23 research outputs found
Conducting Miller-Urey Experiments
In 1953, Stanley Miller reported the production of biomolecules from simple gaseous starting materials, using apparatus constructed to simulate the primordial Earth's atmosphere-ocean system. Miller introduced 200 ml of water, 100 mmHg of H2, 200mmHg of CH4, and 200mmHg of NH3 into the apparatus, then subjected this mixture, under reflux, to an electric discharge for a week, while the water was simultaneously heated. The purpose of this manuscript is to provide the reader with a general experimental protocol that can be used to conduct a Miller-Urey type spark discharge experiment, using a simplified 3 L reaction flask. Since the experiment involves exposing inflammable gases to a high voltage discharge, it is worth highlighting important steps that reduce the risk of explosion. The general procedures described in this work can be extrapolated to design and conduct a wide variety of electric discharge experiments simulating primitive planetary environments
Surface analysis of dental composite and its polymeric overlayer model system by mass spectrometry.
Surface analysis of dental composite and its polymeric overlayer model system by mass spectrometry
Optimization of a Direct Analysis in Real Time/Time-of-Flight Mass Spectrometry Method for Rapid Serum Metabolomic Fingerprinting
Metabolomic fingerprinting of bodily fluids can reveal the underlying causes of metabolic disorders associated with many diseases, and has thus been recognized as a potential tool for disease diagnosis and prognosis following therapy. Here we report a rapid approach in which direct analysis in real time (DART) coupled with time-of-flight (TOF) mass spectrometry (MS) and hybrid quadrupole TOF (Q-TOF) MS is used as a means for metabolomic fingerprinting of human serum. In this approach, serum samples are first treated to precipitate proteins, and the volatility of the remaining metabolites increased by derivatization, followed by DART MS analysis. Maximum DART MS performance was obtained by optimizing instrumental parameters such as ionizing gas temperature and flow rate for the analysis of identical aliquots of a healthy human serum samples. These variables were observed to have a significant effect on the overall mass range of the metabolites detected as well as the signal-to-noise ratios in DART mass spectra. Each DART run requires only 1.2 min, during which more than 1500 different spectral features are observed in a time-dependent fashion. A repeatability of 4.1% to 4.5% was obtained for the total ion signal using a manual sampling arm. With the appealing features of high-throughput, lack of memory effects, and simplicity, DART MS has shown potential to become an invaluable tool for metabolomic fingerprinting
Serum biomarker profiling by solid‐phase extraction with particle‐embedded micro tips and matrix‐assisted laser desorption/ionization mass spectrometry
One of the main challenges in high-throughput serum profiling by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is the development of proteome fractionation approaches that allow the acquisition of reproducible profiles with a maximum number of spectral features and minimum interferences from biological matrices. This study evaluates a new class of solid-phase extraction (SPE) pipette tips embedded with different chromatographic media for fractionation of model protein digests and serum samples. The materials embedded include strong anion exchange (SAX), weak cation exchange (WCX), C18, C8, C4, immobilized metal affinity chromatography (IMAC) and zirconium dioxide particles. Simple and rapid serum proteome profiling protocols based on these SPE micro tips are described and tested using a variety of MALDI matrices. We show that different types of particle-embedded SPE micro tips provide complementary information in terms of the spectral features detected for β-casein digests and control human serum samples. The effect of different sample pretreatments, such as serum dilution and ultrafiltration using molecular weight cut-off membranes, and the reproducibility observed for replicate experiments, are also evaluated. The results demonstrate the usefulness of these simple SPE tips combined with offline MALDI-TOF MS for obtaining information-rich serum profiles, resulting in a robust, versatile and reproducible open-source platform for serum biomarker discovery
Ovarian Cancer Detection from Metabolomic Liquid Chromatography/Mass Spectrometry Data by Support Vector Machines
© 2009 Guan et al; licensee BioMed Central Ltd. This article is available from: http://www.biomedcentral.com/1471-2105/10/259DOI:10.1186/1471-2105-10-259Background: The majority of ovarian cancer biomarker discovery efforts focus on the
identification of proteins that can improve the predictive power of presently available diagnostic
tests. We here show that metabolomics, the study of metabolic changes in biological systems, can
also provide characteristic small molecule fingerprints related to this disease.
Results: In this work, new approaches to automatic classification of metabolomic data produced
from sera of ovarian cancer patients and benign controls are investigated. The performance of
support vector machines (SVM) for the classification of liquid chromatography/time-of-flight mass
spectrometry (LC/TOF MS) metabolomic data focusing on recognizing combinations or "panels" of
potential metabolic diagnostic biomarkers was evaluated. Utilizing LC/TOF MS, sera from 37
ovarian cancer patients and 35 benign controls were studied. Optimum panels of spectral features
observed in positive or/and negative ion mode electrospray (ESI) MS with the ability to distinguish
between control and ovarian cancer samples were selected using state-of-the-art feature selection
methods such as recursive feature elimination and L1-norm SVM.
