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Production and validation of heavy isotope-labeled protein fragments as standards for quantitative MS analysis
KTH, School of Biotechnology (BIO).
2014 (English)Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
Abstract [en]

Quantitative analysis of protein can give a better understanding of protein function as well as finding new biomarkers that can help in diagnostics. Mass spectrometry (MS) is today the main technology for high-throughput protein analysis. To get an absolute concentration of a protein with MS analysis, a good reference is of essence. By uing heavy isotope-labeled proteins or peptides as reference a mass shift is created that enables a quantitative analysis with MS where the intensity fo the exact same peptides can be cmpared. Protein Epitope Signature Tag (PrEST) is a unique protein fragment produced for all human proteins in the course of the Human Protein Atlas (HPA) project to function as antigens in the antibody production. In this project the production of heavy isotope-labeled PrESTs was optimized and their suitability as references examined. By cultivating Escherichia coli cells auxotrophic for arginine and lysine in a minimal medium with heavy isotope-labeled versions of these amino acids, heavy isotope-labeled PrESTs wer produced in a 24-well plate with an average total amount of 0.8 mg that could be used in future reference studies. Only a few different modifications were seen but as many as 11 out of 24 PrESTs had an acetylation that could depend on pore oxygenation in the culture format but need to be verified with more studies with shorter culture time.

Place, publisher, year, edition, pages
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Engineering and Technology
URN: urn:nbn:se:kth:diva-173656OAI: diva2:854287
Available from: 2015-09-18 Created: 2015-09-16 Last updated: 2015-09-18Bibliographically approved

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