液质联用多反应监测法定量目标多肽或蛋白质

为建立优化的血浆内源性多肽提取方法,并且构建目标多肽和蛋白质的质谱定量方 法,本研究考察了超滤法、有机溶剂沉淀法和固相萃取法对血浆内源性多肽的提取效果 ,并通过Tricine-SDS-PAGE对提取效果进行比较.通过液相色谱串联质谱多反应监测 （MRM）分析,建立了多肽标准品ESAT-6定量方法,并将ESAT-6定量建立的液相色谱和质谱条件应用于蛋白质的定量,对多肽和蛋白质MRM定量的标准曲线进行了考 察.Tricine-SDS-PAGE结果表明,乙腈沉淀法是最佳的血浆内源性多肽提取方法,低分子量的多肽可以得到很好的富集,且能有效地去除高分子蛋白质的污染.液相色谱串联 质谱MRM法检测血浆内提取的多肽,标准曲线的线性较好,相关系数为0.999.另外,采 用MRM法对胶内分离的蛋白质进行定量,标准曲线的线性相关系数为0.995.综上所述, 本研究构建了一种简单有效的血浆多肽提取方法,通过液质联用MRM法成功地实现了目标多肽和蛋白质定量测定.该定量方法可以推广应用于复杂样品中的多肽和蛋白质的定 量分析.     

Immobilized Metal Affinity Chromatography Coupled to Multiple Reaction Monitoring Enables Reproducible Quantification of Phospho-signaling

A major goal in cell signaling research is the quantification of phosphorylation pharmacodynamics following perturbations. Traditional methods of studying cellular phospho-signaling measure one analyte at a time with poor standardization, rendering them inadequate for interrogating network biology and contributing to the irreproducibility of preclinical research. In this study, we test the feasibility of circumventing these issues by coupling immobilized metal affinity chromatography (IMAC)-based enrichment of phosphopeptides with targeted, multiple reaction monitoring (MRM) mass spectrometry to achieve precise, specific, standardized, multiplex quantification of phospho-signaling responses. A multiplex immobilized metal affinity chromatography- multiple reaction monitoring assay targeting phospho-analytes responsive to DNA damage was configured, analytically characterized, and deployed to generate phospho-pharmacodynamic curves from primary and immortalized human cells experiencing genotoxic stress. The multiplexed assays demonstrated linear ranges of ≥3 orders of magnitude, median lower limit of quantification of 0.64 fmol on column, median intra-assay variability of 9.3%, median inter-assay variability of 12.7%, and median total CV of 16.0%. The multiplex immobilized metal affinity chromatography- multiple reaction monitoring assay enabled robust quantification of 107 DNA damage-responsive phosphosites from human cells following DNA damage. The assays have been made publicly available as a resource to the community. The approach is generally applicable, enabling wide interrogation of signaling networks.Cell signaling research is faced with the challenging task of interrogating increasingly large numbers of analytes in “systems biology” approaches, while maintaining the high standards of integrity and reproducibility traditionally associated with the scientific approach. For example, studies interrogating complex systems, such as protein signaling networks, require quantification technologies capable of sensitive, specific, multiplexable, and reproducible application. However, recent reports have highlighted alarmingly high rates of irreproducibility in fundamental biological and pre-clinical studies (1, 2), as well as poor performance of affinity reagents used in traditional proteomic assay and detection platforms (3, 4). There is an imminent need for high quality assays, including highly characterized standards and detailed documentation of processes and procedures (5). To improve the translation of cell signaling discoveries into clinical application, we need reproducible and transferable technologies that enable higher throughput quantification of protein phosphorylation.Signaling dynamics through post-translational modifications (e.g. phosphorylation) are predominantly measured by Western blotting. Although this technique has led to many discoveries and is the de facto “gold standard,” it suffers from many drawbacks. Western blotting is a low throughput approach applied to individual analytes (i.e. no multiplexing) and is susceptible to erroneous interpretation when applied quantitatively (6). Alternative immunoassay platforms have emerged (e.g. immunohistochemistry, ELISA, mass cytometry, and bead-based or planar arrays), but suffer from similar limitations, namely specificity issues (because of cross-reactivity of antibodies), poor standardization, and difficulties in multiplexing.One alternative for quantifying phosphorylation is targeted, multiple reaction monitoring (MRM)¹ MS, a widely deployed technique in clinical laboratories for quantification of small molecules (7, 8). MRM is now also well established for precise and specific quantification of endogenous, proteotypic peptides relative to spiked-in stable isotope-labeled internal standards (9–11), and MRM can be applied to phosphopeptides (12–18). MRM assays can be run at high multiplex levels (19–21) and can be standardized to be highly reproducible across laboratories (22–24), even on an international stage (25). Because phosphorylation typically occurs at sub-stoichiometric levels and because phosphopeptides must compete for ionization with more abundant peptides, mass spectrometry-based analysis of phosphorylation requires an analyte enrichment step. Immuno-affinity enrichment approaches using anti-phospho-tyrosine antibodies (26) or panels of antibodies targeting signaling nodes (27) have been implemented with shotgun mass spectrometry. Although anti-peptide antibodies can also be used to enrich individual phosphopeptides upstream of MRM (28), the generation of these reagents is time-consuming and costly, limiting widespread uptake.Phosphopeptide enrichment based on metal affinity chromatography has recently matured into a reproducible approach (29). Immobilized metal affinity chromatography (IMAC) is widely used in discovery phosphoproteomic studies to enrich phosphopeptides upstream of shotgun-based mass spectrometry (30, 31). We hypothesized that a subset of the cellular phosphoproteome with favorable binding characteristics to the IMAC resin might be reproducibly recovered for quantification when coupled with quantitative MRM mass spectrometry, enabling robust IMAC-MRM assays without the need for an antibody.In this report, we: (1) demonstrate the feasibility of generating analytically robust, multiplex IMAC-MRM assays for quantifying cellular phospho-signaling, (2) present a semi-automated, 96-well format magnetic bead-based protocol for IMAC enrichment, (3) provide a catalogue of phosphopeptides that are highly amenable to IMAC-MRM quantification, and (4) make publicly available standard operating protocols (SOP) and fit-for-purpose analytical validation data for IMAC-MRM assays targeting 107 phospho-analytes, providing a community resource for study of the DNA damage response. The data suggest that the IMAC-MRM approach is generally applicable to signaling pathways, enabling wider interrogation of signaling networks.     

