Single Reaction Monitoring Research Articles

Multiclass method for the determination of pharmaceuticals and personal care products in compost from sewage sludge using ultrasound and salt-assisted liquid–liquid extraction followed by ultrahigh performance liquid chromatography-tandem mass spectrometry analysis

An analytical method for the analysis of 16 pharmaceuticals and personal care products in compost from sewage sludge is successfully validated. Ultrasound assisted extraction with a mixture of acetonitrile:ethyl acetate (1:1, v/v) containing 10% (v/v) of acetic acid was carried out. Two cycles of extraction of 10min were applied. A clean-up of the extracts using salt-assisted liquid–liquid extraction (SALLE) was also included. Experimental design was used for the optimization of the main parameters involved in the extraction and cleaned-up steps. The chromatographic separation was carried out by ultrahigh performance liquid chromatography using a mobile phase gradient mixture of a 13mM buffer ammonium formate solution (pH 9.25) (solvent A) and methanol (solvent B). An ACQUITY UPLC® BEH C18 column (1.7μm; 2.1×50mm) column was used. Analytes were separated in less than 11min. The compounds were detected and quantified using single reaction monitoring electrospray tandem mass spectrometry. The limits of detection calculated ranged from 0.5 to 4ngg−1d.w., and the limits of quantification from 2 to 13ngg−1d.w. Recoveries from 93% to 111%, with relative standar deviations lower than 11% in all cases, were obtained. The method was applied to natural compost samples. High concentrations of some analytes were found. Ketoprofen (510ngg−1d.w.), methylparaben (240ngg−1d.w.), diclofenac (175ngg−1d.w.) and flufenamic acid (128ngg−1d.w.) were the most abundant.

The Development of a Sheathless Interface for Capillary Electrophoresis Electrospray Ionization Mass Spectrometry Using a Cellulose Acetate Cast Capillary

The fabrication of a novel sheathless interface for capillary electrophoresis–electrospray–mass spectrometry (CE–ESI–MS) is described. A programmable CO2 laser was used to ablate small channels in the walls of a polyimide capillary near the terminus. Subsequent exposure of the channel region to a cellulose acetate solution followed by drying resulted in the formation of an electrically conductive semi-permeable membrane. Application of an appropriate voltage to the reservoir resulted in the simultaneous establishment of an electrical connection for CE and ESI. Interface viability was demonstrated by conducting a CE separation of a peptide mixture, with detection accomplished via positive ion mode ESI–MS. For the peptide Val-Tyr-Val, a limit of detection of 0.1 femtomole (S/N 3) was achieved using single reaction monitoring. Attributes of the interface include structural robustness, ease of fabrication, minimal interface dead volume, and the ability to alter post-separation analyte ionization status by use of appropriate buffers in the interface reservoir.

A Shotgun Fingerprinting Approach to Document Compliance With an Almond Consumption Intervention

IntroductionAlmonds are an extremely rich source of unsaturated lipids and flavonoids and increased consumption may be recommended to achieve various health benefits. However, there are no known analytical methods to document compliance to chronic almond consumption.ObjectiveTo develop an analytical approach that identifies metabolic profiles associated with long‐term almond intake to ascertain compliance with prescribed consumption.MethodsA shotgun fingerprinting strategy was designed to isolate metabolic changes in erythrocytes after 12 weeks of almond consumption in a weight‐loss study. Representative samples (pools) of erythrocytes from individuals consuming almonds or no almonds were screened by flow injection mass spectrometry. Data based on scan modes for shotgun lipidomics, and m/z values compatible with almond flavonoid content were collected. Specific ion pairs and m/z values detected in pooled samples were combined into single ion monitoring and multiple reaction monitoring methods intended for individual sample screening. Values of ion intensities were normalized by total ion current to obtain relative ion intensities.ResultsOut of the 243 values of m/z monitored by both methods, 5 ratios and combinations of specific ions with receiver operating characteristic (ROC) curves AUC>0.89 had sensitivity of 74.2% and specificity of 90%. Eight out of the 31 participants (25.8%) in the almond group and 3 out of the 30 (10%) participants in the control group were misclassified by all 5 ratios.ConclusionRatios and combinations of specific ions mainly related to membrane lipids were discriminatory of almond consumption from the nut‐free diet. The relationship between these metabolites indicates almond‐induced changes in the metabolomics profile and the misclassifications observed as a result of ratio performance evaluation could be indications of possible non‐compliance as supported by analysis of dietary intake data.Support or Funding InformationAlmond Board of California

