Frakcionacija složenih smjesa peptida na kationskom izmjenjivaču radi poboljšane identifikacije proteina spektrometrijom masa visoke rezolucije

master's thesis

• Title (english): Peptide fractionation by strong cation exchange chromatography for enhanced protein identification by high resolution mass spectrometry
• Author: Dominik Prišćan
• Mentor: Marko Močibob (mentor)
• Committee member: Marko Močibob (predsjednik povjerenstva)
• Committee member: Nives Galić (član povjerenstva)
• Committee member: Đani Škalamera (član povjerenstva)
• Committee member: Morana Dulić (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Chemistry); Zagreb
• Defense date and country: 2024-02-20, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Chemistry

Abstract

Danas je u proteomici za identifikaciju i kvantifikaciju proteina najviše zastupljena metoda temeljena na vezanom sustavu tekućinske kromatografije i tandemne spektrometrije masa, odnosno shotgun‒proteomika. Radi poboljšane identifikacije proteina, postupak može uključivati korak predfrakcionacije peptida. U sklopu ovog rada, napravljena je optimizacija koraka predfrakcionacije peptida, pri čemu je optimiziran gradijent elucije, sastav mobilne faze i broj prikupljenih frakcija. Također, optimizirano je trajanje kromatografskog odjeljivanja pomoću vezanog sustava tekućinske kromatografije i tandemne spektrometrije masa. Optimizirana metoda primijenjena je za predfrakcionaciju realnih uzoraka, pri čemu je metoda korištena za provjeru ekspresije dupliciranih gena za aminoacil‒tRNA‒sintetaze pronađenih u genomu H. aurantiacus i H. geysericola. Nadalje, metoda je korištena za provjeru ekspresije gena za proteine‒nosače i homologe aminoacil‒tRNA‒sintetaza pronađenih u genomu bakterija A. fabrum,
B. diazoefficiens, B. thuringiensis 4AJ1 i 4CC1 te za analizu ekspresije gena koji pokazuju sinteniju s genima proteina‒nosača i homologa aminoacil‒tRNA‒sintetaza.

Abstract (english)

The modern proteomic analyses rely on the coupled system of liquid chromatography and tandem mass spectrometry for comprehensive peptide separation and identification. To further increase the number of identified peptides, improve the identification of proteins and their sequence coverage, the analysis workflow may include a step of pre‒fractionation of peptides. In this thesis, an optimisation of the peptide pre‒fractionation step was conducted, which included the optimisation of the elution gradient, the composition of the mobile phases and the number of fractions collected. In addition, the duration of the chromatographic step in the coupled liquid chromatography‒tandem mass spectrometry system was optimised. The optimised method was applied for the pre‒fractionation of authentic samples, wherein the method was utilized to verify the expression of duplicated genes for aminoacyl‒tRNA synthetases in the genomes of H. auratiacus and H. geysericola. Furthermore, the method was employed to determine the expression of genes for carrier proteins and homologues of aminoacyl‒tRNA synthetases in the genomes of the bacteria A. fabrum, B. diazoefficiens, B. thuringiensis 4AJ1 and 4CC1 as well as to analyse the expression of genes that show synteny with genes for carrier proteins and homologues of aminoacyl‒tRNA‒synthetases.

• Keywords: aminoacil‒tRNA‒sintetaze; kromatografija ionske izmjene; LC-MS/MS; proteomika; UHPLC
• Keywords (english): aminoacyl-tRNA-synthetases; ion exchange chromatography; LC-MS/MS; proteomics; UHPLC
• Language: croatian
• URN:NBN: urn:nbn:hr:217:473613
• Study programme: Title: Graduate university studies in chemistry; specializations in: Graduate university studies in chemistry; Course: Graduate university studies in chemistry; Study programme type: university; Study level: graduate; Academic / professional title: sveučilišni/a magistar/magistra kemije (sveučilišni/a magistar/magistra kemije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2024-03-08 12:26:42