Multiplexed Quantitation of Endogenous Proteins in Dried Blood Spots by Multiple Reaction Monitoring - Mass Spectrometry

Dried blood spot (DBS) sampling, coupled with multiple reaction monitoring mass spectrometry (MRM-MS), is a well-established approach for quantifying a wide range of small molecule biomarkers and drugs. This sampling procedure is simpler and less-invasive than those required for traditional plasma or serum samples enabling collection by minimally trained personnel. Many analytes are stable in the DBS format without refrigeration, which reduces the cost and logistical challenges of sample collection in remote locations. These advantages make DBS sample collection desirable for advancing personalized medicine through population-wide biomarker screening. Here we expand this technology by demonstrating the first multiplexed method for the quantitation of endogenous proteins in DBS samples. A panel of 60 abundant proteins in human blood was targeted by monitoring proteotypic tryptic peptides and their stable isotope-labeled analogs by MRM. Linear calibration curves were obtained for 40 of the 65 peptide targets demonstrating multiple proteins can be quantitatively extracted from DBS collection cards. The method was also highly reproducible with a coefficient of variation of <15% for all 40 peptides. Overall, this assay quantified 37 proteins spanning a range of more than four orders of magnitude in concentration within a single 25 min LC/MRM-MS analysis. The protein abundances of the 33 proteins quantified in matching DBS and whole blood samples showed an excellent correlation, with a slope of 0.96 and an R² value of 0.97. Furthermore, the measured concentrations for 80% of the proteins were stable for at least 10 days when stored at −20 °C, 4 °C and 37 °C. This work represents an important first step in evaluating the integration of DBS sampling with highly-multiplexed MRM for quantitation of endogenous proteins.Dried Blood Spot (DBS)¹ samples have many advantages over blood serum or plasma and are the preferred clinical sample for newborn screening for metabolic diseases (1, 2). These samples are collected by pricking a newborn''s heel and spotting a drop of blood onto specially designed filter paper collection cards. Samples are then dried under ambient conditions and are usually stored with desiccant at room temperature until analysis. This sampling procedure is simpler and less invasive then intravenous blood draws, which require a trained phlebotomist. Not surprisingly, the majority of adult patients prefer the small lancet used in finger-prick blood sampling methods to the larger needles used in intravenous blood draws (3, 4). Unlike plasma or serum samples, which consume ≥250 μl of blood and must be centrifuged within an hour of collection, DBS samples can be prepared using a volume of only 10 μl, and do not require any specialized equipment at the collection site (5). The simplicity and reduced safety risks associated with DBS sampling enables collection by minimally trained staff or by the patients themselves. In addition, many analytes are stable in the DBS format at room temperature, reducing sample transportation and storage costs, as well as the impact on the environment. Finally, DBS samples are safer to transport and are considered exempt from dangerous goods regulations (6, 7). These advantages make DBS sampling very attractive for advancing personalized medicine and population-based biomarker research (8).Numerous biomolecular targets covering genomics, metabolomics, and proteomics applications have been quantified in DBS samples using a wide array of analytical techniques (9). The most common clinical application of DBS sampling is screening newborns for metabolomics disorders by targeting small molecule biomarkers. Early screening programs relied on bacterial inhibition assays and later immunoassays, both of which required a different assay for each target of interest (2). However, the time and cost required to perform each assay independently has limited the number of diseases that could be screened nationwide to only a handful. In addition, a single biomarker often lacked the specificity to produce a definitive diagnosis, requiring extensive secondary testing. Hemoglobin is the only protein that is commonly targeted in DBS samples, and primary screening is accomplished by high-performance liquid chromatography (HPLC) or isoelectric focusing (IEF) methods (2). Similar to small-molecule screening methods, these approaches are low-throughput and are not amendable to multiplexing with additional protein targets. In newborn