Profiling Thiol Redox Proteome Using Isotope Tagging Mass Spectrometry

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on Arabidopsis thaliana, a genetically tractable host plant^1,2. The accumulation of reactive oxygen species (ROS) in cotyledons inoculated with DC3000 indicates a role of ROS in modulating necrotic cell death during bacterial speck disease of tomato³. Hydrogen peroxide, a component of ROS, is produced after inoculation of tomato plants with Pseudomonas³. Hydrogen peroxide can be detected using a histochemical stain 3''-3'' diaminobenzidine (DAB)⁴. DAB staining reacts with hydrogen peroxide to produce a brown stain on the leaf tissue⁴. ROS has a regulatory role of the cellular redox environment, which can change the redox status of certain proteins⁵. Cysteine is an important amino acid sensitive to redox changes. Under mild oxidation, reversible oxidation of cysteine sulfhydryl groups serves as redox sensors and signal transducers that regulate a variety of physiological processes^6,7. Tandem mass tag (TMT) reagents enable concurrent identification and multiplexed quantitation of proteins in different samples using tandem mass spectrometry^8,9. The cysteine-reactive TMT (cysTMT) reagents enable selective labeling and relative quantitation of cysteine-containing peptides from up to six biological samples. Each isobaric cysTMT tag has the same nominal parent mass and is composed of a sulfhydryl-reactive group, a MS-neutral spacer arm and an MS/MS reporter¹⁰. After labeling, the samples were subject to protease digestion. The cysteine-labeled peptides were enriched using a resin containing anti-TMT antibody. During MS/MS analysis, a series of reporter ions (i.e., 126-131 Da) emerge in the low mass region, providing information on relative quantitation. The workflow is effective for reducing sample complexity, improving dynamic range and studying cysteine modifications. Here we present redox proteomic analysis of the Pst DC3000 treated tomato (Rio Grande) leaves using cysTMT technology. This high-throughput method has the potential to be applied to studying other redox-regulated physiological processes.     

Quantitative Mass Spectrometric Profiling of Cancer-cell Proteomes Derived From Liquid and Solid Tumors

In-depth analyses of cancer cell proteomes are needed to elucidate oncogenic pathomechanisms, as well as to identify potential drug targets and diagnostic biomarkers. However, methods for quantitative proteomic characterization of patient-derived tumors and in particular their cellular subpopulations are largely lacking. Here we describe an experimental set-up that allows quantitative analysis of proteomes of cancer cell subpopulations derived from either liquid or solid tumors. This is achieved by combining cellular enrichment strategies with quantitative Super-SILAC-based mass spectrometry followed by bioinformatic data analysis. To enrich specific cellular subsets, liquid tumors are first immunophenotyped by flow cytometry followed by FACS-sorting; for solid tumors, laser-capture microdissection is used to purify specific cellular subpopulations. In a second step, proteins are extracted from the purified cells and subsequently combined with a tumor-specific, SILAC-labeled spike-in standard that enables protein quantification. The resulting protein mixture is subjected to either gel electrophoresis or Filter Aided Sample Preparation (FASP) followed by tryptic digestion. Finally, tryptic peptides are analyzed using a hybrid quadrupole-orbitrap mass spectrometer, and the data obtained are processed with bioinformatic software suites including MaxQuant. By means of the workflow presented here, up to 8,000 proteins can be identified and quantified in patient-derived samples, and the resulting protein expression profiles can be compared among patients to identify diagnostic proteomic signatures or potential drug targets.     

