Last data update: Apr 29, 2024. (Total: 46658 publications since 2009)
Records 1-15 (of 15 Records) |
Query Trace: Rees JC[original query] |
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Stability of lipids in plasma and serum: Effects of temperature-related storage conditions on the human lipidome
Reis GB , Rees JC , Ivanova AA , Kuklenyik Z , Drew NM , Pirkle JL , Barr JR . J Mass Spectrom Adv Clin Lab 2021 22 34-42 Large epidemiological studies often require sample transportation and storage, presenting unique considerations when applying advanced lipidomics techniques. The goal of this study was to acquire lipidomics data on plasma and serum samples stored at potential preanalytical conditions (e.g., thawing, extracting, evaporating), systematically monitoring lipid species for a period of one month. Split aliquots of 10 plasma samples and 10 serum samples from healthy individuals were kept in three temperature-related environments: refrigerator, laboratory benchtop, or heated incubator. Samples were analyzed at six different time points over 28 days using a Bligh & Dyer lipid extraction protocol followed by direct infusion into a lipidomics platform using differential mobility with tandem mass spectrometry. The observed concentration changes over time were evaluated relative to method and inter-individual biological variability. In addition, to evaluate the effect of lipase enzyme levels on concentration changes during storage, we compared corresponding fasting and post-prandial plasma samples collected from 5 individuals. Based on our data, a series of low abundance free fatty acid (FFA), diacylglycerol (DAG), and cholesteryl ester (CE) species were identified as potential analytical markers for degradation. These FFA and DAG species are typically produced by endogenous lipases from numerous triacylglycerols (TAGs), and certain high abundance phosphatidylcholines (PCs). The low concentration CEs, which appeared to increase several fold, were likely mass-isobars from oxidation of other high concentration CEs. Although the concentration changes of the high abundant TAG, PC, and CE precursors remained within method variability, the concentration trends of FFA, DAG, and oxidized CE products should be systematically monitored over time to inform analysts about possible pre-analytical biases due to degradation in the study sample sets. |
Integrated Quantitative Targeted Lipidomics and Proteomics Reveal Unique Fingerprints of Multiple Metabolic Conditions.
Ivanova AA , Rees JC , Parks BA , Andrews M , Gardner M , Grigorutsa E , Kuklenyik Z , Pirkle JL , Barr JR . Biomolecules 2022 12 (10) Aberrations in lipid and lipoprotein metabolic pathways can lead to numerous diseases, including cardiovascular disease, diabetes, neurological disorders, and cancer. The integration of quantitative lipid and lipoprotein profiling of human plasma may provide a powerful approach to inform early disease diagnosis and prevention. In this study, we leveraged data-driven quantitative targeted lipidomics and proteomics to identify specific molecular changes associated with different metabolic risk categories, including hyperlipidemic, hypercholesterolemic, hypertriglyceridemic, hyperglycemic, and normolipidemic conditions. Based on the quantitative characterization of serum samples from 146 individuals, we have determined individual lipid species and proteins that were significantly up- or down-regulated relative to the normolipidemic group. Then, we established protein-lipid topological networks for each metabolic category and linked dysregulated proteins and lipids with defined metabolic pathways. To evaluate the differentiating power of integrated lipidomics and proteomics data, we have built an artificial neural network model that simultaneously and accurately categorized the samples from each metabolic risk category based on the determined lipidomics and proteomics profiles. Together, our findings provide new insights into molecular changes associated with metabolic risk conditions, suggest new condition-specific associations between apolipoproteins and lipids, and may inform new biomarker discovery in lipid metabolism-associated disorders. |
The small HDL particle hypothesis of Alzheimer's disease
Martinez AE , Weissberger G , Kuklenyik Z , He X , Meuret C , Parekh T , Rees JC , Parks BA , Gardner MS , King SM , Collier TS , Harrington MG , Sweeney MD , Wang X , Zlokovic BV , Joe E , Nation DA , Schneider LS , Chui HC , Barr JR , Han SD , Krauss RM , Yassine HN . Alzheimers Dement 2022 19 (2) 391-404 We propose the hypothesis that small high-density lipoprotein (HDL) particles reduce the risk of Alzheimer's disease (AD) by virtue of their capacity to exchange lipids, affecting neuronal membrane composition and vascular and synaptic functions. Concentrations of small HDLs in cerebrospinal fluid (CSF) and plasma were measured in 180 individuals ≥60 years of age using ion mobility methodology. Small HDL concentrations in CSF were positively associated with performance in three domains of cognitive function independent of apolipoprotein E (APOE) ε4 status, age, sex, and years of education. Moreover, there was a significant correlation between levels of small HDLs in CSF and plasma. Further studies will be aimed at determining whether specific components of small HDL exchange across the blood, brain, and CSF barriers, and developing approaches to exploit small HDLs for therapeutic purposes. |
