Analysis of proteinaceous binders in art objects by mass spectrometry methods

[T. Tripković]

152, [88] listova

Hemija-Analitička hemija / Chemistry-Analytical chemistry

Cilj ove doktorske disertacije bio je razvoj metodologije za identifikaciju proteina u uzorcima kulturnog nasleđa koja uključuje primenu masene spektrometrije i bioinformatike. Predloženi postupak je veoma jednostavan i ne zahteva celovitost molekula proteina radi pouzdane identifikacije. Nakon hidrolize tripsinom, peptidi su analizirani primenom MALDITOF masene spektrometrije uz podršku MALDI-TOF/TOF, kao i ESI tandem masene spektrometrije na LTQ-Orbitrap XL instrumentu. Proteini su identifikovani na osnovu generisanih spektara uz pomoć bioinformatičkih alata. U prvom delu disertacije primenjen je PMF pristup baziran na MALDI-TOF masenoj spektrometriji, ali snimljeni su i tandem maseni spektri najintenzivnijih pikova peptida primenom MALDI-TOF/TOF analizatora, na osnovu kojih su identifikovane sekvence aminokiselina u peptidima. Tandem masena spektrometrija pruža uvid ne samo u odnos mase i naelektrisanja peptida, već i u njegovu fragmentaciju koja je visoko specifična. Na taj način moguća je nedvosmislena identifikacija proteina u kompleksnim smešama, čak i iz neočekivanih 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 slikanje tehnikom tempera. Pre primene postupka na analizu uzoraka starih slika, analizirani su i model uzorci kako bi se procenio uticaj pigmenata na uspešnost analize. U analiziranm uzorcima pravoslavnih ikona identifikovani su peptidi kolagena, najverovatnije iz slikarske podloge, koja se tradicionalno pravi od krede ili gipsa i tutkala. Primena tandem masene spektrometrije u analizi proteina, ipak, najčešće podrazumeva primenu tečne hromatografije kuplovane sa jednim ili više masenih analizatora. U drugom delu ove doktorske disertacije uzorci su analizirani pomoću LTQ-Orbitrap XL masenog spektrometra uz prethodno reversno-fazno hromatografsko razdvajanje. Metoda je optimizovana na referentnim i model uzorcima, nakon čega su analizirane mikro količine uzoraka sa starih slika. Superiorne performanse LC/MS pristupa i LTQ-Orbitrap masenog spektrometra, pored peptida kolagena, omogućili su identifikaciju peptida iz žumanceta u dva uzorka bojenog sloja ikona iz XIX veka. Na osnovu toga zaključeno je da je tehnika slikanja jajčana tempera...

The aim of this doctoral dissertation was development of a methodology for identification of proteins in cultural heritage samples, that includes application of mass spectrometry and bioinformatics. The suggested protocol is very simple and the integrity of the protein molecule is not required for the reliable identification. After tripsin hydrolysis, the peptides 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 are identified 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 MALDITOF mass spectrometry, but also, tandem mass spectra of the most intense peptide peaks are collected on MALDI-TOF/TOF mass analyzer, that enabled identification of peptide sequences. 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 protein identification in complex mixtures, even from unexpected sources, if given proteins are sequenced and present in a sequence database. A set of reference proteinaceous materials, traditionally used in tempera technique of painting, are analyzed here. Before applying the procedure for analysis of historical paintings, a number of mock samples are analyzed in order to estimate the influence of pigments on the analysis outcome. Peptides of different collagen chains are identified in analyzed samples of orthodox icons. The collagen is usually present in the ground layer of paintings, that is traditionally made of chalk or gypsum and animal glue. Application of tandem mass spectrometry in protein analysis usually involves combination of liquid chromatography and one or more mass analyzers. In the second part of this dissertation samples are analyzed on LTQ-Orbitrap XL mass spectrometer after chromatographic separation on a reverse phase column. The method is optimized on reference and mock samples before the analysis of micro samples from historical paintings. Superior performances of LC/MS approach and LTQ-Orbitrap mass spectrometer, along with collagen peptides, enabled identification of peptides from egg yolk in two samples from 19th century orthodox icons. Based on that finding it was concluded that the technique of painting was tempera...

proteinska veziva, tempera, analiza proteina, MALDI-TOF, ESI- LTQOrbitrap XL, mapiranje masa peptida, tandem masena spektrometrija, bioinformatika

proteinaceous binders, tempera, pritein analysis, MALDI-TOF, ESILTQ- Orbitrap XL, peptide mass fingerprint, tandem mass spectrometry, bioinformatics

543.42:543.384(043.3)

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