome P450 and gut microbes, while its human microbes-mediated metabolic profiling is still largely obscure due to the endogenous interferences, which makes it extremely difficult to analyze metabolites precisely. In this study, we aim of systematically investigating the biotransformation of naringin mediated by human intestinal microbes through applying stable isotope-labeling method. [2′,3′,5′,6′-D4]naringin was synthesized and incubated anaerobically with human gut microbes. A total of 13 microbial metabolites were detected and identified by UFLC-Q-TOF-MS/MS, among which 5 were reported for the first time. Furthermore, the proposed metabolic pathway revealed that naringin went through extensive phase I metabolism in human intestinal microbes. Of note, diverse metabolic profiles of naringin among human participants were obtained, which could be attributed to the distinct gut microbiota compositions of individuals.

Study of the biochemical formation pathway of aroma compound 1-phenylethanol in tea (Camellia sinensis (L.) O. Kuntze) flowers and other plants

After tea leaves, tea (Camellia sinensis) flowers are becoming a second tea plant resource because they contain not only functional metabolites similar to those found in tea leaves, but also predominant amounts of functional metabolites that only occur in tea leaves in small amounts. 1-Phenylethanol (1PE) is a predominant aroma compound found in tea flowers. A 1PE synthase in tea flowers was isolated, functionally characterized, and shown to have the highest catalytic efficiency for the conversion of acetophenone (AP). To determine why 1PE accumulates more in tea flowers than other plants, we compared their 1PE contents and used a stable isotope labeling method to elucidate the 1PE biosynthetic route. Supplementation with [2H8]l-phenylalanine and [2H5]AP suggested that most plants containing the enzyme/gene catalyzed the conversion of AP to 1PE. Furthermore, the availability of AP derived from l-phenylalanine was responsible for the difference in 1PE accumulation between tea flowers and other plants.

Tauomics and Kinetics in Human Neurons and Biological Fluids

In this issue of Neuron, Sato etal. (2018) combine proteomics and a stable isotope labeling method to examine different forms of tau and their kinetics in human neurons, brain, and cerebrospinal fluid and how these properties relate to clinical state and pathology in Alzheimer's disease.

Stable isotope labeling and 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucopyranoside biosynthetic pathway characterization in Fallopia multiflora.

The THSG biosynthetic pathway in F. multiflora was characterized, and enzymatic activities responsible for the resveratrol synthesis, hydroxylation, and glycosylation reactions involved in THSG biosynthesis were confirmed in vitro. The biosynthetic origin of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucopyranoside (THSG) and the enzymes involved in THSG biosynthesis in Fallopia multiflora were studied using stable isotope labeling and biocatalytic methods. UPLC-MS-based analyses were used to unravel the isotopologue composition of the biosynthetic intermediates and products, as well as to detect the products of the enzyme assay experiments. In this study, 13C-labeled L-phenylalanine (L-PHE), sodium pyruvate (SP), and sodium bicarbonate (SB) were used as putative precursors in the feeding experiment. Labeling of polydatin (PD) and THSG using [13C9]L-PHE and [13C1]L-PHE confirmed that the p-coumaric moiety of PD and THSG was derived from PHE. The results of the feeding experiments with [13C] SB and [2, 3-13C2] SP suggested that PD and THSG were derivatives of resveratrol that were synthesized by glycosylation and hydroxylation. We developed methods using total crude protein extracts (soluble and microsomal) for comprehensive and simultaneous analysis of resveratrol synthase, glycosyltransferase, and hydroxylase activities in various tissue types of wild F. multiflora and callus cultures. The activity of each tested enzyme was confirmed in one or more tissue types or cell cultures in vitro. The results of the enzyme activity experiments and the distributions of PD and THSG were used to determine the main site and pathway of THSG biosynthesis in F. multiflora.

