Related ArticlesA simple and accurate protocol for absolute polar metabolite quantification in cell cultures using q-NMR.
Anal Biochem. 2016 Feb 17;
Authors: Goldoni L, Beringhelli T, Rocchia W, Realini N, Piomelli D
Abstract
Absolute analyte quantification by NMR spectroscopy is rarely pursued in metabolomics, even though this would allow researchers to compare results obtained using different techniques. Here we report on a new protocol that permits, after pH-controlled serum protein removal, the sensitive quantification (LOD 5-25 ?M) of hydrophilic nutrients and metabolites in the extracellular medium of cells in cultures. The method does not require the use of databases and utilizes PULCON (PUlse Length Based Concentration Determination) quantitative NMR to obtain results that are significantly more accurate and reproducible than those obtained by CPMG (Carr-Purcell-Meiboom-Gill) sequence or post processing filtering approaches. Three practical applications of the method highlight its flexibility under different cell culture conditions. We identified and quantified (i) metabolic differences, between genetically engineered human cell lines; (ii) alterations in cellular metabolism induced by differentiation of mouse myoblasts into myotubes; and (iii) metabolic changes caused by activation of neurotransmitter receptors in mouse myoblasts. Thus, the new protocol offers an easily implementable, efficient and versatile tool for the investigation of cellular metabolism and signal transduction.
PMID: 26898303 [PubMed - as supplied by publisher]
A simple protocol for amino acid type selective isotope labeling in insect cells with improved yields and high reproducibility
A simple protocol for amino acid type selective isotope labeling in insect cells with improved yields and high reproducibility
Abstract An easy to use and robust approach for amino acid type selective isotope labeling in insect cells is presented. It relies on inexpensive commercial media and can be implemented in laboratories without sophisticated infrastructure. In contrast to previous protocols, where either high protein amounts or high incorporation ratios were obtained, here we achieve both at the same time. By supplementing media with a well considered amount of yeast extract,...
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10-05-2011 08:57 PM
NMR-based metabolomics of mammalian cell and tissue cultures
NMR-based metabolomics of mammalian cell and tissue cultures
Abstract NMR spectroscopy was used to evaluate growth media and the cellular metabolome in two systems of interest to biomedical research. The first of these was a Chinese hamster ovary cell line engineered to express a recombinant protein. Here, NMR spectroscopy and a quantum mechanical total line shape analysis were utilized to quantify 30 metabolites such as amino acids, Krebs cycle intermediates, activated sugars, cofactors, and others in both media and cell extracts. The impact of bioreactor scale and addition of...
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03-09-2011 04:19 AM
NMR-based metabolomics of mammalian cell and tissue cultures.
NMR-based metabolomics of mammalian cell and tissue cultures.
NMR-based metabolomics of mammalian cell and tissue cultures.
J Biomol NMR. 2011 Mar 4;
Authors: Aranibar N, Borys M, Mackin NA, Ly V, Abu-Absi N, Abu-Absi S, Niemitz M, Schilling B, Li ZJ, Brock B, Russell RJ, Tymiak A, Reily MD
NMR spectroscopy was used to evaluate growth media and the cellular metabolome in two systems of interest to biomedical research. The first of these was a Chinese hamster ovary cell line engineered to express a recombinant protein. Here, NMR spectroscopy and a...
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03-05-2011 01:02 PM
[NMR paper] Cell-free protein production and labeling protocol for NMR-based structural proteomic
Cell-free protein production and labeling protocol for NMR-based structural proteomics.
Related Articles Cell-free protein production and labeling protocol for NMR-based structural proteomics.
Nat Methods. 2004 Nov;1(2):149-53
Authors: Vinarov DA, Lytle BL, Peterson FC, Tyler EM, Volkman BF, Markley JL
Structural proteomics requires robust, scalable methods. Here we describe a wheat germ cell-free platform for protein production that supports efficient NMR structural studies of eukaryotic proteins and offers advantages over cell-based methods....
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11-24-2010 10:01 PM
[NMR paper] A simple protocol to study blue copper proteins by NMR.
A simple protocol to study blue copper proteins by NMR.
Related Articles A simple protocol to study blue copper proteins by NMR.
Eur J Biochem. 2003 Feb;270(4):600-9
Authors: Gelis I, Katsaros N, Luchinat C, Piccioli M, Poggi L
In the case of oxidized plastocyanin from Synechocystis sp. PCC6803, an NMR approach based on classical two and three dimensional experiments for sequential assignment leaves unobserved 14 out of 98 amino acids. A protocol which simply makes use of tailored versions of 2D HSQC and 3D CBCA(CO)NH and CBCANH leads to the...
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11-24-2010 09:01 PM
Rapid, Accurate and Simple Model to Predict NMR Chemical Shifts for Biological Molecu
Rapid, Accurate and Simple Model to Predict NMR Chemical Shifts for Biological Molecules.
Rapid, Accurate and Simple Model to Predict NMR Chemical Shifts for Biological Molecules.
J Phys Chem B. 2010 Nov 18;
Authors: Atieh Z, Aubert-Fre?con M, Allouche AR
We present a new model to predict chemical shifts for biological molecules. It is simple, fast, and involves a limited number of parameters. It is particularly adapted to be used in molecular dynamics studies with a molecular mechanic potential. We test the model for polyamines, which are rather...
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11-20-2010 06:01 PM
[NMR paper] Simple techniques for the quantification of protein secondary structure by 1H NMR spe
Simple techniques for the quantification of protein secondary structure by 1H NMR spectroscopy.
http://www.ncbi.nlm.nih.gov/corehtml/query/egifs/http:--linkinghub.elsevier.com-ihub-images-PubMedLink.gif Related Articles Simple techniques for the quantification of protein secondary structure by 1H NMR spectroscopy.
FEBS Lett. 1991 Nov 18;293(1-2):72-80
Authors: Wishart DS, Sykes BD, Richards FM
Previous work by Wishart et al. (in press) and others has shown a strong tendency for protein secondary structure to be manifested in 1H NMR chemical...
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08-21-2010 11:12 PM
[NMR paper] Simple techniques for the quantification of protein secondary structure by 1H NMR spe
Simple techniques for the quantification of protein secondary structure by 1H NMR spectroscopy.
http://www.ncbi.nlm.nih.gov/corehtml/query/egifs/http:--linkinghub.elsevier.com-ihub-images-PubMedLink.gif Related Articles Simple techniques for the quantification of protein secondary structure by 1H NMR spectroscopy.
FEBS Lett. 1991 Nov 18;293(1-2):72-80
Authors: Wishart DS, Sykes BD, Richards FM
Previous work by Wishart et al. (in press) and others has shown a strong tendency for protein secondary structure to be manifested in 1H NMR chemical...
A simple and accurate protocol for absolute polar metabolite quantification in cell cultures using q-NMR.
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