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. |
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. |
Detection of methicillin-resistant Staphylococcus aureus using phage amplification combined with matrix-assisted laser desorption/ionization mass spectrometry
Rees JC , Barr JR . Anal Bioanal Chem 2016 409 (5) 1379-1386 Antibiotic resistance continues to contribute significantly to morbidity and mortality across the world. Developing new tests for antibiotic-resistant bacteria is a core action to combat resistant infections. We describe a method that uses phage amplification detection (PAD) combined with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) to rapidly identify Staphylococcus aureus and determine phenotypic susceptibility to cefoxitin. Samples tested for S. aureus are incubated together with bacteriophage in the presence and absence of cefoxitin and subjected to rapid trypsin digestion followed by MALDI-MS analysis. Tryptic peptides derived from amplified phage proteins can be detected by MALDI-MS, as validated by time-of-flight (TOF)/TOF analysis of each peptide combined with database searching. Methicillin-resistant S. aureus show significant phage amplification in the presence of cefoxitin, while methicillin-sensitive S. aureus show no phage amplification relative to a no-antibiotic control. We also show that PAD methodology can be implemented on an FDA-approved commercial MALDI-MS bacterial identification system to identify S. aureus and determine antibiotic susceptibility. The novelty of this assay includes the use of phage-derived tryptic peptides as detected by MALDI-MS to monitor the results of PAD on an instrument common to many modern microbiology laboratories. |
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. |
Simultaneous identification and susceptibility determination to multiple antibiotics of Staphylococcus aureus by bacteriophage amplification detection combined with mass spectrometry
Rees JC , Pierce CL , Schieltz DM , Barr JR . Anal Chem 2015 87 (13) 6769-77 The continued advance of antibiotic resistance in clinically relevant bacterial strains necessitates the development and refinement of assays that can rapidly and cost-effectively identify bacteria and determine their susceptibility to a panel of antibiotics. A methodology is described herein that exploits the specificity and physiology of the Staphylococci bacteriophage K to identify Staphylococcus aureus (S. aureus) and determine its susceptibility to clindamycin and cefoxitin. The method uses liquid chromatography-mass spectrometry to monitor the replication of bacteriophage after it is used to infect samples thought to contain S. aureus. Amplification of bacteriophage K indicates the sample contains S. aureus, for it is only in the presence of a suitable host that bacteriophage K can amplify. If bacteriophage amplification is detected in samples containing the antibiotics clindamycin or cefoxitin, the sample is deemed to be resistant to these antibiotics, respectively, for bacteriophage can only amplify in a viable host. Thus, with a single work flow, S. aureus can be detected in an unknown sample and susceptibility to clindamycin and cefoxitin can be ascertained. This Article discusses implications for the use of bacteriophage amplification in the clinical laboratory. |
A proteomic characterization of Bordetella pertussis clinical isolates associated with a California state pertussis outbreak
Williamson YM , Moura H , Whitmon J , Woolfitt AR , Schieltz DM , Rees JC , Guo S , Kirkham H , Bouck D , Ades EW , Tondella ML , Carlone GM , Sampson JS , Barr JR . Int J Proteomics 2015 2015 536537 Bordetella pertussis (Bp) is the etiologic agent of pertussis (whooping cough), a highly communicable infection. Although pertussis is vaccine preventable, in recent years there has been increased incidence, despite high vaccine coverage. Possible reasons for the rise in cases include the following: Bp strain adaptation, waning vaccine immunity, increased surveillance, and improved clinical diagnostics. A pertussis outbreak impacted California (USA) in 2010; children and preadolescents were the most affected but the burden of disease fell mainly on infants. To identify protein biomarkers associated with this pertussis outbreak, we report a whole cellular protein characterization of six Bp isolates plus the pertussis acellular vaccine strain Bp Tohama I (T), utilizing gel-free proteomics-based mass spectrometry (MS). MS/MS tryptic peptide detection and protein database searching combined with western blot analysis revealed three Bp isolates in this study had markedly reduced detection of pertactin (Prn), a subunit of pertussis acellular vaccines. Additionally, antibody affinity capture technologies were implemented using anti-Bp T rabbit polyclonal antisera and whole cellular proteins to identify putative immunogens. Proteome profiling could shed light on pathogenesis and potentially lay the foundation for reduced infection transmission strategies and improved clinical diagnostics. |