Evaluation of Rice Husk for SPE of Fluoroquinolones from Environmental Waters Followed by UHPLC-HESI-MS/MS

Untreated rice husk (RH) was evaluated as solid-phase extraction (SPE) sorbent for Fluoroquinolone antibiotics (FQs). This natural material, made of cellulose, hemicellulose, lignin and silica, and containing various functional groups, i.e., carboxyl, hydroxyl, and amidogen residues, is a potential sorbent material for polar aromatics like FQs. First, the RH capability to adsorb FQ species from water was studied by batch sorption experiments in tap and river waters, and the experimental data, fitted by the Langmuir model, gave adsorption capacities up to 32 mg g−1. Then, RH was tested as column-packed sorbent for pre-concentration of tap and not tampered river waters spiked with six widely employed FQs, i.e., Ciprofloxacin, Danofloxacin, Enrofloxacin, Levofloxacin, Marbofloxacin, and Norfloxacin. The analytes were quantitatively adsorbed on the RH cartridge at the native pH, simultaneously eluted by NH3–MeOH mixture, separated in an 8 min ultrahigh-performance liquid chromatography run and quantified/confirmed by electrospray ionization tandem mass spectrometric detection in single reaction monitoring mode. Recoveries in the range 71–120% were observed (RSD < 15%, n = 3) for 75–1000 ng L−1 spikes. Method detection limits were in the range 25–33 ng L−1. The batch-to-batch reproducibility was assessed, and the analytical procedure was applied to the determination of FQs in actual environmental waters.

Targeted proteomics coming of age - SRM, PRM and DIA performance evaluated from a core facility perspective.

Quantitative mass spectrometry is a rapidly evolving methodology applied in a large number of omics-type research projects. During the past years, new designs of mass spectrometers have been developed and launched as commercial systems while in parallel new data acquisition schemes and data analysis paradigms have been introduced. Core facilities provide access to such technologies, but also actively support the researchers in finding and applying the best-suited analytical approach. In order to implement a solid fundament for this decision making process, core facilities need to constantly compare and benchmark the various approaches. In this article we compare the quantitative accuracy and precision of current state of the art targeted proteomics approaches single reaction monitoring (SRM), parallel reaction monitoring (PRM) and data independent acquisition (DIA) across multiple liquid chromatography mass spectrometry (LC-MS) platforms, using a readily available commercial standard sample. All workflows are able to reproducibly generate accurate quantitative data. However, SRM and PRM workflows show higher accuracy and precision compared to DIA approaches, especially when analyzing low concentrated analytes.

Surface-crosslinked poly(3-mercaptopropyl)methylsiloxane-coatings on silica as new platform for low-bleed mass spectrometry-compatible functionalized stationary phases synthesized via thiol-ene click reaction

