Analiza proteinskih veziva u umetničkim delima metodama masene spektrometrije

Abstract

Cilj ove doktorske disertacije bio je razvoj metodologije za identifikaciju proteina uuzorcima kulturnog nasleđa koja uključuje primenu masene spektrometrije i bioinformatike.Predloženi postupak je veoma jednostavan i ne zahteva celovitost molekula proteina radipouzdane identifikacije. Nakon hidrolize tripsinom, peptidi su analizirani primenom MALDITOFmasene spektrometrije uz podršku MALDI-TOF/TOF, kao i ESI tandem masenespektrometrije na LTQ-Orbitrap XL instrumentu. Proteini su identifikovani na osnovugenerisanih spektara uz pomoć bioinformatičkih alata.U prvom delu disertacije primenjen je PMF pristup baziran na MALDI-TOF masenojspektrometriji, ali snimljeni su i tandem maseni spektri najintenzivnijih pikova peptidaprimenom MALDI-TOF/TOF analizatora, na osnovu kojih su identifikovane sekvenceaminokiselina u peptidima. Tandem masena spektrometrija pruža uvid ne samo u odnos mase inaelektrisanja peptida, već i u njegovu fragmentaciju koja je visoko specifična. Na taj načinmoguća je nedvosmislena identifikacija proteina u kompleksnim smešama, čak i izneočekivanih izvora – pod uslovom da su ti proteini sekvencirani i da se nalaze u bazi podataka.Analiziran je set referentnih proteinskih materijala koji su tradicionalno korišćeni za slikanjetehnikom tempera. Pre primene postupka na analizu uzoraka starih slika, analizirani su i modeluzorci kako bi se procenio uticaj pigmenata na uspešnost analize. U analiziranm uzorcimapravoslavnih ikona identifikovani su peptidi kolagena, najverovatnije iz slikarske podloge, kojase tradicionalno pravi od krede ili gipsa i tutkala.Primena tandem masene spektrometrije u analizi proteina, ipak, najčešće podrazumevaprimenu tečne hromatografije kuplovane sa jednim ili više masenih analizatora. U drugom deluove doktorske disertacije uzorci su analizirani pomoću LTQ-Orbitrap XL masenogspektrometra uz prethodno reversno-fazno hromatografsko razdvajanje. Metoda jeoptimizovana na referentnim i model uzorcima, nakon čega su analizirane mikro količineuzoraka sa starih slika. Superiorne performanse LC/MS pristupa i LTQ-Orbitrap masenogspektrometra, pored peptida kolagena, omogućili su identifikaciju peptida iz žumanceta u dvauzorka bojenog sloja ikona iz XIX veka. Na osnovu toga zaključeno je da je tehnika slikanjajajčana tempera...

The aim of this doctoral dissertation was development of a methodology foridentification of proteins in cultural heritage samples, that includes application of massspectrometry and bioinformatics. The suggested protocol is very simple and the integrity of theprotein molecule is not required for the reliable identification. After tripsin hydrolysis, thepeptides are analyzed by MALDI-TOF mass spectrometry, supported by MALDI-TOF/TOF,as well as ESI tandem mass spectrometry on LTQ-Orbitrap XL instrument. Proteins areidentified with the help of bioinformatic tools, on the basis of the spectra generated.In the first part of this dissertation the PMF approach is applied, based on MALDITOFmass spectrometry, but also, tandem mass spectra of the most intense peptide peaks arecollected on MALDI-TOF/TOF mass analyzer, that enabled identification of peptidesequences. Tandem mass spectrometry provides insight not only in the m/z ratio of peptides,but also in the specific pattern of peptide fragmentation. That way is enabled undoubted proteinidentification in complex mixtures, even from unexpected sources, if given proteins aresequenced and present in a sequence database. A set of reference proteinaceous materials,traditionally used in tempera technique of painting, are analyzed here. Before applying theprocedure for analysis of historical paintings, a number of mock samples are analyzed in orderto estimate the influence of pigments on the analysis outcome. Peptides of different collagenchains are identified in analyzed samples of orthodox icons. The collagen is usually present inthe ground layer of paintings, that is traditionally made of chalk or gypsum and animal glue.Application of tandem mass spectrometry in protein analysis usually involvescombination of liquid chromatography and one or more mass analyzers. In the second part ofthis dissertation samples are analyzed on LTQ-Orbitrap XL mass spectrometer afterchromatographic separation on a reverse phase column. The method is optimized on referenceand mock samples before the analysis of micro samples from historical paintings. Superiorperformances of LC/MS approach and LTQ-Orbitrap mass spectrometer, along with collagenpeptides, enabled identification of peptides from egg yolk in two samples from 19th centuryorthodox icons. Based on that finding it was concluded that the technique of painting wastempera...