Modified MALDI MS fatty acid profiling for bacterial identification
Voorhees KJ , Jensen KR , McAlpin CR , Rees JC , Cody R , Ubukata M , Cox CR . J Mass Spectrom 2013 48 (7) 850-5 Bacterial fatty acid profiling is a well-established technique for bacterial identification. Ten bacteria were analyzed using both positive- and negative-ion modes with a modified matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) approach using CaO as a matrix replacement (metal oxide laser ionization MS (MOLI MS)). The results show that reproducible lipid cleavage similar to thermal in situ tetramethyl ammonium hydroxide saponification/derivatization had occurred. Principal component analysis showed that replicates from each organism grouped in a unique space. Cross validation (CV) of spectra from both ionization modes resulted in greater than 94% validation of the data. When CV results were compared for the two ionization modes, negative-ion data produced a superior outcome. MOLI MS provides clinicians a rapid, reproducible and cost-effective bacterial diagnostic tool. |
Improved detection of botulinum neurotoxin serotype A by Endopep-MS through peptide substrate modification
Wang D , Baudys J , Ye Y , Rees JC , Barr JR , Pirkle JL , Kalb SR . Anal Biochem 2013 432 (2) 115-23 Botulinum neurotoxins (BoNTs) are a family of seven toxin serotypes that are the most toxic substances known to humans. Intoxication with BoNT causes flaccid paralysis and can lead to death if untreated with serotype-specific antibodies. Supportive care, including ventilation, may be necessary. Rapid and sensitive detection of BoNT is necessary for timely clinical confirmation of clinical botulism. Previously, our laboratory developed a fast and sensitive mass spectrometry (MS) method termed the Endopep-MS assay. The BoNT serotypes are rapidly detected and differentiated by extracting the toxin with serotype-specific antibodies and detecting the unique and serotype-specific cleavage products of peptide substrates that mimic the sequence of the BoNT native targets. To further improve the sensitivity of the Endopep-MS assay, we report here the optimization of the substrate peptide for the detection of BoNT/A. Modifications on the terminal groups of the original peptide substrate with acetylation and amidation significantly improved the detection of BoNT/A cleavage products. The replacement of some internal amino acid residues with single or multiple substitutions led to further improvement. An optimized peptide increased assay sensitivity 5-fold with toxin spiked into buffer solution or different biological matrices. |
Modeling bacteriophage amplification as a predictive tool for optimized MALDI-TOF MS-based bacterial detection
Cox CR , Rees JC , Voorhees KJ . J Mass Spectrom 2012 47 (11) 1435-1441 Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a valuable tool for rapid bacterial detection and identification but is limited by the need for relatively high cell count samples, which have been grown under strictly controlled conditions. These requirements can be eliminated by the natural infection of a viable bacterial species of interest with a host-specific phage. This produces a rapid increase in phage protein concentrations in comparison to bacterial concentrations, which can in turn be exploited as a method for signal amplification during MALDI-TOF MS. One drawback to this approach is the requirement for repetitive, time-consuming sample preparation and analysis applied over the course of a phage infection to monitor phage concentrations as a function of time to determine the MALDI-TOF MS detection limit. To reduce the requirement for repeated preparation and analysis, a modified phage therapy model was investigated as a means for predicting the time during a given phage infection when a detectable signal would occur. The modified model used a series of three differential equations composed of predetermined experimental parameters including phage burst size and burst time to predict progeny phage concentrations as a function of time. Using Yersinia pestis with plague diagnostic phage A1122 and Escherichia coli with phage MS2 as two separate, well-characterized model phage-host pairs, we conducted in silico modeling of the infection process and compared it with experimental infections monitored in real time by MALDI-TOF MS. Significant agreement between mathematically calculated phage growth curves and those experimentally obtained by MALDI-TOF MS was observed, thus verifying this method's utility for significant time and labor reduction. (Copyright 2012 John Wiley Sons, Ltd.) |
De novo subtype and strain identification of botulinum neurotoxin type B through toxin proteomics
