Analiza proteinskih veziva u umetničkim delima metodama masene spektrometrije
Analysis of proteinaceous binders in art objects by mass spectrometry methods
Докторанд
Tripković, TatjanaМентор
Nikolić-Mandić, SnežanaЧланови комисије
Baošić, RadaKaradžić, Ivanka
Apostolović, Danijela
Метаподаци
Приказ свих података о дисертацијиСажетак
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
m...oguća 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 peptid...e 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...