Mass Spectrometry-based Quantitative Proteomic Profiling of Human Pancreatic and Hepatic Stellate Cell Lines

The functions of the liver and the pancreas differ; however, chronic inflammation in both organs is associated with fibrosis. Evidence suggests that fibrosis in both organs is partially regulated by organ-specific stellate cells. We explore the proteome of human hepatic stellate cells (hHSC) and human pancreatic stellate cells (hPaSC) using mass spectrometry (MS)-based quantitative proteomics to investigate pathophysiologic mechanisms. Proteins were isolated from whole cell lysates of immortalized hHSC and hPaSC. These proteins were tryptically digested, labeled with tandem mass tags (TMT), fractionated by OFFGEL, and subjected to MS. Proteins significantly different in abundance (P < 0.05) were classified via gene ontology (GO) analysis. We identified 1223 proteins and among them, 1222 proteins were quantifiable. Statistical analysis determined that 177 proteins were of higher abundance in hHSC, while 157 were of higher abundance in hPaSC. GO classification revealed that proteins of relatively higher abundance in hHSC were associated with protein production, while those of relatively higher abundance in hPaSC were involved in cell structure. Future studies using the methodologies established herein, but with further upstream fractionation and/or use of enhanced MS instrumentation will allow greater proteome coverage, achieving a comprehensive proteomic analysis of hHSC and hPaSC.     

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.     

Mapping Accuracy of Short Reads from Massively Parallel Sequencing and the Implications for Quantitative Expression Profiling

Background

Massively parallel sequencing offers an enormous potential for expression profiling, in particular for interspecific comparisons. Currently, different platforms for massively parallel sequencing are available, which differ in read length and sequencing costs. The 454-technology offers the highest read length. The other sequencing technologies are more cost effective, on the expense of shorter reads. Reliable expression profiling by massively parallel sequencing depends crucially on the accuracy to which the reads could be mapped to the corresponding genes.

Methodology/Principal Findings

We performed an in silico analysis to evaluate whether incorrect mapping of the sequence reads results in a biased expression pattern. A comparison of six available mapping software tools indicated a considerable heterogeneity in mapping speed and accuracy. Independently of the software used to map the reads, we found that for compact genomes both short (35 bp, 50 bp) and long sequence reads (100 bp) result in an almost unbiased expression pattern. In contrast, for species with a larger genome containing more gene families and repetitive DNA, shorter reads (35–50 bp) produced a considerable bias in gene expression. In humans, about 10% of the genes had fewer than 50% of the sequence reads correctly mapped. Sequence polymorphism up to 9% had almost no effect on the mapping accuracy of 100 bp reads. For 35 bp reads up to 3% sequence divergence did not affect the mapping accuracy strongly. The effect of indels on the mapping efficiency strongly depends on the mapping software.

Conclusions/Significance

In complex genomes, expression profiling by massively parallel sequencing could introduce a considerable bias due to incorrectly mapped sequence reads if the read length is short. Nevertheless, this bias could be accounted for if the genomic sequence is known. Furthermore, sequence polymorphisms and indels also affect the mapping accuracy and may cause a biased gene expression measurement. The choice of the mapping software is highly critical and the reliability depends on the presence/absence of indels and the divergence between reads and the reference genome. Overall, we found SSAHA2 and CLC to produce the most reliable mapping results.     

Changes of Free Long-Chain Bases in Neuronal Cells During Differentiation and Aging in Culture

Abstract: Changes in the free long-chain base (LCB) composition in rat cerebellar granule cells in culture were studied during differentiation and aging. The total LCB mixtures, extracted from the cells maintained in culture up to 22 days, were derivatized with o -phthalaldehyde and fractionated by reversed-phase HPLC, and each LCB species was quantified. Four main LCBs were components of the total LCB mixtures of cultured cells: C18-sphingosine, C18-sphinganine, C20-sphingosine, and C20-sphinganine. They were found in all the cells analyzed, from 0 to 22 days of culture, with their contents being in the sequence C18-sphingosine> C20-sphinganine and varying from 0.02 ± 0.015 pmol/mg of cell protein for C20-sphinganine at day 0 to 223 ± 22 pmol/mg of cell protein for C18-sphingosine at day 8. Sphinganines were found to be minor components of the total LCB mixture, with C20-sphinganine being particularly scarce in nondifferentiated cells. The cell content of C20-sphinganine progressively increased from day 0 to 22 of culture; that of C18-sphinganine increased up to day 8, when cells are differentiated, and then remained quite constant. The changes of C18- and C20-sphingosine levels during cell culture were qualitatively similar to those of C18- and C20-sphinganine, but the content of the sphingosines was much higher than that of the sphinganines.     

