Scanning of reverse translation via de novo peptide sequencig by tandem mass spectrometry

Sekvenciranje peptida i proteina de novo korišteno je kao alat za reverzno iščitavanje translacije. Pri tome je nedvojbeno sekvenciranje de novo omoguceno selektivnim obilježavanjem N-kraja peptida 5-formil-1,3-benzen-di-sulfonicnom kiselinom prije analize tandemnom spektrometrijom masa. Opisana kemijska modifikacija usmjerava fragmentaciju peptidnih iona ka disocijaciji peptidne veze prilikom kolizijom inducirane disocijacije u spektrometru masa. Nastali fragmentni ioni b-serije detektiraju se u negativnom načinu rada spektrometra masa, a fragmentni ioni y-serije detektiraju se u pozitivnom načinu spektrometra masa. Stoga je, za isti peptid, aminokiselinski slijed iščitan iz spektra snimljenog u pozitivnom načinu rada spektrometra masa moguće dodatno provjeriti iščitavanjem iz spektra snimljenog pri negativnom načinu rada spektrometra masa (tzv. nedvojbeno sekvenciranje peptida de novo). Jedna od primjena ovakvog načina sekvenciranja de novo je i identifikacija proteina, koja se provodi pretragom, iz spektra iščitane sekvence peptida, u bazi podataka, odnosno iščitavanjem reverzne translacije. Disertacija prikazuje primjenjivost, pouzdanost i točnost kao i komparativnu prednost novorazvijene de novo tehnike nad klasičnim tehnikama identifikacije proteina.De novo peptide and protein sequencing was used as a tool for reverse translation scanning. Unambiguous de novo sequencing is achived by selective derivatization of peptide N-terminus by 5-formyl-1,3-benzendisulfonic acid before tandem mass spectrometry analysis. Described chemical modification of peptide directs fragmentation pathway to the peptide bound dissociation during collision-induced dissociation in the mass spectrometer. The resulting b-series of fragmentation ions is detected in the negative ion mode of the mass spectrometer, and y-series of fragmentation ions is detected in the positive ione mode of the mass spectrometer. Therefore, for the same peptide, amino acid sequence read out from the spectrum recorded in the positive ione mode can be additionally verified by reading out from the spectrum obtained in the negative ion mode of mass spectrometer (the so-called unambiguous de novo sequencing). One application of the described de novo sequencing technique is protein identification, which is performed by searching for previously spectral obtained peptide sequence, in the protein database ie. by scanning of reverse translation. This thesis presents applicability, reliability and accuracy as well as the comparative advantage of the newly developed de novo sequencing technique over the conventional protein identification techniques