A thin functional film of poly(3-mercaptopropyl)methylsiloxane was coated onto vinyl-modified silica particles (5μm, 100Å pore size) and chemically crosslinked to the surface. Excess of thiol functionalities allow bonding of alkene containing ligands by thiol-ene click reaction in a second step (QN-VII). Besides that a single step surface modification procedure was established in which alkene functional ligands were directly added to the polysiloxane coating solution and thus, after evaporation of the solvent, crosslinking to the vinylized surface and bonding of chromatographic ligand to the thiolated polysiloxane film occur simultaneously in one step (QN-VI). Successful bonding of the polysiloxane film was confirmed for both approaches by 29Si cross-polarization/magic angle spinning NMR spectra. The new surface functionalization concept can be utilized as a new platform for the preparation of various low-bleed, mass spectrometry-compatible stationary phases with a variety of functional ligands. The concept was demonstrated by thiol-ene click reaction with quinine carbamate and its subsequent use for enantiomer separation by HPLC-UV and HPLC-ESI-QTOF-MS of acidic chiral analytes. Chromatographic enantioselectivities were similar to a comparable brush-type CSP (QN-V0). The greatly reduced background signal in LC-MS, however, comes at expense of somewhat lower chromatographic efficiencies (C-term by factor of 2 larger compared to brush-type CSP). For quantitative analysis in single reaction monitoring (MRMHR) in high sensitivity mode, limit of detection and limit of quantification results are comparable for both surface-polymer modified CSPs, with only slightly higher values for the conventional brush-type CSP (QN-V0).

In silico verification and parallel reaction monitoring prevalidation of potential prostate cancer biomarkers.

Targeted proteomics of potential biomarkers is often challenging. Hence, we developed an intermediate workflow to streamline potential urinary biomarkers of prostate cancer (PCa). Using previously discovered potential PCa biomarkers; we selected proteotypic peptides for targeted validation. Preliminary in silico immunohistochemical and single reaction monitoring (SRM) verification was performed. Successful PTPs were then prevalidated using parallel reaction monitoring (PRM) and reconfirmed in 15 publicly available databases. Stringency-based targetable potential biomarkers were shortlisted following in silico screening. PRM reveals top 12 potential biomarkers including the top ranking seven in silico verification-based biomarkers. Database reconfirmation showed differential expression between PCa and benign/normal prostatic urine samples. The pragmatic penultimate screening step, described herein, would immensely improve targeted proteomics validation of potential disease biomarkers.

Fluoroquinolone residues in compost by green enhanced microwave-assisted extraction followed by ultra performance liquid chromatography tandem mass spectrometry

A novel, simple and straightforward method for determination of fluoroquinolones (FQs) in compost has been developed. The procedure entails a low-pressurized microwave-assisted extraction (MAE) carried out by a high performance instrument, in alkaline aqueous solution containing magnesium ions as FQs complexing agent, followed by ultra performance liquid chromatography tandem mass spectrometry (UPLC–MS). Ciprofloxacin (CIP), Enrofloxacin (ENR), Levofloxacin (LEV) and Norfloxacin (NOR), four widely used FQ antibiotics, were simultaneously extracted from compost by a single MAE cycle (20min, 135°C). The method was validated in terms of linearity, selectivity, sensitivity and accuracy. Quantitative absolute recovery (70–112%, n=3) and suitable precision (RSD<15%, n=3) were observed, at concentration levels ranging from 25 ng g−1 to 2500 ng g−1. Analytes were separated in a 10min chromatographic run and quantified/confirmed in single reaction monitoring (SRM) mode. UPLC coupled to SRM-MS detection allowed to achieve improved sensitivity, and selective detection. Method detection and quantification limits, MDLs and MQLs, were in the range 2.2–3.0 ng g−1 and 6.6–9.0 ng g−1, respectively. The high-performance microwave system here used strongly improved the extraction efficiency with respect to a conventional apparatus. The procedure proved to be simpler, less expensive, faster, and more green with respect to the few methods currently described in literature, providing at the same time suitable recovery and reproducibility. The analytical method has been applied to the analysis of actual compost samples, wherein FQs have been quantified at concentrations up to 88 ng g−1.