screening programs, these challenges associated with small molecule analysis were overcome with the introduction of multiple reaction monitoring mass spectrometry (MRM-MS) into the clinical laboratories (1, 10). The specificity of MRM enables hundreds of analytes to be monitored during a single experiment to facilitate the development of highly multiplexed assays. The addition of stable isotope-labeled internal standards (SIS) enables the acquisition of highly reproducible results across a variety of instrumentation at different institutions. It is now common for 20–30 small molecule targets including amino acids, fatty acid acylcarnitines, and organic acid acylcarnitines to be analyzed by flow injection MRM-MS, at a cost of $10–20 USD per patient sample (11). Expansion of the screening panel to include additional small-molecule biomarkers on an existing platform may cost less than $1 each. In addition to newborn screening, DBS sampling combined with MS is also gaining acceptance in small-molecule drug development (12, 13). Here the collection of smaller blood volumes allows serial sampling from mice reducing the total number of animals required to generate preclinical toxicology and pharmacokinetic data (5).Despite the successful use of DBS samples in MS-based experiments for small molecule analysis, there have been few reports of using this technology for protein targets (13). Daniel and coworkers reported a screening method for identifying β-thalassemia using the well-known biomarker HbA₂, a hemoglobin variant composed of two alpha- and two delta-globin subunits (14). Proteins were extracted in an aqueous solution, digested with trypsin in 30 min and infused for MRM-MS analysis. Multiple hemoglobin peptides were targeted to measure the abundance of the delta-globin chain, using peptides from the beta-globin chain as an internal standard. This ratio correlated well with the abundance of intact HbA₂ as determined by a well-established HPLC method. The shorter acquisition time and the increased specificity of the MS-based method showed promise for improving population-wide screening. Boemer et al. used a similar flow injection MRM-MS strategy to screen newborns for hemoglobin variants associated with sickle cell disease (15). They analyzed more than 2000 DBS samples by targeting tryptic peptides that were unique to four different beta-globin mutations and compared their results with a standard IEF method that measured the intact proteins obtained from corresponding whole blood samples. Their flow injection MRM approach was able to identify the correct phenotype for all targeted variants. Recently, deWilde et al. reported a method for screening newborn DBS samples for ceruloplasmin, a protein linked to Wilson''s disease (16). Their method combined SIS peptides with LC/MRM-MS and produced results similar to those from an immunoassay for the analysis of seven patient samples. The monitoring of a therapeutic protein in rat blood was demonstrated with Kehler et al. to evaluate the suitability of this approach for supporting preclinical trials (17). Finally, a multiplexed approach was presented by Sleczka et al. for the simultaneous quantitation of two therapeutic proteins in spiked DBS samples collected from several animal models (18).In all previous methods, only one to two proteins were targeted in DBS samples and therefore the true multiplexing capabilities of MRM were not realized. MRM-based methods using SIS peptides have already proven proficient at highly multiplexed quantitation of proteins in plasma and serum samples (19–21). Our current work demonstrates the potential for integrating DBS sampling with LC/MRM-MS for highly multiplexed quantitation of endogenous proteins. Many of the 60 proteins that we have targeted have been cleared or approved by FDA, and are already being analyzed one at a time in clinical laboratories. The assay developed in this study includes a highly reproducible method for extracting multiple proteins from DBS samples followed by trypsin digestion and surrogate peptides, along with their SIS analogs, were analyzed by a standard-flow LC/MRM-MS platform that has previously been shown to give accurate, sensitive, and robust analysis of proteotypic peptides in human plasma (21, 22). The quantitative results from whole blood and the corresponding DBS samples were compared, and the integrity of DBS samples stored under various temperatures has been evaluated.     

Megadalton Complexes in the Chloroplast Stroma of Arabidopsis thaliana Characterized by Size Exclusion Chromatography,Mass Spectrometry,and Hierarchical Clustering

