Mass spectrum types revision Flashcards
What is the principle of TTP mass spectrum is based off?
based off the fact that ligand bound proteins have altered thermostability so can produce a melting curve when subjected to a range of temperatures and comparison with a non ligand bound curve can show which temperature of solube proteins have altered stability
What is CETSA
cellular thermal shift assay developed by Norland et al in 2013 at karlolinksa insitute.
What bioinformatic software can be used to to do the mass spec analysis in TTP mass spec?
IsobarQuant
Why do we use an orbitrap mass spec in TTP?
the differences in proximal isobaric tandem mass tags are very small (6kda) so less precise methods wont be able to resolve these differences.
what happens when the proteins in solution denature which allows for the process of TTP to be achived?
they denature and precipitate so are removed from the protein solution upon centrifugation allowing for tagging of only the protiens left in solution.
when a drug binds to a protein in TTP-MS what can be expected of the melting curve?
the temperature that contains the proteins that bind to the drug will show an altered thermostability. will denature slower than the ones without being bound. (expect hightend stability)
what are isobaric tandem mass tags?
TMT belongs to a family of reagents referred to as isobaric mass tags which are a set of molecules with the same mass, but yield reporter ions of differing mass after fragmentation.
at what residues are the TMT tags addded?
added at lysine residues
what is HDX mass spec?
mass spec technique that measures the exchange rate of hydrogen atoms in a protein with deuterium. exchange rate is based off the accessibility of the different regions of a protein to the deuterated solvent.
What sort of info can HDX obtain>
secondary and tertiary structures, stability and conformational dynamics, such as short lived intermediate states.
what type of mass spec is HDX commonly used with?
ESI mass spec as allows for the intact proteins or peptides ot be analysed wihtout significant fragmentation
what are the steps involved in a HDX mass spec
sample of proteins/peptides are introduced to a deuterated solvent and incubated under specific temperatures and pH.
The sample is then quenched to stop HDX echange.
protylitic digenstion occurs to generate a mixture of labelled peptides.
Labelled peptides are ionised by ESI and anlysed in Mass spec to find M/Z
compared to unlabeled sample to measure the degree of deuterium uptake and calculate the exchange rate at difrernt regions of the protein.
What do the exchange rates in HDX imply?
reflect the changes in protein conformation and stability as exhange occurs at the surfaces exposed to the solvent.
What is pulse labelling?
when the protein is exposed to deuterium for short periods, labelled and rapildy quenhced. creates a snapshot of the protein conformation at a specific timepoint so can be used to track changes in conformation over time by repeated experimentation.
