Razvoj multidimenzionalne kromatografije za razdvajanje peptida pomoću automatizirane mikroekstrakcije na čvrstoj fazi

master's thesis

• Title (english): Development of multidimensional chromatography for separation of peptides using automated solid-phase microextraction
• Author: Nika Bilić
• Mentor: Bojan Žunar (mentor)
• Mentor: Mario Cindrić (komentor)
• Mentor: Renata Biba (pomoć pri izradi, neposredni voditelj)
• Committee member: Renata Teparic (predsjednik povjerenstva)
• Committee member: Mario Cindrić (član povjerenstva)
• Committee member: Igor Slivac (član povjerenstva)
• Granter: University of Zagreb; Faculty of Food Technology and Biotechnology; (Department of Chemistry and Biochemistry); (Laboratory for Biochemistry); Zagreb
• Defense date and country: 2024-07-18, Croatia
• Scientific / art field, discipline and subdiscipline: BIOTECHNICAL SCIENCES; Biotechnology

Abstract

Multidimenzionalna tekućinska kromatografija (LC) spregnuta sa spektrometrijom masa (MS) ima ključnu ulogu u proteomskom pristupu odozdo-nagore zbog povećanja osjetljivosti metode smanjenja kompleksnosti uzorka. Mikroekstrakcija čvrstom fazom (µSPE) je jednostavna i učinkovita metoda pripreme uzoraka za MS. Cilj ovog rada bio je ispitati ortogonalnost nekonsekutivnog sustava za multidimenzionalno frakcioniranje peptida BSA kako bi se utvrdilo koja kombinacija tehnika pokazuje najveću nezavisnost. Optimizirano je i ispitano frakcioniranje kombinacijom 6 metoda odjeljivanja na µSPE uređaju s pozitivnim pritiskom: i) izmjenjivač kombiniranog načina jake anionske izmjene i izmjene obrnutih faza, ii) jaki kationski i iii) jaki anionski izmjenjivač, iv) hidrofilno-lipofilni izmjenjivač i izmjenjivač obrnutih faza pri v) niskom pH i vi) visokom pH. Šest nastalih frakcija je analizirano LC-MS sustavom, a sirovi podaci su obrađeni alatom za pretragu baza podataka. Rezultati su pokazali da za frakcioniranje peptida BSA najveću ortogonalnost daje kombinacija hidrofilno-lipofilnog izmjenjivača ili jakih ionskih izmjenjivača u prvoj dimenziji te jakih ionskih izmjenjivača u drugoj dimenziji.

Abstract (english)

Multidimensional liquid chromatography (LC) coupled with mass spectrometry (MS) is pivotal in the bottom-up proteomic approach for its enhanced sensitivity through the reduction of peptide complexity. Solid-phase microextraction (µSPE) is a simple and efficient sample preparation method for MS. The aim of this study was to optimize and evaluate the orthogonality of offline multidimensional fractionation methods for BSA peptides to determine the combination of fractionation methods which provides the highest degree of independence, using a positive pressure µSPE platform. The fractionation capabilities of 6 separation methods were examined: i) mixed-mode strong anion-exchange and reversed phase, ii) strong cation-exchange, iii) hydrophilic-lipophilic balance, iv) strong anion-exchange, and v) low pH and vi) high pH reverse phase. Six obtained fractions of each fractionation were analyzed on an LC-MS system, with raw data processed using a protein database search engine. The results showed that for BSA peptides, the highest orthogonality was achieved with a combination of hydrophilic-lipophilic balance or strong ion-exchange chromatography (IEC) in the first dimension and strong IEC in the second dimension.

• Keywords: multidimenzionalnost; mikroekstrakcija na čvrstoj fazi; ortogonalnost; nepokretne faze; spektrometrija masa
• Keywords (english): multidimensionality; solid phase microextraction; orthogonality; stationary phase; mass spectrometry
• Language: croatian
• URN:NBN: urn:nbn:hr:159:789331
• Study programme: Title: Molecular Biotechnology; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra inženjer/inženjerka molekularne biotehnologije (magistar/magistra inženjer/inženjerka molekularne biotehnologije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2024-08-27 09:23:07