Conclusion: Three evaluation processes (leave-one-out-cross-validation, 12-fold-cross-validation,
52-20-split-validation) were used to examine the SVM models based on the selected panels in terms
of their ability for differentiating control vs. disease serum samples. The statistical significance for
these feature selection results were comprehensively investigated. Classification of the serum
sample test set was over 90% accurate indicating promise that the above approach may lead to the
development of an accurate and reliable metabolomic-based approach for detecting ovarian cancer
A tiered analytical approach for investigating poor quality emergency contraceptives.
Reproductive health has been deleteriously affected by poor quality medicines. Emergency contraceptive pills (ECPs) are an important birth control method that women can use after unprotected coitus for reducing the risk of pregnancy. In response to the detection of poor quality ECPs commercially available in the Peruvian market we developed a tiered multi-platform analytical strategy. In a survey to assess ECP medicine quality in Peru, 7 out of 25 different batches showed inadequate release of levonorgestrel by dissolution testing or improper amounts of active ingredient. One batch was found to contain a wrong active ingredient, with no detectable levonorgestrel. By combining ultrahigh performance liquid chromatography-ion mobility spectrometry-mass spectrometry (UHPLC-IMS-MS) and direct analysis in real time MS (DART-MS) the unknown compound was identified as the antibiotic sulfamethoxazole. Quantitation by UHPLC-triple quadrupole tandem MS (QqQ-MS/MS) indicated that the wrong ingredient was present in the ECP sample at levels which could have significant physiological effects. Further chemical characterization of the poor quality ECP samples included the identification of the excipients by 2D Diffusion-Ordered Nuclear Magnetic Resonance Spectroscopy (DOSY 1H NMR) indicating the presence of lactose and magnesium stearate
Biomarkers of Whale Shark Health: A Metabolomic Approach
<div><p>In a search for biomarkers of health in whale sharks and as exploration of metabolomics as a modern tool for understanding animal physiology, the metabolite composition of serum in six whale sharks (<em>Rhincodon typus</em>) from an aquarium collection was explored using <sup>1</sup>H nuclear magnetic resonance (NMR) spectroscopy and direct analysis in real time (DART) mass spectrometry (MS). Principal components analysis (PCA) of spectral data showed that individual animals could be resolved based on the metabolite composition of their serum and that two unhealthy individuals could be discriminated from the remaining healthy animals. The major difference between healthy and unhealthy individuals was the concentration of homarine, here reported for the first time in an elasmobranch, which was present at substantially lower concentrations in unhealthy whale sharks, suggesting that this metabolite may be a useful biomarker of health status in this species. The function(s) of homarine in sharks remain uncertain but it likely plays a significant role as an osmolyte. The presence of trimethylamine oxide (TMAO), another well-known protective osmolyte of elasmobranchs, at 0.1–0.3 mol L<sup>−1</sup> was also confirmed using both NMR and MS. Twenty-three additional potential biomarkers were identified based on significant differences in the frequency of their occurrence between samples from healthy and unhealthy animals, as detected by DART MS. Overall, NMR and MS provided complementary data that showed that metabolomics is a useful approach for biomarker prospecting in poorly studied species like elasmobranchs.</p> </div
TLC analysis.
<p>Results for a levonorgestrel standard, sample 1 (positive control), and sample 2a observed under 254 nm light.</p
Feasibility of Detecting Prostate Cancer by Ultraperformance Liquid Chromatography–Mass Spectrometry Serum Metabolomics
Prostate
cancer (PCa) is the second leading cause of cancer-related
mortality in men. The prevalent diagnosis method is based on the serum
prostate-specific antigen (PSA) screening test, which suffers from
low specificity, overdiagnosis, and overtreatment. In
this work, untargeted metabolomic profiling of age-matched serum samples
from prostate cancer patients and healthy individuals was performed
using ultraperformance liquid chromatography coupled to high-resolution
tandem mass spectrometry (UPLC-MS/MS) and machine learning methods.
A metabolite-based in vitro diagnostic multivariate index assay
(IVDMIA) was developed to predict the presence of PCa in serum samples
with high classification sensitivity, specificity, and accuracy. A
panel of 40 metabolic spectral features was found to be differential
with 92.1% sensitivity, 94.3% specificity, and 93.0% accuracy. The
performance of the IVDMIA was higher than the prevalent PSA test.
Within the discriminant panel, 31 metabolites were identified by MS
and MS/MS, with 10 further confirmed chromatographically by
standards. Numerous discriminant metabolites were mapped in the steroid
hormone biosynthesis pathway. The identification of fatty acids, amino
acids, lysophospholipids, and bile acids provided further
insights into the metabolic alterations associated with the disease.
With additional work, the results presented here show great potential
toward implementation in clinical settings