Abstract P6-03-10: Targeting voltage-gated sodium channels with the antiepileptic drug phenytoin inhibits breast tumor growth and metastasis

Abstract Voltage-gated Na+ channels (VGSCs) are heteromeric protein complexes containing pore-forming α subunits and smaller, non-pore-forming β subunits. The β subunits are multifunctional channel modulators and are members of the immunoglobulin superfamily of cell adhesion molecules (CAMs). VGSCs are classically expressed in electrically excitable cells, e.g. neurons, where they mediate action potential firing, neurite outgrowth and migration during development. An increasing body of evidence indicates that VGSCs are also expressed in metastatic cells from a number of cancers, including breast cancer. The Nav1.5 α subunit (encoded by SCN5A) is expressed in cancer cell lines, where it enhances migration and invasion. SCN5A is up-regulated in tumor samples in several published datasets, associating with recurrence, metastasis, and reduced overall survival. We have previously shown that the VGSC-blocking antiepileptic drug phenytoin inhibits the migration and invasion of metastatic MDA-MB-231 cells in vitro. In addition, we have recently shown that the VGSC β1 subunit enhances breast tumor growth and metastasis in a xenograft model of breast cancer. The purpose of the present study was to establish whether or not VGSCs might be a viable therapeutic target by testing the effect of phenytoin on tumor growth and metastasis in vivo. We found that Nav1.5 expression was retained on MDA-MB-231 cells in orthotopic xenografts in immune-deficient mice. Mice were treated with phenytoin (60 mg/kg) or vehicle by daily intraperitoneal injection for 3 weeks, starting 1 week following implantation of tumor cells. Plasma phenytoin concentration was measured at the end of the experiment by liquid chromatography-mass spectrometry with single reaction monitoring (LC-SRM-MS) using metaxalone as an internal standard. Phenytoin significantly reduced tumor growth, detected by in vivo bioluminescent imaging and caliper measurements, without affecting animal weight. Phenytoin reduced the density of Ki67-positive cycling cells in the primary tumors, but did not affect the density of apoptotic cells expressing activated caspase-3, or the density of CD31-positive vessel structures. Phenytoin also reduced local invasion and the density of MMP9-positive cells within primary tumors. Finally, phenytoin significantly reduced metastasis to the liver, lungs and spleen, detected by bioluminescent imaging and GFP immunohistochemistry. This is the first study showing that phenytoin reduces tumor growth and metastasis in vivo. Together, our data support the hypothesis that VGSCs are up-regulated in breast cancer, favoring an invasive phenotype, and may thus be promising targets for therapeutic intervention. We propose that pharmacologically targeting VGSCs should be further studied as a potentially novel, cost-effective, anti-cancer therapy. Repurposing FDA-approved oral VGSC-blocking antiepileptic or antiarrhythmic drugs, e.g. phenytoin, may improve patient outcomes in the adjuvant setting. Citation Format: William J Brackenbury, Michaela Nelson, Ming Yang, Adam A Dowle, Jerry R Thomas. Targeting voltage-gated sodium channels with the antiepileptic drug phenytoin inhibits breast tumor growth and metastasis [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-03-10.

Plasma QconCATs reveal a gender-specific proteomic signature in apheresis platelet plasma supernatants.

Gender differences in the proteome composition of apheresis platelet supernatants have always been postulated, and might underlie a higher risk of adverse reactions when transfusing apheresis products from female donors. Preliminary proteomic studies provided an overview of gender-dependent relative compositional differences in the proteome of apheresis platelet supernatants during routine storage in the blood bank. Here we apply a proteomics approach for absolute quantitation of approximately 100 proteins in apheresis platelet supernatants from male and female donors at storage days 1 and 5. Absolute quantitative proteomic analyses allowed us to confirm and expand on previous observations about gender and storage-dependency of platelet supernatant protein profiles.

Accurate quantitation of MHC-bound peptides by application of isotopically labeled peptide MHC complexes.