To characterize MDa-sized macromolecular chloroplast stroma protein assemblies and to extend coverage of the chloroplast stroma proteome, we fractionated soluble chloroplast stroma in the non-denatured state by size exclusion chromatography with a size separation range up to ∼5 MDa. To maximize protein complex stability and resolution of megadalton complexes, ionic strength and composition were optimized. Subsequent high accuracy tandem mass spectrometry analysis (LTQ-Orbitrap) identified 1081 proteins across the complete native mass range. Protein complexes and assembly states above 0.8 MDa were resolved using hierarchical clustering, and protein heat maps were generated from normalized protein spectral counts for each of the size exclusion chromatography fractions; this complemented previous analysis of stromal complexes up to 0.8 MDa (Peltier, J. B., Cai, Y., Sun, Q., Zabrouskov, V., Giacomelli, L., Rudella, A., Ytterberg, A. J., Rutschow, H., and van Wijk, K. J. (2006) The oligomeric stromal proteome of Arabidopsis thaliana chloroplasts. Mol. Cell. Proteomics 5, 114–133). This combined experimental and bioinformatics analyses resolved chloroplast ribosomes in different assembly and functional states (e.g. 30, 50, and 70 S), which enabled the identification of plastid homologues of prokaryotic ribosome assembly factors as well as proteins involved in co-translational modifications, targeting, and folding. The roles of these ribosome-associating proteins will be discussed. Known RNA splice factors (e.g. CAF1/WTF1/RNC1) as well as uncharacterized proteins with RNA-binding domains (pentatricopeptide repeat, RNA recognition motif, and chloroplast ribosome maturation), RNases, and DEAD box helicases were found in various sized complexes. Chloroplast DNA (>3 MDa) was found in association with the complete heteromeric plastid-encoded DNA polymerase complex, and a dozen other DNA-binding proteins, e.g. DNA gyrase, topoisomerase, and various DNA repair enzymes. The heteromeric ≥5-MDa pyruvate dehydrogenase complex and the 0.8–1-MDa acetyl-CoA carboxylase complex associated with uncharacterized biotin carboxyl carrier domain proteins constitute the entry point to fatty acid metabolism in leaves; we suggest that their large size relates to the need for metabolic channeling. Protein annotations and identification data are available through the Plant Proteomics Database, and mass spectrometry data are available through Proteomics Identifications database.Chloroplasts are essential plant organelles of prokaryotic origin that perform a variety of metabolic and signaling functions. Best known for their role in photosynthesis, they also carry out the biosynthesis of many primary and secondary metabolites like lipids, amino acids, vitamins, nucleotides, tetrapyrroles, and hormones (1). Subcellular localization prediction by TargetP (2) combined with a correction for false positive and false negative rates suggested that all non-green plastid types and chloroplasts together contain some 3500 proteins in Arabidopsis thaliana (3). More than 95% of the chloroplast proteins are nucleus-encoded and post-translationally imported into the chloroplast (4–6). Over the last decade, several studies were published that aimed to identify (subfractions of) the Arabidopsis chloroplast proteome (e.g. Refs. 7–10). The precise number of bona fide chloroplast proteins from these proteomics studies is probably somewhere around 1000–1300; comparing this number with the predicted chloroplast proteome indicates that ∼50% of the proteome has still not been observed. Recently, we concluded that when compared with the predicted Arabidopsis chloroplast proteome the chloroplast proteome identified to date is particularly underrepresented (40–70%) for proteins involved in signaling, stress, development, unassigned function, and DNA/RNA metabolism (9). To probe deeper into the chloroplast proteome, enrichment for low abundance proteins prior to MS analysis is required.Many biochemical functions are executed by protein assemblies. Several studies have catalogued the assembly states of chloroplast proteins in plants. Separation of the oligomeric Arabidopsis stromal proteome by two-dimensional native gel electrophoresis (CN¹-PAGE) profiled 240 non-redundant proteins and captured information for 124 complexes (11). However, native gel electrophoresis has a practical size limit, and only protein complexes below ∼1000 kDa can be effectively separated, thereby missing megadalton-sized complexes. Several megadalton-sized complexes in plants have been characterized by targeted purification schemes, including the spinach 30 and 50 S ribosomal particles (12–14), cytosolic ribosomes (15, 16), the tobacco plastid-encoded RNA polymerase (PEP) complex (17), maize mitochondrial pyruvate dehydrogenase complex (PDC) (18), and pea chloroplast acetyl-CoA carboxylase (ACCase) complex (19). Proteome characterization of a membrane-depleted, Triton-insoluble, and high density pellet from pea plastids was highly enriched for the chloroplast PDC as well as proteins involved in plastid gene expression and carbon fixation (20). However, because no subsequent fractionation was performed, specific protein associations could not be resolved.To extend chloroplast proteome coverage and characterize MDa-sized macromolecular assemblies to complement the previous CN-PAGE analysis of complexes up to 0.8 MDa, we fractionated the soluble chloroplast stroma by size exclusion chromatography (SEC) with a particular focus on complexes greater than 0.8 MDa. Proteins were identified by mass spectrometry analysis using an LTQ-Orbitrap, a high accuracy and high sensitivity hybrid instrument (21, 22). SEC migration profiles for identified proteins were generated from matched spectral counts. Hierarchical clustering and protein heat maps of the SEC migration profiles revealed that the identified protein complexes include 30, 50, and 70 S ribosomal particles; PDC; PEP; and ACCase, indicating successful MDa size fractionation. In addition, many “new” proteins were detected, and they were enriched for functions in plastid gene expression, in particular putative ribosomal biogenesis factors. Finally, protein annotations and identification data are available via the Plant Proteomics Database (PPDB) at http://ppdb.tc.cornell.edu/, and mass spectrometry data with their metadata were deposited in the Proteomics Identifications database (PRIDE) (http://www.ebi.ac.uk/pride/) under accession numbers 11459–11568.The concept of using chromatography (or other continuous fractionation techniques) of protein complexes (or other types of cellular protein fractions) with mass spectrometry-based quantification to determine co-localization has been applied using stable isotope labeling (23, 24) or label-free techniques (25, 26). When combined with cluster analysis (this study and Ref. 24), principle component analysis (23), or correlation of normalized elution profiles (this study and Refs. 25 and 26), this strategy is clearly a powerful tool and is widely applicable to other subcellular proteomes.     