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.     

Evaluation of Multi-tRNA Synthetase Complex by Multiple Reaction Monitoring Mass Spectrometry Coupled with Size Exclusion Chromatography

Eight aminoacyl-tRNA synthetases (M, K, Q, D, R, I, EP and LARS) and three auxiliary proteins (AIMP1, 2 and 3) are known to form a multi-tRNA synthetase complex (MSC) in mammalian cells. We combined size exclusion chromatography (SEC) with reversed-phase liquid chromatography multiple reaction monitoring mass spectrometry (RPLC-MRM-MS) to characterize MSC components and free ARS proteins in human embryonic kidney (HEK 293T) cells. Crude cell extract and affinity-purified proteins were fractionated by SEC in non-denaturing state and ARSs were monitored in each fraction by MRM-MS. The eleven MSC components appeared mostly in earlier SEC fractions demonstrating their participation in complex formation. TARSL2 and AIMP2-DX2, despite their low abundance, were co-purified with KARS and detected in the SEC fractions, where MSC appeared. Moreover, other large complex-forming ARS proteins, such as VARS and FARS, were detected in earlier fractions. The MRM-MS results were further confirmed by western blot analysis. Our study demonstrates usefulness of combined SEC-MRM analysis for the characterization of protein complexes and in understanding the behavior of minor isoforms or variant proteins.     

Targeted Selected Reaction Monitoring Mass Spectrometric Immunoassay for Insulin-like Growth Factor 1

Insulin-like growth factor 1 (IGF1) is an important biomarker of human growth disorders that is routinely analyzed in clinical laboratories. Mass spectrometry-based workflows offer a viable alternative to standard IGF1 immunoassays, which utilize various pre-analytical preparation strategies. In this work we developed an assay that incorporates a novel sample preparation method for dissociating IGF1 from its binding proteins. The workflow also includes an immunoaffinity step using antibody-derivatized pipette tips, followed by elution, trypsin digestion, and LC-MS/MS separation and detection of the signature peptides in a selected reaction monitoring (SRM) mode. The resulting quantitative mass spectrometric immunoassay (MSIA) exhibited good linearity in the range of 1 to 1,500 ng/mL IGF1, intra- and inter-assay precision with CVs of less than 10%, and lowest limits of detection of 1 ng/mL. The linearity and recovery characteristics of the assay were also established, and the new method compared to a commercially available immunoassay using a large cohort of human serum samples. The IGF1 SRM MSIA is well suited for use in clinical laboratories.     

Analysis of Phosphorylated Sphingolipid Long-Chain Bases Reveals Potential Roles in Heat Stress and Growth Control in Saccharomyces

Sphingolipid long-chain bases and their phosphorylated derivatives, for example, sphingosine-1-phosphate in mammals, have been implicated as signaling molecules. The possibility that Saccharomyces cerevisiae cells also use long-chain-base phosphates to regulate cellular processes has only recently begun to be examined. Here we present a simple and sensitive procedure for analyzing and quantifying long-chain-base phosphates in S. cerevisiae cells. Our data show for the first time that phytosphingosine-1-phosphate (PHS-1-P) is present at a low but detectable level in cells grown on a fermentable carbon source at 25°C, while dihydrosphingosine-1-phosphate (DHS-1-P) is only barely detectable. Shifting cells to 37°C causes transient eight- and fivefold increases in levels of PHS-1-P and DHS-1-P, respectively, which peak after about 10 min. The amounts of both compounds return to the unstressed levels by 20 min after the temperature shift. These data are consistent with PHS-1-P and DHS-1-P being signaling molecules. Cells unable to break down long-chain-base phosphates, due to deletion of DPL1 and LCB3, show a 500-fold increase in PHS-1-P and DHS-1-P levels, grow slowly, and survive a 44°C heat stress 10-fold better than parental cells. These and other data for dpl1 or lcb3 single-mutant strains suggest that DHS-1-P and/or PHS-1-P act as signals for resistance to heat stress. Our procedure should expedite experiments to determine how the synthesis and breakdown of these compounds is regulated and how the compounds mediate resistance to elevated temperature.     