Knowledge of the accurate copy number of HLA class I presented ligands is important in fundamental and clinical immunology. Currently, the best copy number determinations are based on mass spectrometry, employing single reaction monitoring (SRM) in combination with a known amount of isotopically labeled peptide. The major drawback of this approach is that the losses during sample pretreatment, i.e. immunopurification and filtration steps, are not well defined and must, therefore, be estimated. In addition, such losses can vary for individual peptides. Therefore, we developed a new approach in which isotopically labeled peptide-MHC monomers (hpMHC) are prepared and added directly after cell lysis, i.e. before the usual sample processing. Using this approach, all losses during sample processing can be accounted for and allows accurate determination of specific MHC class I-presented ligands. Our study pinpoints the immunopurification step as the origin of the rather extreme losses during sample pretreatment and offers a solution to account for these losses. Obviously, this has important implications for accurate HLA-ligand quantitation. The strategy presented here can be used to obtain a reliable view of epitope copy number and thus allows improvement of vaccine design and strategies for immunotherapy.

A liquid chromatography-mass spectrometry assay for quantification of Exendin[9-39] in human plasma

Exendin[9-39] is a glucagon-like peptide-1 receptor (GLP-R) antagonist and a potential therapeutic drug for treatment of congenital hyperinsulism by lowering insulin concentration in plasma. A specific and sensitive LC-MS/MS method was validated for quantification of Exendin[9-39] in human plasma. Exendin[9-39] and the stable isopically labeled internal standard eluted at 9.2 min and were analyzed by single reaction monitoring (SRM) of the transitions m/z 842.9→991.8 and 848.2→998.8, respectively. The calibration curve was linear in the range 15-1260 ng/mL with a limit of detection of 1.3 ng/mL. The CVs of the standards were 2.7-13.1% within-run and 3.1-13.2% between-run. The matrix effect was >100% and the SPE recovery was 98.4±12.9%. In absence of protease inhibitors, short-term stability at room temperature was only one hour. Accordingly, samples were kept on ice and sample processing was kept below 1h. Human plasma samples from a clinical pilot study in which Exendin[9-39] was administered intravenously were analyzed and concentrations up to 600 ng/mL were reported Plasma samples from the study were stored at -80 °C with internal standard and successfully reanalyzed after 12 months.

Ultra-fast cyclosporin A quantitation in whole blood by Laser Diode Thermal Desorption – Tandem Mass Spectrometry; comparison with High Performance Liquid Chromatography–Tandem Mass Spectrometry

In the last decade the quantitation of immunosuppressive drugs has seen vast improvements in analytical methods, optimizing time, accuracy of analysis and cost. Laser Diode Thermal Desorption (LDTD) coupled to Atmospheric Pressure Chemical ionization–tandem mass spectrometry (APCI-MS/MS) represents a technological breakthrough that removes the chromatographic separation step and thereby significantly increases the analytical throughput for the quantitation of cyclosporin A (CsA) in whole blood for therapeutic drug monitoring (TDM).A simple protein precipitation step was used prior to depositing 5μL of the extract on a 96-well LazWell™ plate and CsA was quantified by LDTD-APCI-MS/MS.The laser pattern was set to ramp from 0 to 45% laser power within 2s. The APCI parameters were set to negative needle voltage (−2μA), carrier gas temperature (30°C) and air flow rate (3Lmin−1). The negative ion single reaction monitoring transitions for CsA and its internal standard cyclosporin D (CsD) were respectively m/z 1201.1/1088.9 and m/z 1214.8/1102.8; obtained with a collision energy of −40V.The analysis was achieved within 9s from sample to sample.The extraction procedure yielded high recovery (92%; RSD=9.4%, n=6). The lower limit of quantitation was fixed at the first level of calibration: 23.5ngmL−1 (accuracy=112.3%; RSD=9.6%; n=6) and a blank+6 point linear regression up to 965ngmL−1 was used. Using 4 levels of quality control (QC), intra-day assays (n=6) ranged from 93.5 to 95.7% (bias) and from 3.4 to 13.1% (RSD) while inter-day assays (n=6) ranged from 92.9 to 105.3% (bias) and from 4.9 to 7.5% (RSD). An inter-sample contamination of CsA of 2.3% was calculated that was considered negligible with respect to the range of CsA concentrations. Whole blood samples (120) from patients under CsA treatment were analyzed by LDTD-APCI-MS/MS and HPLC–ESI-MS/MS, the gold standard reference method for CsA quantification. Both methods agreed (P≥0.99), with a coefficient of correlation of 0.99 (95% confidence interval 0.982–0.991). The Passing–Bablok regression revealed no significant deviation from linearity (Cusum test, P=0.11). This method seems suitable for use in TDM of CsA.