Identification of Microalgae by Laser Desorption/Ionization Mass Spectrometry Coupled with Multiple Nanomatrices

In this study, we demonstrate a simple method to identify microalgae by surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS) using three different substrates: HgSe, HgTe, and HgTeSe nanostructures. The fragmentation/ionization processes of complex molecules in algae varied according to the heat absorption and transfer efficiency of the nanostructured matrices (NMs). Therefore, the mass spectra obtained for microalgae showed different patterns of m/z values for different NMs. The spectra contained both significant and nonsignificant peaks. Constructing a Venn diagram with the significant peaks obtained for algae when using HgSe, HgTe, and HgTeSe NMs in m/z ratio range 100–1000, a unique relationship among the three sets of values was obtained. This unique relationship of sets is different for each species of microalgae. Therefore, by observing the particular relationship of sets, we successfully identified different algae such as Isochrysis galbana, Emiliania huxleyi, Thalassiosira weissflogii, Nannochloris sp., Skeletonema cf. costatum, and Tetraselmis chui. This simple and cost-effective SALDI-MS analysis method coupled with multi-nanomaterials as substrates may be extended to identify other microalgae and microorganisms in real samples.

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Graphical Abstract Identification of microalgae by surface-assisted laser desorption/ionization mass spectrometry coupled with three different mercury-based nanosubstrates

     

Mathematical Modeling of Size Exclusion Chromatography

A mathematical model of the size exclusion chromatography (SEC) process in chromatographic columns has been developed. It considers the following three mass transfer processes in the SEC column: axial dispersion in the bulk‐fluid phase, interfacial film mass‐transfer between the stationary and mobile phases, and diffusion of solutes within the macro pores of the packing particles. Differential equations of the process model were solved by the finite difference method. Characteristics of the column and the packing particles (bed void volume fraction, particle porosity, accessible particle porosity) were obtained experimentally, as well as retention times of different molecules with known molecular weights. Experiments were performed with two different columns containing two different packing materials, Superdex 75 HR 10/30 and BioSep SEC S2000, respectively. The model has been validated by comparing theoretical and experimental retention times for the different columns.     

蛋白质的排阻色谱复性的新进展

外源蛋白在大肠杆菌中高效表达时 ,常常形成不溶的、无活性的包涵体 ,包涵体蛋白的复性是重组蛋白生产过程中的一个技术难题。排阻色谱 (sizeexclusionchromatography ,SEC)用于蛋白复性是一种较新的、适用于任何一种蛋白的方法 ,与常用的稀释复性法相比 ,它能在高的起始蛋白浓度下对蛋白进行复性 ,活性回收率较高 ,同时又能使目标蛋白得到一定程度的纯化。对使用SEC复性的进展进行了评述 ,其内容包括SEC复性的原理及其复性过程中的影响因素 ,并对其未来发展进行了展望。     

Simultaneous Quantification of Multiple Urinary Naphthalene Metabolites by Liquid Chromatography Tandem Mass Spectrometry

Naphthalene is an environmental toxicant to which humans are exposed. Naphthalene causes dose-dependent cytotoxicity to murine airway epithelial cells but a link between exposure and human pulmonary disease has not been established. Naphthalene toxicity in rodents depends on P450 metabolism. Subsequent biotransformation results in urinary elimination of several conjugated metabolites. Glucuronide and sulfate conjugates of naphthols have been used as markers of naphthalene exposure but, as the current studies demonstrate, these assays provide a limited view of the range of metabolites generated from the parent hydrocarbon. Here, we present a liquid chromatography tandem mass spectrometry method for measurement of the glucuronide and sulfate conjugates of 1-naphthol as well as the mercapturic acids and N-acetyl glutathione conjugates from naphthalene epoxide. Standard curves were linear over 2 log orders. On column detection limits varied from 0.91 to 3.4 ng; limits of quantitation from 1.8 to 6.4 ng. The accuracy of measurement of spiked urine standards was -13.1 to + 5.2% of target and intra-day and inter-day variability averaged 7.2 (± 4.5) and 6.8 (± 5.0) %, respectively. Application of the method to urine collected from mice exposed to naphthalene at 15 ppm (4 hrs) showed that glutathione-derived metabolites accounted for 60-70% of the total measured metabolites and sulfate and glucuronide conjugates were eliminated in equal amounts. The method is robust and directly measures several major naphthalene metabolites including those derived from glutathione conjugation of naphthalene epoxide. The assays do not require enzymatic deconjugation, extraction or derivatization thus simplifying sample work up.     

