Kemijski aktivirana fragmentacija proteina i peptida obilježenih 4-formilbenzensulfonskom kiselinom

doctoral thesis

• Title (english): Chemically activated fragmentation of proteins and peptides labeled with 4-formylbenzensulfonic acid
• Author: Luka Ozdanovac
• Mentor: Mario Cindrić (mentor)
• Committee member: Vesna Petrović Peroković (predsjednik povjerenstva)
• Committee member: Iva Juranović Cindrić (član povjerenstva)
• Committee member: Ivana Varenina (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Chemistry); Zagreb
• Defense date and country: 2024-07-17, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Chemistry
• Universal decimal classification (UDC): 54 - Chemistry. Crystallography. Mineralogy

Abstract

Provedeno je istraživanje induciranja usmjerenog fragmentiranja peptida i proteina tandemnom spektrometrijom masa uz pomoć 4-formilbenzensulfonske kiseline (FBSA) i 4'-formilbenzo-18-kruna-6-etera (4fb18C6). Preduvjet postizanja usmjerene fragmentacije je kovalentno obilježavanje N-kraja peptida. Analiza nastalih produkata i fragmentnih iona provedena je vezanim sustavom nanotekućinske kromatografije i tandemne spektrometrije masa uz ionizaciju elektroraspršenjem. Reakcije derivatizacije, tj. reduktivne aminacije, provedene su u vodenom mediju pri pH 6 uz pomoć mikrovalnog zračenja. Selektivnim obilježavanjem N-kraja peptida s FBSA omogućena je kemijski aktivirana fragmentacija u pozitivnom i negativnom načinu rada spektrometra masa uz primjenu uvjeta za redukciju naboja. Stopa neželjenih reakcija je gotovo zanemariva, a dobiveni skup podataka osigurao je pouzdano sekvenciranje peptida de novo. S druge strane, obilježavanjem peptida 4fb18C6 reagensom i preklapanjem fragmentnih iona dobivenih tandemnom spektrometrijom masa istovjetnih intaktnih i derivatiziranih peptida omogućena je redukcija kemijskog šuma, te identifikacija proteina pomoću algoritma BLASTp pretragom NCBInr i UniProtKB/Swiss-Prot baza podataka. Razvijena metodologija derivatizacije uz sekvenciranje peptida de novo pouzdana je metoda za točno određivanje primarnog slijeda aminokiselina u peptidima i predstavlja novi alat u identifikaciji vrsta pretragom slovnih baza podataka.

Abstract (english)

A research was conducted to on the induction of directed fragmentation of peptides and proteins by tandem mass spectrometry using 4-formylbenzenesulfonic acid (FBSA) and 4'-formylbenzo-18-crown-6-ether (4fb18C6). A prerequisite for achieving directed fragmentation is the covalent labeling of the N-terminus of the peptide. The analysis of the resulting products and fragment ions was carried out using a coupled system of nanoliquid chromatography and tandem mass spectrometry with electrospray ionization. The derivatization reactions, i.e. reductive amination, were carried out in an aqueous medium at pH 6 under microwave irradiation. Selective labeling of the N-terminus of peptides with FBSA enabled chemically activated fragmentation in both positive and negative modes of the mass spectrometer under charge reduction conditions. The rate of side reactions is almost negligible and the resulting data set ensured reliable de novo peptide sequencing. On the other hand, the labeling of peptides with the reagent 4fb18C6 and the overlap of fragment ions obtained from identical intact and derivatized peptides in tandem mass spectrometry allowed the reduction of chemical noise and the identification of proteins using the BLASTp algorithm by searching the NCBInr and UniProtKB/Swiss-Prot databases. The developed derivatization method with de novo peptide sequencing is a reliable method for the accurate determination of the primary amino acid sequence in peptides and represents a new tool for the identification of species by searching sequence databases.

• Keywords: 4-formilbenzensulfonska kiselina; derivatizacija; krunasti eter; sekvenciranje de novo; spektrometrija masa
• Keywords (english): 4-formylbenzenesulfonic acid; crown ether; de novo sequencing; derivatization; mass spectrometry
• Language: croatian
• URN:NBN: urn:nbn:hr:217:964410
• Promotion: 2024
• Project: Number: KK.01.1.1.07.0023; Title: Qua/Qua Protein: Kvantitativna i kvalitativna analiza proteina za potrebe biomedicine i biotehnološke industrije; Acronym: Qua/Qua Protein; Leader: Mario Cindrić; Jurisdiction: eu; Funder: Europska komisija; Funding stream: European Regional Development Fund
• Study programme: Title: Doctoral study; Study programme type: university; Study level: postgraduate; Academic / professional title: doktor/doktorica u području prirodnih znanosti (doktor/doktorica u području prirodnih znanosti)
• Type of resource: Text
• Extent: 153 str. ; 30 cm
• File origin: Born digital
• Access conditions: Embargoed access; Embargo expiration date: 2025-07-17
• Created on: 2024-09-06 10:20:12