Cellular uptake and antiproliferative effects of 11-oxo-eicosatetraenoic acid

Cyclooxygenases (COX) metabolize arachidonic acid (AA) to hydroxyeicosatetraenoic acids (HETE), which can then be oxidized by dehydrogenases, such as 15-hydroxyprostaglandin dehydrogenase (15-PGDH), to oxo-eicosatetraenoic acids (ETE). We have previously established that 11-oxo-eicosatetraenoic acid (oxo-ETE) and 15-oxo-ETE are COX-2/15-PGDH-derived metabolites. Stable isotope dilution (SID) chiral liquid chromatography coupled with electron capture atmospheric pressure chemical ionization (ECAPCI) single reaction monitoring (SRM) MS has been used to quantify uptake of 11-oxo-ETE and 15-oxo-ETE in both LoVo cells and human umbilical vein endothelial cells (HUVEC). Intracellular 11-oxo- and 15-oxo-ETE concentrations reached maximum levels within 1 h and declined rapidly, with significant quantitative differences in uptake between the LoVo cells and the HUVECs. Maximal intracellular concentrations of 11-oxo-ETE were 0.02 ng/4 × 105 cells in the LoVo cells and 0.58 ng/4 × 105 cells in the HUVECs. Conversely, maximal levels of 15-oxo-ETE were 0.21 ng/4 × 105 in the LoVo cells and 0.01 ng/4 × 105 in the HUVECs. The methyl esters of both 11-oxo- and 15-oxo-ETE increased the intracellular concentrations of the corresponding free oxo-ETEs by 3- to 8-fold. 11-oxo-ETE, 15-oxo-ETE, and their methyl esters inhibited proliferation in both HUVECs and LoVo cells at concentrations of 2–10 μM, with 11-oxo-ETE methyl ester being the most potent inhibitor. Cotreatment with probenecid, an inhibitor of multiple drug resistance transporters (MRP)1 and 4, increased the antiproliferative effect of 11-oxo-ETE methyl ester in LoVo cells and increased the intracellular concentration of 11-oxo-ETE from 0.05 ng/4 × 105 cells to 0.18 ng/4 × 105 cells. Therefore, this study has established that the COX-2/15-PGDH-derived eicosanoids 11-oxo- and 15-oxo-ETE enter target cells, that they inhibit cellular proliferation, and that their inhibitory effects are modulated by MRP exporters.

Receptor-based high-throughput screening and identification of estrogens in dietary supplements using bioaffinity liquid-chromatography ion mobility mass spectrometry

