Last data update: May 06, 2024. (Total: 46732 publications since 2009)
Records 1-10 (of 10 Records) |
Query Trace: McWilliams LG[original query] |
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Development and application of a high throughput one-pot extraction protocol for quantitative LC-MS/MS analysis of phospholipids in serum and lipoprotein fractions in normolipidemic and dyslipidemic subjects
Gardner MS , Kuklenyik Z , Lehtikoski A , Carter KA , McWilliams LG , Kusovschi J , Bierbaum K , Jones JI , Rees J , Reis G , Pirkle JL , Barr JR . J Chromatogr B Analyt Technol Biomed Life Sci 2019 1118-1119 137-147 Progress toward better diagnosis and treatment of lipid metabolism-related diseases requires high throughput approaches for multiplexed quantitative analysis of structurally diverse lipids, including phospholipids (PLs). This work demonstrates a simplified "one-pot" phospholipid extraction protocol, as an alternative to conventional liquid-liquid extraction. Performed in a 96-well format, the extraction was coupled with high throughput UPLC and multiplexed tandem mass spectrometry (MS/MS) detection, allowing non-targeted quantification of phosphatidylcholines (PC), sphingomyelins (SM), lysophosphatidylcholines (LPC), phosphatidylethanolamines (PE), and phosphatidylinositols (PI). Using 50muL aliquots of serum samples from 110 individuals, lipoproteins were fractionated by size, and analyzed for phospholipids and non-polar lipids including free cholesterol (FC), cholesteryl esters (CEs) and triglycerides (TGs). Analysis of serum samples with wide range of Total-TG levels showed significant differences in PL composition. The correlations of molar ratios in lipoprotein size fractions, SM/PL with FC/PL, PE/PL with TG/CE, and PE/PL with PI/PL, demonstrate the applicability of the method for quantitative composition analysis of high, low and very-low density lipoproteins (HDL, LDL and VLDL), and characterization of lipid metabolism related disease states. |
Core lipid, surface lipid and apolipoprotein composition analysis of lipoprotein particles as a function of particle size in one workflow integrating asymmetric flow field-flow fractionation and liquid chromatography-tandem mass spectrometry
Kuklenyik Z , Jones JI , Gardner MS , Schieltz DM , Parks BA , Toth CA , Rees JC , Andrews ML , Carter K , Lehtikoski AK , McWilliams LG , Williamson YM , Bierbaum KP , Pirkle JL , Barr JR . PLoS One 2018 13 (4) e0194797 Lipoproteins are complex molecular assemblies that are key participants in the intricate cascade of extracellular lipid metabolism with important consequences in the formation of atherosclerotic lesions and the development of cardiovascular disease. Multiplexed mass spectrometry (MS) techniques have substantially improved the ability to characterize the composition of lipoproteins. However, these advanced MS techniques are limited by traditional pre-analytical fractionation techniques that compromise the structural integrity of lipoprotein particles during separation from serum or plasma. In this work, we applied a highly effective and gentle hydrodynamic size based fractionation technique, asymmetric flow field-flow fractionation (AF4), and integrated it into a comprehensive tandem mass spectrometry based workflow that was used for the measurement of apolipoproteins (apos A-I, A-II, A-IV, B, C-I, C-II, C-III and E), free cholesterol (FC), cholesterol esters (CE), triglycerides (TG), and phospholipids (PL) (phosphatidylcholine (PC), sphingomyelin (SM), phosphatidylethanolamine (PE), phosphatidylinositol (PI) and lysophosphatidylcholine (LPC)). Hydrodynamic size in each of 40 size fractions separated by AF4 was measured by dynamic light scattering. Measuring all major lipids and apolipoproteins in each size fraction and in the whole serum, using total of 0.1 ml, allowed the volumetric calculation of lipoprotein particle numbers and expression of composition in molar analyte per particle number ratios. Measurements in 110 serum samples showed substantive differences between size fractions of HDL and LDL. Lipoprotein composition within size fractions was expressed in molar ratios of analytes (A-I/A-II, C-II/C-I, C-II/C-III. E/C-III, FC/PL, SM/PL, PE/PL, and PI/PL), showing differences in sample categories with combinations of normal and high levels of Total-C and/or Total-TG. The agreement with previous studies indirectly validates the AF4-LC-MS/MS approach and demonstrates the potential of this workflow for characterization of lipoprotein composition in clinical studies using small volumes of archived frozen samples. |
Simultaneous quantification of free cholesterol, cholesteryl esters, and triglycerides without ester hydrolysis by UHPLC separation and in-source collision induced dissociation coupled MS/MS