Qualitative Profiling and Quantification of Neonicotinoid Metabolites in Human Urine by Liquid Chromatography Coupled with Mass Spectrometry

Neonicotinoid pesticides have been widely applied for the production of fruits and vegetables, and occasionally detected in conventionally grown produce. Thus oral exposure to neonicotinoid pesticides may exist in the general population; however, neonicotinoid metabolites in human body fluids have not been investigated comprehensively. The purpose of this study is the qualitative profiling and quantitative analysis of neonicotinoid metabolites in the human spot urine by liquid chromatography coupled with mass spectrometry (LC/MS). Human urine samples were collected from three patients suspected of subacute exposure to neonicotinoid pesticides. A qualitative profiling of urinary metabolites was performed using liquid chromatography/time-of-flight mass spectrometry (LC/TOFMS) with a database of nominal molecular weights of 57 known metabolites of three neonicotinoid pesticides (acetamiprid, Imidacloprid, and clothianidin), as well as the parent compounds. Then a quantitative analysis of selected urinary metabolites was performed using liquid chromatography/tandem mass spectrometry (LC/MS/MS) with a standard pesticide and metabolite, which were detected by the qualitative profiling. The result of qualitative profiling showed that seven metabolites, i.e. an acetamiprid metabolite, N-desmethyl-acetamiprid; three Imidacloprid metabolites, 5-hydroxy-Imidacloprid, 4,5-dihydroxy-imidacloprid, 4,5-dehydro-Imidacloprid; a common metabolite of acetamiprid and Imidacloprid, N-(6-chloronicotinoyl)-glycine; and two clothianidin metabolites, N-desmethyl-clothianidin, N-(2-(methylsulfanyl)thiazole-5-carboxyl)-glycine, as well as acetamiprid, were detected in the urine of three cases. The result of the quantitative analysis showed N-desmethyl-acetamiprid was determined in the urine of one case, which had been collected on the first visit, at a concentration of 3.2 ng/mL. This is the first report on the qualitative and quantitative detection of N-desmethyl-acetamiprid in the human urine. The results suggest that the one case with detection of N-desmethyl-acetamiprid was exposed to acetamiprid through the consumption of contaminated foods. Urinary N-desmethyl-acetamiprid, as well as 5-hydroxy-Imidacloprid and N-desmethyl-clothianidin, may be a good biomarker for neonicotinoid exposure in humans and warrants further investigation.     

Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This protocol provides step-by-step instructions for the development and application of quantitative assays using selected reaction monitoring (SRM) mass spectrometry (MS). First, likely quantotypic target peptides are identified based on numerous criteria. This includes identifying proteotypic peptides, avoiding sites of posttranslational modification, and analyzing the uniqueness of the target peptide to the target protein. Next, crude external peptide standards are synthesized and used to develop SRM assays, and the resulting assays are used to perform qualitative analyses of the biological samples. Finally, purified, quantified, heavy isotope labeled internal peptide standards are prepared and used to perform isotope dilution series SRM assays. Analysis of all of the resulting MS data is presented. This protocol was used to accurately assay the absolute abundance of proteins of the chemotaxis signaling pathway within RAW 264.7 cells (a mouse monocyte/macrophage cell line). The quantification of G_i2 (a heterotrimeric G-protein α-subunit) is described in detail.     

Urinary Prognostic Biomarkers and Classification of IgA Nephropathy by High Resolution Mass Spectrometry Coupled with Liquid Chromatography

IgA nephropathy is the most common cause of primary glomerulonephritis. There are different pathologic biopsy-based scoring systems in use, but there is no consensus among nephrologists yet regarding the best classification method. Our aim was to test urine proteomics as a non-invasive method for classification of IgA nephropathy. This aim was pursued by discovering novel prognostic protein biomarkers in urine, and linking them to pathogenesis of the disease through known signaling and metabolic pathways. 13 urine samples of the patients with biopsy-proven IgA nephropathy were analyzed via two proteomics approaches: nanoflow LC-MS/MS and GeLC-MS/MS. The results of label-free quantification were subjected to multivariate statistical analysis, which could classify patients into two groups, broadly corresponding to the primary and advance stages. The proteome classification correlated well with biopsy-based scoring systems, especially endocapillary hypercellularity score of the Oxford’s classification. Differentially excreted candidate proteins were found as potential prognostic biomarkers: afamin, leucine-rich alpha-2-glycoprotein, ceruloplasmin, alpha-1-microgolbulin, hemopexin, apolipoprotein A-I, complement C3, vitamin D-binding protein, beta-2-microglobulin, and retinol-binding protein 4. Pathway analysis suggested impairment of Extra Cellular Matrix (ECM)-Receptor Interaction pathways as well as activation of complement and coagulation pathway in progression of IgA nephropathy.     

