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Identification of Trypsin Digested Transferrin using HPLC and MALDI-MS
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH).
2019 (English)Independent thesis Basic level (professional degree), 10 credits / 15 HE creditsStudent thesisAlternative title
Identifiering av trypsin-klyvt transferrin med HPLC och MALDI-MS (Swedish)
Abstract [en]

In this project, separation of trypsin digested transferrin (Tf) has been studied, using a RP HPLC- UV system equipped with a C18 column. 0.1% TFA/MQ-water and 90% MeOH were used as mobile phase A and mobile phase B, respectively. For economic reasons, the protein cytochrome c (cyt-C) was used to optimize the digestion procedure and LC system, before analysis of Tf. Four digestion methods were applied for analyzing cyt-C and Tf. The first method was digestion with no denaturing, reducing or alkylating agent. The other digestion methods used urea or heating as a denaturing agent, and lastly dithiothreitol (DTT) and iodoacetamide (IAA) as reducing and alkylating agent, respectively. The results from HPLC-UV showed that a gradient elution with a high concentration of organic solvent is favorable for the separation of cyt-C peptides. MALDI-MS was used to identify peptides, and the outcomes showed that denaturation by heat before digestion gave the best results.

Abstract [sv]

I detta projekt har separation av trypsin-klyvt transferrin (Tf) studerats, med användning av ett RP HPLC-UV system, som bestod av en C18 kolonn. 0,1% TFA/MQ-vatten och 90% MeOH användes som mobilfas A respektive mobilfas B. Av ekonomiska skäl användes proteinet cytokrom c (cyt-C) före analys av Tf för att optimera klyvningsprocessen och LC systemet. Fyra klyvningsmetoder studerades för analysering av cyt-C och Tf. Den första metoden innehöll inget denaturerande, reducerande eller alkylerande medel. De andra klyvningsmetoderna innehöll urea eller värme som denaturerande medel, och slutligen ditiotreitol (DTT) och jodacetamid (IAA) som reducerande respektive alkylerande medel. Resultaten från HPLC-UV visade att en gradienteluering med en hög koncentration av den organiska lösningen är gynnsam för separationen av peptiderna från cyt-C. MALDI-MS användes för att identifiera peptiderna, och resultaten visade att denaturering med värme före klyvning gav bäst resultat.

Place, publisher, year, edition, pages
2019.
Keywords [en]
Transferrin, Glycopeptides, Digestion, HPLC, MALDI-MS
National Category
Chemical Engineering
Identifiers
URN: urn:nbn:se:kth:diva-266157OAI: oai:DiVA.org:kth-266157DiVA, id: diva2:1382130
Educational program
Bachelor of Science in Engineering - Chemical Engineering
Available from: 2020-01-02 Created: 2020-01-02 Last updated: 2020-01-02Bibliographically approved

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