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Methods in post-translational modification studies: protein purification and chemical digestion optimization

Arribas Díez, Ignacio LU (2017) KEMT30 20171
Department of Chemistry
Abstract
The study of post-translational modification has driven scientist to have a better understanding of protein function, activity and behavior in the metabolism that could later be useful for developing new drugs and treatments targeting key proteins in diseases, like cancer or neuronal disorders. In this project we tried to optimize the Acyl-Biotin Exhange of proteins coming from Chinese Hamster Ovary cells to purify the ones with fatty acids bound to their cysteines, also known as palmitoylated proteins. We managed to obtain a small amount of them after changing the composition of the buffers and reducing the complexity of the mixture by subcellular fractionation. We also tested alternative chemicals for protein digestion that cleave at PTM... (More)
The study of post-translational modification has driven scientist to have a better understanding of protein function, activity and behavior in the metabolism that could later be useful for developing new drugs and treatments targeting key proteins in diseases, like cancer or neuronal disorders. In this project we tried to optimize the Acyl-Biotin Exhange of proteins coming from Chinese Hamster Ovary cells to purify the ones with fatty acids bound to their cysteines, also known as palmitoylated proteins. We managed to obtain a small amount of them after changing the composition of the buffers and reducing the complexity of the mixture by subcellular fractionation. We also tested alternative chemicals for protein digestion that cleave at PTM sites like phosphorylation and glycosylation: S-Ethyl trifluorothioacetate and pentafluoropropionic acid. These chemicals were tested on different conditions and compared with CNBr+Tryp digestion, where we were able to find optimal conditions for protein cleavage with pentafluropropionic acid and designed an especial glass system for gas-phase digestion with S-Ethyl trifluorothioacetate. (Less)
Popular Abstract
Proteins have an essential role in biology. They perform numerous functions in the organism: from sending the signals from the brain, to breaking down the nutrients we eat and many more. They are made out of chains composed of building blocks called amino acids and their constructors are the ribosomes. But not all protein are completely functional once they’ve been produced, some of them require certain changes. These rearrangements are called post-translational modifications (PTMs) and they usually involve linking other molecules, like sugars or fats, to the proteins.
In this project we focused on the study of methods for studying PTMs than could help us understand them and helps us in the fight against diseases like cancer or neuronal... (More)
Proteins have an essential role in biology. They perform numerous functions in the organism: from sending the signals from the brain, to breaking down the nutrients we eat and many more. They are made out of chains composed of building blocks called amino acids and their constructors are the ribosomes. But not all protein are completely functional once they’ve been produced, some of them require certain changes. These rearrangements are called post-translational modifications (PTMs) and they usually involve linking other molecules, like sugars or fats, to the proteins.
In this project we focused on the study of methods for studying PTMs than could help us understand them and helps us in the fight against diseases like cancer or neuronal disorders.
We worked on a purification protocol named Acyl-Biotin Exchange (ABE) for obtaining proteins that have fats attached to them, named palmitoylated proteins (pps). We also tested two different molecules that are able to cut proteins at specific amino acids that are related to other modifications: pentafluoropropionic acid (PFPA) and S-ethyl trifluorothioacetate (SeT). In the future these chemicals could help localize were the PTMs are in the protein. We can see this by mass spectrometry analysis, which measures the mass of the fragments produced by the molecules and can give us the exact arrangement of the sequence, identifying the protein it belongs to.
We were able to obtain some palmitoylated proteins after we tested different components of the cells by separate instead of all together: a less complex sample helps the ABE to be more efficient at capturing pps. We also managed to find the best conditions for PFPA to cut proteins and discovered that in combination with another protein cutting agent, trypsin, we are able to identify many more proteins from complex mixtures. As for SeT, we designed a glass system that creates the conditions necessary for it to work, as it is a liquid that needs to transform into gas to be able to cleave proteins. The glass system seems to recreate the conditions for SeT to be transformed into a gas, but we could not obtain proper cleavage of the proteins at the end. (Less)
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author
Arribas Díez, Ignacio LU
supervisor
organization
course
KEMT30 20171
year
type
H2 - Master's Degree (Two Years)
subject
keywords
trifluorothioacetate, pentafluoropropionic, digestion, chemical, palmitoylation, acyl, biotin, exchange, protein science, proteinvetenskap
language
English
id
8915936
date added to LUP
2017-07-13 11:27:28
date last changed
2017-07-13 11:27:28
@misc{8915936,
  abstract     = {The study of post-translational modification has driven scientist to have a better understanding of protein function, activity and behavior in the metabolism that could later be useful for developing new drugs and treatments targeting key proteins in diseases, like cancer or neuronal disorders. In this project we tried to optimize the Acyl-Biotin Exhange of proteins coming from Chinese Hamster Ovary cells to purify the ones with fatty acids bound to their cysteines, also known as palmitoylated proteins. We managed to obtain a small amount of them after changing the composition of the buffers and reducing the complexity of the mixture by subcellular fractionation. We also tested alternative chemicals for protein digestion that cleave at PTM sites like phosphorylation and glycosylation: S-Ethyl trifluorothioacetate and pentafluoropropionic acid. These chemicals were tested on different conditions and compared with CNBr+Tryp digestion, where we were able to find optimal conditions for protein cleavage with pentafluropropionic acid and designed an especial glass system for gas-phase digestion with S-Ethyl trifluorothioacetate.},
  author       = {Arribas Díez, Ignacio},
  keyword      = {trifluorothioacetate,pentafluoropropionic,digestion,chemical,palmitoylation,acyl,biotin,exchange,protein science,proteinvetenskap},
  language     = {eng},
  note         = {Student Paper},
  title        = {Methods in post-translational modification studies: protein purification and chemical digestion optimization},
  year         = {2017},
}