体积排阻液相色谱法质粒的含量测定

首次以体积排阻液相色谱法进行质粒提取液浓度的测定研究,通过纯质粒溶液在260 nm处的紫外吸峰面积与质粒浓度之间数据的线性最小二乘拟合,得到了反映质粒浓度和紫外吸收峰峰面积之间关系的标准曲线:Y=2×10~(-5) X,通过验证实验,确定了质粒提取液中不同组分,如核糖核酸,蛋白质等在色谱图中的出峰位置.结合标准曲线测定了自制的质粒提取液的质粒浓度为0.32 mg/mL.该方法方便,快捷,准确.     

Symmetric Assembly of a Decameric Subcomplex in Human Multi-tRNA Synthetase Complex Via Interactions between Glutathione Transferase-Homology Domains and Aspartyl-tRNA Synthetase

Aminoacyl-tRNA synthetases (AARSs) ligate amino acids to their cognate tRNAs during protein synthesis. In humans, eight AARSs and three non-enzymatic AARS-interacting multifunctional proteins (AIMP1–3), which are involved in various biological processes, form a multi-tRNA synthetase complex (MSC). Elucidation of the structures and multiple functions of individual AARSs and AIMPs has aided current understanding of the structural arrangement of MSC components and their assembly processes. Here, we report the crystal structure of a complex comprising a motif from aspartyl-tRNA synthetase (DRS) and the glutathione transferase (GST)-homology domains of methionyl-tRNA synthetase (MRS), glutamyl-prolyl-tRNA synthetase (EPRS), AIMP2, and AIMP3. In the crystal structure, the four GST domains are assembled in the order of MRS-AIMP3-EPRS-AIMP2, and the GST domain of AIMP2 binds DRS through the β-sheet in the GST domain. The C-terminus of AIMP3 enhances the binding of DRS to the tetrameric GST complex. A DRS dimer and two GST tetramers binding to the dimer with 2-fold symmetry complete a decameric complex. The formation of this complex enhances the stability of DRS and enables it to retain its reaction intermediate, aspartyl adenylate. Since the catalytic domains of MRS and EPRS are connected to the decameric complex through their flexible linker peptides, and lysyl-tRNA synthetase and AIMP1 are also linked to the complex via the N-terminal region of AIMP2, the DRS–GST tetramer complex functions as a frame in the MSC.     

Metabolite Profiling of a Diverse Collection of Wheat Lines Using Ultraperformance Liquid Chromatography Coupled with Time-of-Flight Mass Spectrometry

Genetic differences among major types of wheat are well characterized; however, little is known about how these distinctions affect the small molecule profile of the wheat seed. Ethanol/water (65% v/v) extracts of seed from 45 wheat lines representing 3 genetically distinct classes, tetraploid durum (Triticum turgidum subspecies durum) (DW) and hexaploid hard and soft bread wheat (T. aestivum subspecies aestivum) (BW) were subjected to ultraperformance liquid chromatography coupled with time-of-flight mass spectrometry (UPLC-TOF-MS). Discriminant analyses distinguished DW from BW with 100% accuracy due to differences in expression of nonpolar and polar ions, with differences attributed to sterol lipids/fatty acids and phospholipids/glycerolipids, respectively. Hard versus soft BW was distinguished with 100% accuracy by polar ions, with differences attributed to heterocyclic amines and polyketides versus phospholipid ions, respectively. This work provides a foundation for identification of metabolite profiles associated with desirable agronomic and human health traits and for assessing how environmental factors impact these characteristics.     

Targeted Quantitation of Site-Specific Cysteine Oxidation in Endogenous Proteins Using a Differential Alkylation and Multiple Reaction Monitoring Mass Spectrometry Approach

