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KIT QUANTA -standardization kit for absolute protein quantitation: monitoring of methionine oxidation induced by liquid chromatography separation
Baumans, France; Baiwir, Dominique; Colombo, Maria et al.
2019XXIVèmes Rencontre du club jeunes de la Société française de Spectrométrie de masse
 

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Abstract :
[en] In the context of biomarker discovery and their absolute quantification in complex samples, a standardization strategy aiming to control the entire sample preparation process before LC-MS analysis would be extremely valuable. Our approach involves the use of a chimeric protein and different levels of its heavy peptides spiked at opportune moment in the sample during sample processing. Among these peptides, some containing a methionine are inserted. Methionine oxidation is a well-known protein modification occurring in vivo but also in vitro and may result in structural and functional protein alteration. Controlling this artefactual modification is then of great interest and will be possible thanks to our strategy. To address the origin of methionine oxidation, we sought differential oxidation levels comparing direct infusion, LC-MS using new columns and LC-MS using old ones. The direct infusion of pure synthetic peptides in a Q ExactiveTM Hybrid Quadrupole-OrbitrapTM showed a low percentage of oxidized peptide (less than 2%). Once injected onto the LC system coupled online with the Q Exactive, this percentage jumps to higher value (50-60% dependant of the peptide injected). The same peptides have been injected on the same LC system on which both trapping and analytical columns were replaced by new ones. Methionine oxidation in this case, was as low as in the direct infusion, meaning that the previous use of columns has a huge influence on the oxidation of methionine in pure synthetic peptides. This effect is certainly due to the presence of metal ions in the columns that promote methionine oxidation during chromatography separation. The same effect was observed on complex samples ((i.e. commercial HeLa protein digest and plasma digest). Indeed, the percentage of oxidized peptides varies from 1 to 8% when using the new columns and increases to 20% to 80% when the same samples were injected on the old columns. The use history of a column greatly impacts the oxidation degree of peptides and has to be taken into account when studying methionine oxidation. Future works will be dedicated to the implementation of strategies that could stabilize the oxidation during chromatography separation.
Disciplines :
Chemistry
Author, co-author :
Baumans, France ;  Université de Liège - ULiège > Département de chimie (sciences) > Laboratoire de spectrométrie de masse (L.S.M.)
Baiwir, Dominique  ;  Université de Liège - ULiège > GIGA Platforms
Colombo, Maria;  Kaneka Eurogentec S.A.
Allain, Camille;  Université de Mons - UMONS > Laboratoire de Protéomique et Microbiologie
Tavernier, Vincent;  Université de Mons - UMONS > Laboratoire de Protéomique et Microbiologie
Leroy, Baptiste;  Université de Mons - UMONS > Laboratoire de Protéomique et Microbiologie
Wattiez, Ruddy;  Université de Mons - UMONS > Laboratoire de Protéomique et Microbiologie
De Pauw, Edwin  ;  Université de Liège - ULiège > Département de chimie (sciences) > Chimie analytique inorganique
Eppe, Gauthier  ;  Université de Liège - ULiège > Département de chimie (sciences) > Chimie analytique inorganique
Mazzucchelli, Gabriel  ;  Université de Liège - ULiège > Département de chimie (sciences) > Laboratoire de spectrométrie de masse (L.S.M.)
Language :
English
Title :
KIT QUANTA -standardization kit for absolute protein quantitation: monitoring of methionine oxidation induced by liquid chromatography separation
Publication date :
March 2019
Event name :
XXIVèmes Rencontre du club jeunes de la Société française de Spectrométrie de masse
Event place :
Quiberon, France
Event date :
du 18 mars au 22 mars 2019
Available on ORBi :
since 08 July 2020

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