proteomics lecture 3 & 4 Flashcards
what is proteomics?
identification and characterization of proteins and all their properties
what can you answer using proteomics?
- which proteins are present at a certain location and time
- with which other proteins do they interact?
- Are they post-translationally modified?
- what is their 3 Dstructure
- function?
challenges of working with proteins?
- present in large range of concentrations
- undergo post-translational modifications
- When is it active
- proteases are everywhere
different types of chromatography and protein purification?
- size exclusion
- ian exchange
- hydrophobic interaction
- Affinity
- Reversed phase
what is MS?
analytic method to measure the molecular weight of molecules
how does peptide mass fingerprinting work?
trypsini ze protein → ms → compare to proteins in database
Where does trypsin c leave?
behind R or k
General outline MS?
seperation of ions based on m/z ratio followed b ydetection
ion source → mass analyzer → detector
mass analyser uses electromagnetic field to accelerate and deflect ions
undisired molecules removed b yvacuum
ionization techniques?
- makes sure ions move te detection unit and can be seperated
- Remoud er addition of elections
- addition of charged molecules
- soft or hard ionization
ionization in Lc-MS?
electrospray ionization
production of charged droplets - production gas phase ions - addition more charges
principle Of MALDI?
- Dissolve compound and add matrix, then add Sample
- Evacuate air from sample chamber
- laser shoots pulses of light at sample
types of Mass analysers in LC-MS
- Trapping analysers
- Quadropole
- Time of flight
how does TOF analyse?
- longer travel → big ions and less charge
- Time required to hit detector depends specific for each m/z combi
how works the ion trap Mass analyser?
accumulation ions → ejection scanning ions oot of the trap b yincreasing the Rf amplitudes and measere Respens → detection
how does the quadruple mass analyzer work?
- Both DC an Ff applied to rods
- opposite rods connected
- Mas sjust right stable= pass
- big ions are slower → deflected by DC fields
- smaller ions are fast → gain amplitude and are lost by Rf fields
