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A High Throughput, ...
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Heywood, Wendy E
(author)
A High Throughput, Multiplexed and Targeted Proteomic CSF Assay to Quantify Neurodegenerative Biomarkers and Apolipoprotein E Isoforms Status.
- Article/chapterEnglish2016
Publisher, publication year, extent ...
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2016-10-20
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MyJove Corporation,2016
Numbers
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LIBRIS-ID:oai:gup.ub.gu.se/246427
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https://gup.ub.gu.se/publication/246427URI
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https://doi.org/10.3791/54541DOI
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Subject category:ref swepub-contenttype
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Subject category:art swepub-publicationtype
Notes
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Many neurodegenerative diseases arestill lacking effective treatments. Reliable biomarkers for identifying and classifying these diseases will be important in the development of future novel therapies. Oftenpotential new biomarkers do not make itinto the clinicdue to limitationsin their development andhigh costs.However,targeted proteomics using Multiple Reaction Monitoring Liquid Chromatography-tandem/Mass Spectrometry (MRM LC-MS/MS), specifically using triple quadrupole mass spectrometers, is one method that can be used to rapidly evaluate and validate biomarkersfor clinical translation into diagnostic laboratories. Traditionally, this platform has been used extensively for measurement of small moleculesin clinical laboratories, but it is the potential to analyze proteins, that makes it an attractive alternative to ELISA (Enzyme-Linked Immunosorbent Assay)-based methods. We describe here how targeted proteomics can be used to measure multiplexed markers of dementia, including the detection and quantitation of the known risk factor apolipoprotein E isoform 4 (ApoE4). In order to make the assay suitable for translation, it is designed to be rapid, simple, highly specific and cost effective. To achieve this, every step in the development of the assay must be optimized for the individual proteins and tissues they are analyzed in. This method describes a typical workflow including various tips and tricks to developing a targeted proteomics MRM LC-MS/MS for translation. The method development is optimized using custom synthesized versions of tryptic quantotypic peptides, which calibratethe MS for detection and then spiked into CSF to determine correct identification of the endogenous peptide in the chromatographic separation prior to analysis in the MS. To achieve absolute quantitation, stable isotope-labeled internal standard versions of the peptides with short amino acid sequence tags and containing a trypsin cleavage site, are included in the assay.
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Baud, Anna
(author)
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Bliss, Emily
(author)
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Sirka, Ernestas
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Schott, Jonathan M
(author)
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Zetterberg, Henrik,1973Gothenburg University,Göteborgs universitet,Institutionen för neurovetenskap och fysiologi, sektionen för psykiatri och neurokemi,Institute of Neuroscience and Physiology, Department of Psychiatry and Neurochemistry(Swepub:gu)xzethe
(author)
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Galimberti, Daniela
(author)
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Sebire, Neil J
(author)
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Mills, Kevin
(author)
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Göteborgs universitetInstitutionen för neurovetenskap och fysiologi, sektionen för psykiatri och neurokemi
(creator_code:org_t)
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In:Journal of visualized experiments : JoVE: MyJove Corporation:1161940-087X
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