Gardner MS , McWilliams LG , Jones JI , Kuklenyik Z , Pirkle JL , Barr JR . J Am Soc Mass Spectrom 2017 28 (11) 2319-2329 We demonstrate the application of in-source nitrogen collision-induced dissociation (CID) that eliminates the need for ester hydrolysis before simultaneous analysis of esterified cholesterol (EC) and triglycerides (TG) along with free cholesterol (FC) from human serum, using normal phase liquid chromatography (LC) coupled to atmospheric pressure chemical ionization (APCI) tandem mass spectrometry (MS/MS). The analysis requires only 50 muL of 1:100 dilute serum with a high-throughput, precipitation/evaporation/extraction protocol in one pot. Known representative mixtures of EC and TG species were used as calibrators with stable isotope labeled analogs as internal standards. The APCI MS source was operated with nitrogen source gas. Reproducible in-source CID was achieved with the use of optimal cone voltage (declustering potential), generating FC, EC, and TG lipid class-specific precursor fragment ions for multiple reaction monitoring (MRM). Using a representative mixture of purified FC, CE, and TG species as calibrators, the method accuracy was assessed with analysis of five inter-laboratory standardization materials, showing -10% bias for Total-C and -3% for Total-TG. Repeated duplicate analysis of a quality control pool showed intra-day and inter-day variation of 5% and 5.8% for FC, 5.2% and 8.5% for Total-C, and 4.1% and 7.7% for Total-TG. The applicability of the method was demonstrated on 32 serum samples and corresponding lipoprotein sub-fractions collected from normolipidemic, hypercholesterolemic, hypertriglyceridemic, and hyperlipidemic donors. The results show that in-source CID coupled with isotope dilution UHPLC-MS/MS is a viable high precision approach for translational research studies where samples are substantially diluted or the amounts of archived samples are limited. |
High throughput quantification of apolipoproteins A-I and B-100 by isotope dilution mass spectrometry targeting fast trypsin releasable peptides without reduction and alkylation
Parks BA , Schieltz DM , Andrews ML , Gardner MS , Rees JC , Toth CA , Jones JI , McWilliams LG , Kuklenyik Z , Pirkle JL , Barr JR . Proteomics Clin Appl 2017 11 PURPOSE: Apolipoprotein A-I (ApoA-I) and Apolipoprotein B-100 (ApoB-100) are amphipathic proteins that are strong predictors of cardiovascular disease risk. The traceable calibration of apolipoprotein assays is a persistent challenge, especially for ApoB-100, which cannot be solubilized in purified form. EXPERIMENTAL DESIGN: Simultaneous quantitation method for ApoA-I and ApoB-100 was developed using tryptic digestion without pre-digestion reduction and alkylation, followed by liquid chromatography separation coupled with isotope dilution mass spectrometry (IDMS) analysis. The accuracy of the method was assured by selecting structurally exposed signature peptides, optimal choice of detergent, protein:enzyme ratio, and incubation time. Peptide calibrators were value assigned by isobaric-tagging-IDMS amino acid analysis. RESULTS: The method reproducibility was validated in technical repeats of three serum samples, giving 2-3% intra-day CVs (N = 5) and <7% inter-day CVs (N = 21). The repeated analysis of inter-laboratory harmonization standards showed -1% difference for ApoA-I and -12% for ApoB-100 relative to the assigned value. The applicability of the method was demonstrated by repeated analysis of 24 patient samples with a wide range of total cholesterol and triglyceride levels. CONCLUSIONS AND CLINICAL RELEVANCE: The method is applicable for simultaneous analysis of ApoA-I and ApoB-100 in patient samples, and for characterization of serum pool calibrators for other analytical platforms. |
On-column trypsin digestion coupled with LC-MS/MS for quantification of apolipoproteins
Toth CA , Kuklenyik Z , Jones JI , Parks BA , Gardner MS , Schieltz DM , Rees JC , Andrews ML , McWilliams LG , Pirkle JL , Barr JR . J Proteomics 2016 150 258-267 Apolipoproteins measured in plasma or serum are potential biomarkers for assessing metabolic irregularities that are associated with the development of cardiovascular disease (CVD). LC-MS/MS allows quantitative measurement of multiple apolipoproteins in the same sample run.However, the accuracy and precision of the LC-MS/MS measurement depends on the reproducibility of the enzymatic protein digestion step. With the application of an immobilized enzyme reactor (IMER), the reproducibility of the trypsin digestion can be controlled with high precision via flow rate, column volume and temperature. In this report, we demonstrate the application of an integrated IMER-LC-MS/MS platform for the simultaneous quantitative analysis of eight apolipoproteins. Using a dilution series of a characterized serum pool as calibrator, the method was