Quantitative O-glycomics based on improvement of the one-pot method for nonreductive O-glycan release and simultaneous stable isotope labeling with 1-(d0/d5)phenyl-3-methyl-5-pyrazolone followed by mass spectrometric analysis

Rapid, simple and versatile methods for quantitative analysis of glycoprotein O-glycans are urgently required for current studies on protein O-glycosylation patterns and the search for disease O-glycan biomarkers. Relative quantitation of O-glycans using stable isotope labeling followed by mass spectrometric analysis represents an ideal and promising technique. However, it is hindered by the shortage of reliable nonreductive O-glycan release methods as well as the too large or too small inconstant mass difference between the light and heavy isotope form derivatives of O-glycans, which results in difficulties during the recognition and quantitative analysis of O-glycans by mass spectrometry. Herein we report a facile and versatile O-glycan relative quantification strategy, based on an improved one-pot method that can quantitatively achieve nonreductive release and in situ chromophoric labeling of intact mucin-type O-glycans in one step. In this study, the one-pot method is optimized and applied for quantitative O-glycan release and tagging with either non-deuterated (d0-) or deuterated (d5-) 1-phenyl-3-methyl-5-pyrazolone (PMP). The obtained O-glycan derivatives feature a permanent 10-Da mass difference between the d0- and d5-PMP forms, allowing complete discrimination and comparative quantification of these isotopically labeled O-glycans by mass spectrometric techniques. Moreover, the d0- and d5-PMP derivatives of O-glycans also have a relatively high hydrophobicity as well as a strong UV adsorption, especially suitable for high-resolution separation and high-sensitivity detection by RP-HPLC-UV. We have refined the conditions for the one-pot reaction as well as the corresponding sample purification approach. The good quantitation feasibility, reliability and linearity of this strategy have been verified using bovine fetuin and porcine stomach mucin as model O-glycoproteins. Additionally, we have also successfully applied this method to the quantitative O-glycomic comparison between perch and salmon eggs by ESI-MS, MS/MS and online RP-HPLC-UV-ESI-MS/MS, demonstrating its excellent applicability to various complex biological samples. Biological significanceO-Linked glycoproteins, generated via a widely existing glycosylation modification process on serine (Ser) or threonine (Thr) residues of nascent proteins, play essential roles in a series of biological processes. As a type of informational molecule, the O-glycans of these glycoproteins participate directly in these biological mechanisms. Thus, the characteristic differences or changes of O-glycans in expression level usually relate to pathologies of many diseases and represent an important opportunity to uncover the functional mechanisms of various glycoprotein O-glycans. The novel strategy introduced here provides a simple and versatile analytical method for the precise quantitation of glycoprotein O-glycans by mass spectrometry, enabling rapid evaluation of the differences or changes of O-glycans in expression level. It is attractive for the field of quantitative/comparative O-glycomics, which has great significance for exploring the complex structure-function relationship of O-glycans, as well as for the search of O-glycan biomarkers of some major diseases and O-glycan related targets of some drugs.

NMR spectral analysis using prior knowledge

Signal assignment is a fundamental step for analyses of protein structure and dynamics with nuclear magnetic resonance (NMR). Main-chain signal assignment is achieved with a sequential assignment method and/or an amino-acid selective stable isotope labeling (AASIL) method. Combinatorial selective labeling (CSL) methods, as well as our labeling strategy, stable isotope encoding (SiCode), were developed to reduce the required number of labeled samples, since one of the drawbacks of AASIL is that many samples are needed. Signal overlapping in NMR spectra interferes with amino-acid determination by CSL and SiCode. Since spectral deconvolution by peak fitting with a gradient method cannot resolve closely overlapped signals, we developed a new method to perform both peak fitting and amino acid determination simultaneously, with a replica exchange Monte Carlo method, incorporating prior knowledge of stable-isotope labeling ratios and the amino-acid sequence of the protein.

Transformation kinetics of silver nanoparticles and silver ions in aquatic environments revealed by double stable isotope labeling

A double stable isotope labeling method (107AgNPs and 109AgNO3) was used to track the transformation kinetics of AgNPs and Ag+ in aquatic environments.