|Title:||Method development for quantifying protein modifications using liquid chromatography hyphenated to tandem mass spectrometry||Other Titles:||Etablierung einer LC-MS/MS Methode zur Quantitativen Bestimmung von Peptidmodifikationen||Language:||English||Authors:||Soher, Elisabeth||Qualification level:||Diploma||Advisor:||Marchetti-Deschmann, Martina||Issue Date:||2016||Number of Pages:||167||Qualification level:||Diploma||Abstract:||
Zearalenone (ZEN) is a mycotoxin, which is produced by various species of the fungus Fusarium, such as F. graminearum, F. equiseti and F.culmorum. Since this fungus is common to grow on crops, food and feed are often contaminated with the mycotoxin. Romer Labs developed a quick test, in form of a lateral flow device (LFD), to determine the amount of contamination on grain. An important constituent for this LFD is zearalenone - carboxymethyl oxime (ZEN-CMO) coupled to conalbumin (CON). Since it is always desirable to produce at the lowest possible cost, it is of interest whether different synthesis strategies can lead to the same results in terms of modification efficiency on the protein, while reducing various synthesis steps or chemicals in use. Therefore the goal was to establish a liquid chromatography - tandem mass spectrometry method (LC-MS/MS) to quantify ZEN-CMO modifications introduced onto the target protein. For detailed protein characterization, the proteins were digested and analyzed on a nano-LC-electrospray ionization (ESI)-ion trap mass spectrometer performing collision induced dissociation (CID) fragmentation for peptide identification and modification localization. In order to maximize protein sequence coverage, two proteases were tested. Immobilized trypsin was compared to a trypsin/LysC mix. Further method development for quantification was performed on an ultra high performance liquid chromatography (UPLC) - ESI - triple quadrupole instrument. We found trypsin/LysC to perform best in terms of sequence coverage after in solution digestion and peptide desalting. It was possible to identify 27 out of 59 possible Lysine modification sites and it was found that most of the identified protein modifications were located on the protein-s surface. Furthermore, fragmentation mass spectra gave insight into fragmentation mechanisms of ZEN-CMO and ZEN-CMO modified peptides using an ion trap mass analyzer, exhibiting reporter fragment ions which were further used to develop a quantification method applying triple quadrupole MS technology.
Mass Spectrometry; Protein Conjugates
|Library ID:||AC13093715||Organisation:||E164 - Institut für Chemische Technologien und Analytik||Publication Type:||Thesis
|Appears in Collections:||Thesis|
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checked on May 16, 2021
checked on May 16, 2021
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