validated by repeated analysis of pooled sera and individual serum samples with a wide range of lipid profiles, all showing intra-assay CV<4.4% and inter-assay CV<8%. In addition, the method was compared with traditional homogeneous digestion coupled LC-MS/MS for the quantification of apoA-I and apoB-100. Applied in large scale human population studies, this method can serve the translation of a wider panel of apolipoprotein biomarkers from research to clinical application. SIGNIFICANCE: Currently, the translation of apolipoprotein biomarkers to clinical application is impaired because of the high cost of large cohort studies using traditional single-analyte immunoassays. The application of on-line tryptic digestion coupled with LC-MS/MS analysis is an effective way to address this problem. In this work we demonstrate a high throughput, multiplexed, automated proteomics workflow for the simultaneous analysis of multiple proteins. |
Quantification of ricin, RCA and comparison of enzymatic activity in 18 Ricinus communis cultivars by isotope dilution mass spectrometry
Schieltz DM , McWilliams LG , Kuklenyik Z , Prezioso SM , Carter AJ , Williamson YM , McGrath SC , Morse SA , Barr JR . Toxicon 2015 95 72-83 The seeds of the Ricinus communis (Castor bean) plant are the source of the economically important commodity castor oil. Castor seeds also contain the proteins ricin and R. communis agglutinin (RCA), two toxic lectins that are hazardous to human health. Radial immunodiffusion (RID) and the enzyme linked immunosorbent assay (ELISA) are two antibody-based methods commonly used to quantify ricin and RCA; however, antibodies currently used in these methods cannot distinguish between ricin and RCA due to the high sequence homology of the respective proteins. In this study, a technique combining antibody-based affinity capture with liquid chromatography and multiple reaction monitoring (MRM) mass spectrometry (MS) was used to quantify the amounts of ricin and RCA independently in extracts prepared from the seeds of eighteen representative cultivars of R. communis which were propagated under identical conditions. Additionally, liquid chromatography and MRM-MS was used to determine rRNA N-glycosidase activity for each cultivar and the overall activity in these cultivars was compared to a purified ricin standard. Of the cultivars studied, the average ricin content was 9.3 mg/g, the average RCA content was 9.9 mg/g, and the enzymatic activity agreed with the activity of a purified ricin reference within 35% relative activity. |
Detection and quantification of ricin in beverages using isotope dilution tandem mass spectrometry
McGrath SC , Schieltz DM , McWilliams LG , Pirkle JL , Barr JR . Anal Chem 2011 83 (8) 2897-905 The toxic plant protein ricin has gained notoriety due to wide availability and potential use as a bioterrorism agent, with particular concern for food supply contamination. We have developed a sensitive and selective mass spectrometry-based method to detect ricin in tap water, 2% milk, apple juice, and orange juice. Ricin added to beverage matrices was extracted using antibody-bound magnetic beads and digested with trypsin. Absolute quantification was performed using isotope dilution mass spectrometry with a linear ion trap operating in product-ion-monitoring mode. The method allows for identification of ricin A chain and B chain and for distinction of ricin from ricin agglutinin within a single analytical run. Ricin-bound beads were also tested for deadenylase activity by incubation with a synthetic ssDNA oligomer. Depurination of the substrate by ricin was confirmed by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOFMS). This method was used successfully to extract ricin from each beverage matrix. The activity of recovered ricin was assessed, and quantification was achieved, with a limit of detection of 10 fmol/mL (0.64 ng/mL). |
Analysis of active ricin and castor bean proteins in a ricin preparation, castor bean extract, and surface swabs from a public health investigation
Schieltz DM , McGrath SC , McWilliams LG , Rees J , Bowen MD , Kools JJ , Dauphin LA , Gomez-Saladin E , Newton BN , Stang HL , Vick MJ , Thomas J , Pirkle JL , Barr JR . Forensic Sci Int 2011 209 70-9 In late February 2008, law enforcement officials in Las Vegas, Nevada, discovered in a hotel room, a copy of The Anarchist Cookbook, suspected castor beans and a "white powder" thought to be a preparation of ricin. Ricin is a deadly toxin from the seed of the castor bean plant (Ricinus communis). The United States regulates the possession, use, and transfer of ricin and it is the only substance considered a warfare agent in both the Chemical and the Biological Weapons Conventions. Six samples obtained from the hotel room were analyzed by laboratories at the Centers for Disease