Reactive oxygen species (ROS) are both physiological intermediates in cellular signaling and mediators of oxidative stress. The cysteine-specific redox-sensitivity of proteins can shed light on how ROS are regulated and function, but low sensitivity has limited quantification of the redox state of many fundamental cellular regulators in a cellular context. Here we describe a highly sensitive and reproducible oxidation analysis approach (OxMRM) that combines protein purification, differential alkylation with stable isotopes, and multiple reaction monitoring mass spectrometry that can be applied in a targeted manner to virtually any cysteine or protein. Using this approach, we quantified the site-specific cysteine oxidation status of endogenous p53 for the first time and found that Cys182 at the dimerization interface of the DNA binding domain is particularly susceptible to diamide oxidation intracellularly. OxMRM enables analysis of sulfinic and sulfonic acid oxidation levels, which we validate by assessing the oxidation of the catalytic Cys215 of protein tyrosine phosphatase-1B under numerous oxidant conditions. OxMRM also complements unbiased redox proteomics discovery studies as a verification tool through its high sensitivity, accuracy, precision, and throughput.Oxidation of cysteine residues plays a critical role in modifying the structure and function of many proteins. Although cysteine oxidation is a tightly regulated biological process, nonenzymatic processes can contribute substantially to its levels, such as during oxidative stress. Regulatory oxidation states such as disulfide bonding and S-nitrosylation are readily modulated (1) and play an essential role in many physiological processes, including cell cycle, growth, death, and differentiation (2). In contrast, prolonged accumulation of reactive oxygen species is associated with many pathological conditions and leads to stable overoxidized states (sulfinic and sulfonic acid) that may disrupt redox regulation and protein function (3) and, in most cases, are thought to be nonregenerative.Assays capable of comprehensively assessing the dynamic changes in site-specific oxidation states are especially critical to understanding the contribution of redox status to many diseases. Numerous redox-sensitive proteins, including essential cellular regulators such as p53, have been described previously (for review, see ref. 4). However, technical factors have hampered the identification of specific site(s) of modification and characterization of their redox status in cells. Site-directed mutagenesis is often employed to determine whether specific cysteines have redox-regulated functional roles (1), but this approach provides no information on the oxidation status of the endogenous protein. In addition, cysteine oxidation is dynamically dependent on the concentration, location, and specificity of small-molecule oxidants (5) and regulators of various antioxidant enzymes (6). Thiol pKa (7), solvent accessibility, and subcellular compartment (8, 9) also contribute to the dynamics of cysteine oxidation. Because the interface between chronic oxidative stress and disruption of essential cellular signaling has substantial biological relevance to disease and age-related pathological conditions (10–13), there is a strong need to develop sensitive and flexible assays capable of quantifying dynamic changes in the redox status of specific endogenous proteins.Direct analysis of most regulatory cysteine modifications is not suitable for robust quantitation because the modifications tend to be labile and susceptible to artifactual changes. Accurate preservation of the thiol oxidation state is commonly achieved with a three-step differential alkylation labeling strategy in which nonoxidized cysteines are 1) labeled with a tag, 2) chemically reduced, and 3) labeled with a distinguishing tag. The value of this process is that it replaces the labile oxidation-modified cysteines with highly stable alkylated forms (1). Differential alkylation specifically targets cysteine oxidation species that are susceptible to reaction with chemical reductants (DTT or TCEP¹) because higher oxidation states such as sulfinic and sulfonic acid are chemically irreversible. Fluorescent or epitope tags have been employed to evaluate redox sensitivity at the protein level (14, 15); however, combining differential alkylation using stable isotope-labeled reagents with mass spectrometry simultaneously identifies the specifically oxidized site and quantifies its reversible oxidation status. Although labeled iodoacetic acid (16) and N-ethylmaleimide (17) (NEM) have often been used, commercial ICAT reagents have become the preferred stable isotope label for redox analysis (18–21). A recent ICAT-based study identified and quantified the reversible oxidation of 120 redox-sensitive cysteines in Escherichia coli in an unbiased manner (18). However, because of the limited sensitivity, dynamic range, number of testable conditions, and stochastic sampling inherent in unbiased proteomics experiments, this approach has limited utility for interrogating targeted moderate- to low-abundance proteins or comprehensively characterizing multiple cysteines within a single protein. The use of an ICAT-based approach for targeted analysis of specific proteins is significantly limited by both the difficulty in scaling down the ICAT protocol and the disruption of protein structure that occurs after alkylation with numerous ICAT adducts, each over a kilodalton in size, which may occlude antibody epitopes useful for immunoaffinity enrichment.To overcome these limitations, we developed a highly sensitive method, OxMRM, that integrates protein purification, differential alkylation using a generic d₅ stable isotope-labeled NEM, and multiple reaction monitoring (MRM). OxMRM can quantify the oxidation status, both reversible and irreversible, of virtually any targeted cysteine or protein, even if in low abundance. We validate the OxMRM approach using the low-abundance tumor suppressor protein p53 and an established overoxidizable signaling regulator, protein tyrosine phosphatase-1B (PTP1B) (22, 23). These proteins serve as benchmark examples of nuclear and cytoplasmic redox-regulated proteins bearing reversibly and irreversibly oxidized cysteines. The flexibility of the OxMRM method allows it to be applied to essentially any protein or cysteine of interest with equal ease, allowing in-depth, site-specific analysis of putative and established redox-sensitive proteins. This makes OxMRM an ideal complement to large-scale redox proteomics studies as a verification tool with high sensitivity, precision, accuracy, and capacity to assess numerous oxidation conditions.