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Investigating the use of isotope-labeled standards as calibrants in label-free quantification
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Biology Education Centre.
2018 (English)Independent thesis Advanced level (professional degree), 20 credits / 30 HE creditsStudent thesis
Abstract [en]

The ability to accurately identify and quantify proteins in complexsamples is of great importance in the field of proteomics. Using massspectrometry, samples can be analysed and quantified either by theincorporation of a labelled standard of known concentration, or bylabel free quantification. Label free quantification has manybenefits, including time, cost, and ease of use, but is not asaccurate as the use of isotope label standards. In this project, thepossibility of increasing accuracy in quantification results from LFQusing a set of isotope labelled standards, QPrESTs, is investigated.The standards were produced by metabolic incorporation of heavyLysine and Arginine during expression inE. coli. They were then qualitycontrolled using SDS-PAGE for purity analysis, and LC-MS/MS forquantification and confirmation of MW. Human cell lysate samplesspiked with a set of 21 QPrEST standards were analysed by LC-MS/MSand quantified by QPrEST-H/L intensity ratios and intensity basedLFQ. In the LFQ protein quantification indices obtained from MaxQuantwere combined with BCA results, or with calibration curves obtainedfrom spiked in QPrEST standards. The LFQ results that best matchedthose obtained from QPrEST-H/L were those that used the calibrationcurves for quantification, which were found in a ~3-fold range, witha correlation coefficient varying from 0.67 to 1. Assuming thatQPrEST-H/L is the most accurate quantification method used, thisindicates that the use of QPrEST standards as calibrants can bebeneficial when it comes to increasing the accuracy in LFQ.

Place, publisher, year, edition, pages
2018.
Series
UPTEC X ; 18 018
National Category
Natural Sciences Engineering and Technology
Identifiers
URN: urn:nbn:se:uu:diva-355130OAI: oai:DiVA.org:uu-355130DiVA, id: diva2:1225243
Educational program
Molecular Biotechnology Engineering Programme
Supervisors
Examiners
Available from: 2018-06-27 Created: 2018-06-26 Last updated: 2018-06-27Bibliographically approved

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CiteExportLink to record
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