Control and Prevention using a panel of biological and mass spectrometric assays. The biological assays (real time-PCR, time resolved fluorescence and cytotoxicity) provided presumptive evidence of active ricin in each of the samples. This initial screen was followed by an in-depth analysis using a novel, state-of-the-art mass spectrometry-based ricin functional assay and high sensitivity tandem mass spectrometry for protein identification. Mass spectrometric analysis positively identified ricin and confirmed that in each of the samples it was enzymatically active. The tandem mass spectrometry analysis used here is the most selective method available to detect ricin toxin. In each sample, ricin was unequivocally identified along with other R. communis plant proteins, including the highly homologous protein RCA120. Although database searches using tandem mass spectra acquired from the samples indicated that additional controlled substances were not present in these samples, the mass spectrometric results did provide extensive detail about the sample contents. To the best of our knowledge following a review of the available literature, this report describes the most detailed analysis of a white powder for a public health or forensic investigation involving ricin. |
Studies on botulinum neurotoxins type/C1 and mosaic/DC using endopep-MS and proteomics
Moura H , Terilli RR , Woolfitt AR , Gallegos-Candela M , McWilliams LG , Solano MI , Pirkle JL , Barr JR . FEMS Immunol Med Microbiol 2010 61 (3) 288-300 Botulinum neurotoxins (BoNTs) are very potent toxins and category A biological threat agents. BoNT serotypes/C1 and/D affect birds and mammals and can be potentially lethal to humans. We have previously described the usefulness of the Endopep-MS method to detect the activity of BoNT A through G. This report was followed by the application of the method to clinical samples. The activity of the BoNT serotypes associated with human disease (/A,/B,/E, and/F) was successfully detected. However, BoNT/C and/D require different conditions for fast substrate cleavage and a comprehensive description of a method to study BoNT/C and/D has not yet been reported. This work describes a new, optimized version of the Endopep-MS method to detect BoNTs/C1 and/DC either spiked directly in 20 muL of reaction buffer or spiked in a larger volume of buffer and further extracted using antibody-coated magnetic beads. It was found that the incubation temperature at 42 degrees C was more effective for both toxin serotypes, but each toxin serotype has an optimum cleavage pH. Additionally, we describe for the first time a proteomics study using a fast trypsin digestion method and label-free quantification of these toxin serotypes. |
Kinetics of lethal factor and poly-D-glutamic acid antigenemia during inhalation anthrax in rhesus macaques
Boyer AE , Quinn CP , Hoffmaster AR , Kozel TR , Saile E , Marston CK , Percival A , Plikaytis BD , Woolfitt AR , Gallegos M , Sabourin P , McWilliams LG , Pirkle JL , Barr JR . Infect Immun 2009 77 (8) 3432-41 Systemic anthrax manifests as toxemia, rapidly disseminating septicemia, immune collapse, and death. Virulence factors include the anti-phagocytic gamma-linked poly-d-glutamic acid (PGA) capsule and two binary toxins, complexes of protective antigen (PA) with lethal factor (LF) and edema factor. We report the characterization of LF, PA, and PGA levels during the course of inhalation anthrax in five rhesus macaques. We describe bacteremia, blood differentials, and detection of the PA gene (pagA) by PCR analysis of the blood as confirmation of infection. For four of five animals tested, LF exhibited a triphasic kinetic profile. LF levels (mean +/- standard error [SE] between animals) were low at 24 h postchallenge (0.03 +/- 1.82 ng/ml), increased at 48 h to 39.53 +/- 0.12 ng/ml (phase 1), declined at 72 h to 13.31 +/- 0.24 ng/ml (phase 2), and increased at 96 h (82.78 +/- 2.01 ng/ml) and 120 h (185.12 +/- 5.68 ng/ml; phase 3). The fifth animal had an extended phase 2. PGA levels were triphasic; they were nondetectable at 24 h, increased at 48 h (2,037 +/- 2 ng/ml), declined at 72 h (14 +/- 0.2 ng/ml), and then increased at 96 h (3,401 +/- 8 ng/ml) and 120 h (6,004 +/- 187 ng/ml). Bacteremia was also triphasic: positive at 48 h, negative at 72 h, and positive at euthanasia. Blood neutrophils increased from preexposure (34.4% +/- 0.13%) to 48 h (75.6% +/- 0.08%) and declined at 72 h (62.4% +/- 0.05%). The 72-h declines may establish a "go/no go" turning point in infection, after which systemic bacteremia ensues and the host's condition deteriorates. This study emphasizes the value of LF detection as a tool for early diagnosis of inhalation anthrax before the onset of fulminant systemic infection. |
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