Use of DNA-Aptamers for Enrichment of Low Abundant Proteins in Cellular Extracts for Quantitative Detection by Selected Reaction Monitoring

The relationship between the amount of a target protein in a complex biological sample and its amount measured by selected reaction monitoring (SRM) mass spectrometry upon the affinity enrichment of the target protein with aptamers immobilized on a solid phase has been investigated. Human thrombin added in known concentrations to cellular extracts derived from bacterial cells was used as a model target protein. The affinity enrichment of thrombin in cellular extracts by means of the thrombin-binding aptamer immobilized on the surface of magnetic microbeads resulted in an approximately 10-fold increase of the concentration of the target protein and a 100-fold decrease of the low limit of a target protein concentration range where its quantitative detection by SRM was possible without interference from other peptides present in the tryptic digest.     

OLIgo Mass Profiling (OLIMP) of Extracellular Polysaccharides

The direct contact of cells to the environment is mediated in many organisms by an extracellular matrix. One common aspect of extracellular matrices is that they contain complex sugar moieties in form of glycoproteins, proteoglycans, and/or polysaccharides. Examples include the extracellular matrix of humans and animal cells consisting mainly of fibrillar proteins and proteoglycans or the polysaccharide based cell walls of plants and fungi, and the proteoglycan/glycolipid based cell walls of bacteria. All these glycostructures play vital roles in cell-to-cell and cell-to-environment communication and signalling.An extraordinary complex example of an extracellular matrix is present in the walls of higher plant cells. Their wall is made almost entirely of sugars, up to 75% dry weight, and consists of the most abundant biopolymers present on this planet. Therefore, research is conducted how to utilize these materials best as a carbon-neutral renewable resource to replace petrochemicals derived from fossil fuel. The main challenge for fuel conversion remains the recalcitrance of walls to enzymatic or chemical degradation due to the unique glycostructures present in this unique biocomposite.Here, we present a method for the rapid and sensitive analysis of plant cell wall glycostructures. This method OLIgo Mass Profiling (OLIMP) is based the enzymatic release of oligosaccharides from wall materials facilitating specific glycosylhydrolases and subsequent analysis of the solubilized oligosaccharide mixtures using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS)¹ (Figure 1). OLIMP requires walls of only 5000 cells for a complete analysis, can be performed on the tissue itself², and is amenable to high-throughput analyses³. While the absolute amount of the solubilized oligosaccharides cannot be determined by OLIMP the relative abundance of the various oligosaccharide ions can be delineated from the mass spectra giving insights about the substitution-pattern of the native polysaccharide present in the wall.OLIMP can be used to analyze a wide variety of wall polymers, limited only by the availability of specific enzymes⁴. For example, for the analysis of polymers present in the plant cell wall enzymes are available to analyse the hemicelluloses xyloglucan using a xyloglucanase^{5, 11, 12, 13}, xylan using an endo-β-(1-4)-xylanase ^6,7, or for pectic polysaccharides using a combination of a polygalacturonase and a methylesterase ⁸. Furthermore, using the same principles of OLIMP glycosylhydrolase and even glycosyltransferase activities can be monitored and determined ⁹.     

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry (CCMS)

