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Find video protocols related to scientific articles indexed in Pubmed.
Stability assessment of different chelating moieties used for elemental labeling of bio-molecules.
Metallomics
PUBLISHED: 09-16-2011
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Integrating elemental labeling in quantitative LC-ICP-MS based bio-analysis requires fundamental experiments concerning the stability of complexes during analysis. In a competitive approach complex stability of the chelating moieties 1,4,7,10-tetraazacyclododecane-N,N,N,N-tetraaceticacid (DOTA), 1,4,7-triazacyclononane-N,N,N-triacetic acid (NOTA) and diethylenetriaminepentaacetic dianhydride (DTPA) in combination with 11 different lanthanides was investigated under typical chromatographic conditions. Measurements were carried out via LC-ICP-QMS using a novel mixed mode separation method. The influence of chromatographic separation, pH and temperature on complex stability constants was assessed regarding further applications of multiplexing in bio-analytical assays. The limit of detection (LOD) for LC-ICP-QMS was 0.03 nM for all investigated Tm complexes (0.15 fmol absolute). Quantification of the complexes was performed via external, flow injection based calibration. For all investigated complexes the stability was significantly decreased by the chromatographic conditions. Moreover, complexation by DOTA revealed two different signals suggesting the presence of a stable intermediate product. Ln(3+)-DOTA and Ln(3+)-NOTA complexes provided high stability at 5 °C and 37 °C over a time of 12 hours, whereas Ln(3+)-DTPA complexes showed significant degradation at 37 °C.
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High-throughput flow injection analysis of labeled peptides in cellular samples - ICP-MS analysis versus fluorescence based detection.
Int J Mass Spectrom
PUBLISHED: 07-15-2011
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A high throughput method based on flow injection analysis was developed and validated for the quantification of the peptide B?(15-42) in cellular samples comparing different labeling strategies and detection methods. The used labels were 1,4,7,10-tetraazacyclododecane-N, N, N, N-tetraaceticacid (In-DOTA) and 2-(4-isothiocyanatobenzyl) - 1,4,7,10-tetraazacyclododecane-N, N, N, N-tetraacetic acid (In-DOTA-Bn) for elemental labeling. 6-Hydroxy-9-(2-carboxyphenyl)- (3H)-xanthen-3-on (fluorescein) was employed as fluorescence label. The explored peptide (mass = 3 kD) is a novel candidate drug, which shows an anti-inflammatory effect after an event of myocardial infarction. The analysed samples were fractioned cell compartments of human umbilical cord vein endothelial cells (HUVEC) maintained via lysis with Triton X buffer. In order to enhance sensitivity and selectivity of peptide quantification via flow injection the peptide was labeled prior to incubation using elemental and fluorescence labels. Quantification of the elemental and fluorescence labeled peptide was performed via flow injection analysis combined with inductive coupled plasma sector field mass spectrometry (FIA-ICP-SFMS) or fluorescence detection (FIA-FLD), respectively. The employed quantification strategies were external calibration in the case of fluorescence detection and external calibration with and without internal standardization and on-line IDMS in the case of ICP-MS detectionThe limit of detection (LOD) for FIA-ICP-MS was 9 pM In-DOTA-B?(15-42) (0.05 fmol absolute) whereas FIA-FLD showed a LOD of 100 pM (3 fmol absolute) for the fluorescein labeled peptide. Short term precision of FIA-ICP-MS was superior for all ICP-MS based quantification strategies compared to FIA-FLD (FIA-ICP-SFMS: 0.3-3.3%; FIA-FLD: 6.5%). Concerning long term precision FIA-ICP-SFMS with on-line IDMS and internal standardization showed the best results (3.1 and 4.6%, respectively) whereas the external calibration of both applied methodological approaches was only in the range of 10 %.The concentrations in the Triton X soluble fraction relative to the applied amount of Indium in the cell culture were in the range of 0.75-1.8% for In-DOTA or 0.30-0.79% for the 2-(4-isothiocyanatobenzyl) - 1,4,7,10-tetraazacyclododecane-N, N, N, N-tetraacetic acid (In-DOTA-Bn) labeled peptide B?(15-42). In the Triton X insoluble fraction the relative concentrations of Indium were 0.03-0.18% for the In-DOTA labeled peptide and 0.03-0.13% for B?(15-42)-In-DOTA-Bn.
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Elemental labelling combined with liquid chromatography inductively coupled plasma mass spectrometry for quantification of biomolecules: a review.
Anal. Chim. Acta
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This article reviews novel quantification concepts where elemental labelling is combined with flow injection inductively coupled plasma mass spectrometry (FI-ICP-MS) or liquid chromatography inductively coupled plasma mass spectrometry (LC-ICP-MS), and employed for quantification of biomolecules such as proteins, peptides and related molecules in challenging sample matrices. In the first sections an overview on general aspects of biomolecule quantification, as well as of labelling will be presented emphasizing the potential, which lies in such methodological approaches. In this context, ICP-MS as detector provides high sensitivity, selectivity and robustness in biological samples and offers the capability for multiplexing and isotope dilution mass spectrometry (IDMS). Fundamental methodology of elemental labelling will be highlighted and analytical, as well as biomedical applications will be presented. A special focus will lie on established applications underlining benefits and bottlenecks of such approaches for the implementation in real life analysis. Key research made in this field will be summarized and a perspective for future developments including sophisticated and innovative applications will given.
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What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

Video X seems to be unrelated to Abstract Y...

In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.