A high-throughput bioaffinity liquid chromatography-mass spectrometry (BioMS) approach was developed and applied for the screening and identification of recombinant human estrogen receptor α (ERα) ligands in dietary supplements. For screening, a semi-automated mass spectrometric ligand binding assay was developed applying (13)C2, (15) N-tamoxifen as non-radioactive label and fast ultra-high-performance-liquid chromatography-electrospray ionisation-triple-quadrupole-MS (UPLC-QqQ-MS), operated in the single reaction monitoring mode, as a readout system. Binding of the label to ERα-coated paramagnetic microbeads was inhibited by competing estrogens in the sample extract yielding decreased levels of the label in UPLC-QqQ-MS. The label showed high ionisation efficiency in positive electrospray ionisation (ESI) mode, so the developed BioMS approach is able to screen for estrogens in dietary supplements despite their poor ionisation efficiency in both positive and negative ESI modes. The assay was performed in a 96-well plate, and all these wells could be measured within 3 h. Estrogens in suspect extracts were identified by full-scan accurate mass and collision-cross section (CCS) values from a UPLC-ion mobility-Q-time-of-flight-MS (UPLC-IM-Q-ToF-MS) equipped with a novel atmospheric pressure ionisation source. Thanks to the novel ion source, this instrument provided picogram sensitivity for estrogens in the negative ion mode and an additional identification point (experimental CCS values) next to retention time, accurate mass and tandem mass spectrometry data. The developed combination of bioaffinity screening with UPLC-QqQ-MS and identification with UPLC-IM-Q-ToF-MS provides an extremely powerful analytical tool for early warning of ERα bioactive compounds in dietary supplements as demonstrated by analysis of selected dietary supplements in which different estrogens were identified.

Abstract 1931: Development of a functional assay for the determination of BRCA1 carrier status.

Abstract Germline mutations in the breast cancer susceptibility gene BRCA1 confer an overall lifetime risk of developing breast cancer of up to 80%. Given the high risk of cancer development associated with BRCA1 mutation carriers, it is important that they be identified early and accurately. However, identification is impeded by the number of Variants of Unknown Significance (VUS), therefore there is compelling rationale for a functional assay that will identify pathogenic mutations. Previous work in our lab developed a novel functional assay to predict BRCA1 status based on gene expression profiles. That study analyzed EBV-transformed lymphoblastoid cell lines (LCLs) of BRCA1 mutation carriers (BRCA1+/−) and controls (BRCA1+/+) and found that lymphocytes from BRCA1 carriers could be distinguished with high fidelity (∼90% accuracy) from individuals with two wild type copies of the gene. Interestingly, carriers were recognized through their basal gene expression profiles. Global gene expression level changes following ionizing radiation (IR) to induce the DNA damage response (DDR) did not support the development of an accurate class predictor. The final predictor included a set of 43 genes. Many of these genes were also markers of differentiation in blood cells. A subset of these genes have now been validated in LCLs using qRT-PCR and protein-based methods including single reaction monitoring mass spec (SRM-MS) and flow cytometry. These assays were able to distinguish BRCA1 mutation carriers from controls based on the expression levels of specific markers, including genes with known roles in lymphocyte differentiation. Using these techniques, ongoing work is being done to test the ability of these markers to discriminate BRCA1 mutation carriers from controls using lymphocytes isolated from fresh blood samples. Citation Format: Lauren Bathurst, Paolo Uy, Harriet Feilotter, Scott Davey. Development of a functional assay for the determination of BRCA1 carrier status. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1931. doi:10.1158/1538-7445.AM2013-1931

Selective and sensitive detection of chromium(VI) in waters using electrospray ionization mass spectrometry

From 2000 through 2011, there were 14 criminal cases of violations of the Clean Water Act involving the discharge of chromium, a toxic heavy metal, into drinking and surface water sources. As chromium(VI), a potential carcinogen present in the environment, represents a significant safety concern, it is currently the subject of an EPA health risk assessment. Therefore, sensitive and selective detection of this species is highly desired. This study reports the analysis of chromium(VI) in water samples by electrospray ionization mass spectrometry (ESI-MS) following its reduction and complexation with ammonium pyrrolidinedithiocarbamate (APDC). The reduction and subsequent complexation produce a characteristic [Cr(III)O]-PDC complex which can be detected as a protonated ion of m/z 507 in the positive ion mode. The detection is selective to chromium(VI) under acidic pH, even in the presence of chromium(III) and other metal ions, providing high specificity. Different water samples were examined, including deionized, tap, and river waters, and sensitive detection was achieved. In the case of deionized water, quantification over the concentration range of 3.7 to 148ppb gave an excellent correlation coefficient of 0.9904 using the enhanced MS mode scan. Using the single-reaction monitoring (SRM) mode (monitoring the characteristic fragmentation of m/z 507 to m/z 360), the limit of detection (LOD) was found to be 0.25ppb. The LOD of chromium(VI) for both tap and river water samples was determined to be 2.0ppb. A preconcentration strategy using simple vacuum evaporation of the aqueous sample was shown to further improve the ESI signal by 15 fold. This method, with high sensitivity and selectivity, should provide a timely solution for the real-world analysis of toxic chromium(VI).