Kalb SR , Baudys J , Rees JC , Smith TJ , Smith LA , Helma CH , Hill K , Kull S , Kirchner S , Dorner MB , Dorner BG , Pirkle JL , Barr JR . Anal Bioanal Chem 2012 403 (1) 215-26 Botulinum neurotoxins (BoNTs) cause the disease botulism, which can be lethal if untreated. There are seven known serotypes of BoNT, A-G, defined by their response to antisera. Many serotypes are distinguished into differing subtypes based on amino acid sequence, and many subtypes are further differentiated into toxin variants. Previous work in our laboratory described the use of a proteomics approach to distinguish subtype BoNT/A1 from BoNT/A2 where BoNT identities were confirmed after searching data against a database containing protein sequences of all known BoNT/A subtypes. We now describe here a similar approach to differentiate subtypes BoNT/B1, /B2, /B3, /B4, and /B5. Additionally, to identify new subtypes or hitherto unpublished amino acid substitutions, we created an amino acid substitution database covering every possible amino acid change. We used this database to differentiate multiple toxin variants within subtypes of BoNT/B1 and B2. More importantly, with our amino acid substitution database, we were able to identify a novel BoNT/B subtype, designated here as BoNT/B7. These techniques allow for subtype and strain level identification of both known and unknown BoNT/B rapidly with no DNA required. |
Viable Staphylococcus aureus quantitation using 15N metabolically-labeled bacteriophage amplification coupled with a multiple reaction monitoring proteomic workflow
Pierce CL , Rees JC , Fernandez FM , Barr JR . Mol Cell Proteomics 2011 11 (1) M111 012849 A multiple reaction monitoring liquid chromatography (LC) method with tandem mass spectrometric detection (MS/MS) for quantitation of Staphylococcus aureus via phage amplification detection (PAD) is described. This PAD method enables rapid and accurate quantitation of viable S. aureus by detecting an amplified capsid protein from a specific phage. A known amount of metabolically-labeled (15)N reference bacteriophage, utilized as the input phage and as the internal standard for quantitation, was spiked into S. aureus samples. Following a 2-h incubation, the sample was subjected to a 3-min rapid trypsin digest and analyzed by high-throughput LC-MS/MS targeting peptides unique to both the (15)N (input phage) and (14)N (progeny phage) capsid proteins. Quantitation was achieved by comparing peak areas of target peptides from the metabolically labeled (15)N bacteriophage peptide internal standard with that of the wild-type (14)N peptides that were produced by phage amplification and subsequent digestion when the host bacteria was present. This approach is based on the fact that a labeled species differs from the unlabeled one in terms of its mass but exhibits almost identical chemical properties such as ion yields and retention times. A 6-point calibration curve for S. aureus concentration was constructed with standards ranging from 5.0x10(4) CFU mL(-1) to 2.0x10(6) CFU mL(-1), with the (15)N reference phage spiked at a concentration of 1.0x10(9) PFU mL(-1). Amplification with (15)N bacteriophage coupled with LC-MS/MS detection offers speed (3 h total analysis time), sensitivity (LOD: < 5.0 x 10(4) CFU mL(-1)), accuracy, and precision for quantitation of S. aureus. |
Detection of Staphylococcus aureus using (15)N-labeled bacteriophage amplification coupled with matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry
Pierce CL , Rees JC , Fernandez FM , Barr JR . Anal Chem 2011 83 (6) 2286-93 A novel approach to rapid bacterial detection using an isotopically labeled (15)N bacteriophage and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) is introduced. Current phage amplification detection (PAD) via mass spectrometric analysis is limited because host bacteria must be inoculated with low phage titers in such a way that initial infecting phage concentrations must be below the detection limit of the instrument, thus lengthening incubation times. Additionally, PAD techniques cannot distinguish inoculate input phage from output phage which can increase the possibility of false positive results. Here, we report a rapid and accurate PAD approach for identification of Staphylococcus aureus via detection of bacteriophage capsid proteins. This approach uses both a wild-type (14)N and a (15)N-isotopically labeled S. aureus-specific bacteriophage. High (15)N phage titers, above our instrument's detection limits, were used to inoculate S. aureus. MALDI-TOF MS detection of the (14)N progeny capsid proteins in the phage-amplified culture indicated the presence of the host bacteria. Successful phage amplification was observed after 90 min of incubation. The amplification was observed by both MALDI-TOF MS analysis and by standard plaque assay measurements. This method overcomes current limitations by improving analysis times while increasing selectivity when compared to previously reported PAD methodologies. |
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