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity chromatography ¹ or Activity Based Protein Profiling ². Trifunctional Capture Compounds (CCs, Figure 1A) ³ are the basis for a generic approach, in which the initial equilibrium-driven interaction between a small molecule probe (the selectivity function, here S-adenosyl-_L-homocysteine, SAH, Figure 1A) and target proteins is irreversibly fixed upon photo-crosslinking between an independent photo-activable reactivity function (here a phenylazide) of the CC and the surface of the target proteins. The sorting function (here biotin) serves to isolate the CC - protein conjugates from complex biological mixtures with the help of a solid phase (here streptavidin magnetic beads). Two configurations of the experiments are possible: "off-bead" ⁴ or the presently described "on-bead" configuration (Figure 1B). The selectivity function may be virtually any small molecule of interest (substrates, inhibitors, drug molecules). S-Adenosyl-_L-methionine (SAM, Figure 1A) is probably, second to ATP, the most widely used cofactor in nature ^{5, 6}. It is used as the major methyl group donor in all living organisms with the chemical reaction being catalyzed by SAM-dependent methyltransferases (MTases), which methylate DNA ⁷, RNA ⁸, proteins ⁹, or small molecules ¹⁰. Given the crucial role of methylation reactions in diverse physiological scenarios (gene regulation, epigenetics, metabolism), the profiling of MTases can be expected to become of similar importance in functional proteomics as the profiling of kinases. Analytical tools for their profiling, however, have not been available. We recently introduced a CC with SAH as selectivity group to fill this technological gap (Figure 1A).SAH, the product of SAM after methyl transfer, is a known general MTase product inhibitor ¹¹. For this reason and because the natural cofactor SAM is used by further enzymes transferring other parts of the cofactor or initiating radical reactions as well as because of its chemical instability ¹², SAH is an ideal selectivity function for a CC to target MTases. Here, we report the utility of the SAH-CC and CCMS by profiling MTases and other SAH-binding proteins from the strain DH5α of Escherichia coli (E. coli), one of the best-characterized prokaryotes, which has served as the preferred model organism in countless biochemical, biological, and biotechnological studies. Photo-activated crosslinking enhances yield and sensitivity of the experiment, and the specificity can be readily tested for in competition experiments using an excess of free SAH.Download video file.^{(106M, mov)}     

Specificity and Commonality of the Phosphoinositide-Binding Proteome Analyzed by Quantitative Mass Spectrometry

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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.     

Condensation Reaction Between 2,2-Diphenylthio-2,3-Dideoxyribose and Silylated Pyrimidine Bases

Abstract

The condensation reaction between 2,2-diphenylthio-2,3-dideoxyribose and silylated pyrimidine bases was examined. In the presence of TMSOTf as a catalyst, this reaction proceeded to give the nucleosides in the ratio of α: β = 2:8. Each β-anomer was converted to protected 2′,3′-dideoxynucleosides.     

Protein Profiling in Hepatocellular Carcinoma by Label-Free Quantitative Proteomics in Two West African Populations

Background

Hepatocellular Carcinoma is the third most common cause of cancer related death worldwide, often diagnosed by measuring serum AFP; a poor performance stand-alone biomarker. With the aim of improving on this, our study focuses on plasma proteins identified by Mass Spectrometry in order to investigate and validate differences seen in the respective proteomes of controls and subjects with LC and HCC.

Methods

Mass Spectrometry analysis using liquid chromatography electro spray ionization quadrupole time-of-flight was conducted on 339 subjects using a pooled expression profiling approach. ELISA assays were performed on four significantly differentially expressed proteins to validate their expression profiles in subjects from the Gambia and a pilot group from Nigeria. Results from this were collated for statistical multiplexing using logistic regression analysis.

Results

Twenty-six proteins were identified as differentially expressed between the three subject groups. Direct measurements of four; hemopexin, alpha-1-antitrypsin, apolipoprotein A1 and complement component 3 confirmed their change in abundance in LC and HCC versus control patients. These trends were independently replicated in the pilot validation subjects from Nigeria. The statistical multiplexing of these proteins demonstrated performance comparable to or greater than ALT in identifying liver cirrhosis or carcinogenesis. This exercise also proposed preliminary cut offs with achievable sensitivity, specificity and AUC statistics greater than reported AFP averages.

Conclusions

The validated changes of expression in these proteins have the potential for development into high-performance tests usable in the diagnosis and or monitoring of HCC and LC patients. The identification of sustained expression trends strengthens the suggestion of these four proteins as worthy candidates for further investigation in the context of liver disease. The statistical combinations also provide a novel inroad of analyses able to propose definitive cut-offs and combinations for evaluation of performance.