Optimizing dimodular nonribosomal peptide synthetases and natural dipeptides in an Escherichia coli heterologous host

Nonribosomal peptides are an important class of natural products that have a broad range of biological activities. Their structural complexity often prevents simple chemical synthesis, and production from the natural producer is often low, which deters pharmaceutical development. Expression of biosynthetic machinery in heterologous host organisms like Escherichia coli is one way to access these structures, and subsequent optimization of these systems is critical for future development. We utilized the aureusimine biosynthetic gene cluster as a model system to identify the optimal conditions to produce nonribosomal peptides in the isopropyl β-d-1-thiogalactopyranoside (IPTG)-inducible T7 promoter system of pET28. Single reaction monitoring of nonribosomal products was used to find the optimal concentration of IPTG, postinduction temperature, and the effect of amino acid precursor supplementation. In addition, principle component analysis of these extracts identified 3 previously undiscovered pyrazine products of the aureusimine biosynthetic locus, highlighting the utility of heterologously expressing nonribosomal peptide synthetases to find new products.

Chloroquine Interference with Hemoglobin Endocytic Trafficking Suppresses Adaptive Heme and Iron Homeostasis in Macrophages: The Paradox of an Antimalarial Agent

The CD163 scavenger receptor pathway for Hb:Hp complexes is an essential mechanism of protection against the toxicity of extracellular hemoglobin (Hb), which can accumulate in the vasculature and within tissues during hemolysis. Chloroquine is a lysosomotropic agent, which has been extensively used as an antimalarial drug in the past, before parasite resistance started to limit its efficacy in most parts of the world. More recent use of chloroquine is related to its immunomodulatory activity in patients with autoimmune diseases, which may also involve hemolytic disease components. In this study we examined the effects of chloroquine on the human Hb clearance pathway. For this purpose we developed a new mass-spectrometry-based method to specifically quantify intracellular Hb peptides within the endosomal-lysosomal compartment by single reaction monitoring (SRM). We found that chloroquine exposure impairs trafficking of Hb:Hp complexes through the endosomal-lysosomal compartment after internalization by CD163. Relative quantification of intracellular Hb peptides by SRM confirmed that chloroquine blocked cellular Hb:Hp catabolism. This effect suppressed the cellular heme-oxygenase-1 (HO-1) response and shifted macrophage iron homeostasis towards inappropriately high expression of the transferrin receptor with concurrent inhibition of ferroportin expression. A functional deficiency of Hb detoxification and heme-iron recycling may therefore be an adverse consequence of chloroquine treatment during hemolysis.

Single nanoparticle plasmonics

Interest in nanotechnology is driven by the unique and novel properties of nanoscale materials such as the strong interaction of metal particles with light, caused by localized surface plasmon resonances (LSPRs). In this perspective article we review and discuss prominent advantages and advances in single particle studies of plasmonic nanostructures. Common techniques and recent improvements in spatial and spectral resolution will first be outlined, covering both far-field and near-field phenomena. Then, new insight and information uniquely obtained from single particle approaches will be overviewed, including several fundamental studies of plasmonic behaviour, as well as applications using single particle tracking and chemical reaction monitoring. Finally, highly interdisciplinary future directions and experiments are discussed.