L13 : Mass Spec and Biosignatures Search Flashcards
(31 cards)
What are the key advantages to mass spectrometry?
- Very sensitive
- Fast, reliable, accurate
- Can analyse small molecules to large biological molecules
- Gives info on atoms present within mol
What are the three core components of a mass spectrometer?
Ionisation source
Mass analyser
Detector
What are the basic components and options of a mass spectrometer?
- Sample induction
- Direct infusion or chromatography - Ionisation source
- Hard or soft ionisation - Mass analyser
- Quadrupole, TOF, ion trap etc. - Detector
- Measure m/z ratio - Data analysis
- Database search or manual interpretation
What are the two methods of sample induction and type of analysis?
Direct infusion
- Top down (whole)
Chromatography
- Bottom up (digested)
Why digest a protein in bottom up proteomics?
Protein mixtures or complexes
Can be difficult to analyse so digestion simplifies sample into smaller peptides
Peptide fingerprints can be matched to databases
Information lost on higher order structure and some PTMs
Compare the two methods of sample induction?
Direct infusion:
- Simple samples (small mol, single protein)
- Top down analysis
- Faster but less identified
Chromatography:
- Complex mixture (whole cell lysates)
- Bottom up analysis
- Slower but more identified
What are hard and soft ionisation techniques?
Hard:
- Electron ionisation
- Chemical ionisation
Significant fragmentation, informative
Soft:
- Electrospray ionisation
- MALDI
Impart minimal energy, preferred for large/fragile
Briefly summarise ESI and MALDI?
ESI
- Liquid phase ionisation technique
MALDI
- Solid phase sample mixed with matrix
- Laser based ionisation method
What types of molecules is MALDI used for?
MALDI
- Large, fragile biomolecules
- eg. proteins, NAs
Explain how electrospray ionisation works?
- Proteins passed through metal needle at high potential (typically 3-5 kV)
- Build up charge at needle tip overcomes surface tension
- Produces fine mist of droplets emerging with net charge
- Counter current drying gases aid desolvation and droplet evaporation
- Production of solvent free molecular ions
Explain how matrix assisted laser/desorption ionisation (MALDI) works?
- Sample (analyte of interest) diluted with large excess of matrix solution
- Deposited on metal sample plates to dry
- Ionisation takes place from solid state
- Matrix absorbs most energy from laser, preventing damage/fragmentation of large ion
What are common MALDI matrices and their common characteristics?
- Nicotinic acid
- 2,5 - dihydroxybenzoic acid
Often contain phenolic (OH) and/or COOH groups
Efficiently absorb UV light (UV lasers often used)
What do mass analysers do?
Separate ions with different mass to charge ratios (m/z)
What determines performance of mass analyser?
- Mass resolution (distinguish two peaks of similar m/z)
- Mass accuracy (how close experimental to actual mass)
What makes up a quadrupole mass analyser?
Consists of 4 metal rods with opposite polarities applied to adjacent rods
Uses 2 voltage types:
- DC (direct current) - amplitude U
- RF (radio frequency) - amplitude V)
How does quadrupole mass analyser work?
Creates complex electric field that cause ions to oscillate as they travel
- Only ions with specific m/z ratio will have stable paths and successfully reach detector
- Others are deflected or destabilised
How does linear TOF analyser work?
Time taken for ion to reach detector at known distance is measured
Time depends on m/z of particle (larger = longer)
Experimental parameters used to find m/z
What is fourier transform ion cyclotron resonance (FTICR)?
Highly accurate MS technique that determines m/z based on cyclotron frequency of ions in fixed magnetic field
How does FTICR generate a mass spectrum?
- Ions trapped in Penning trap
- Excited at resonant cyclotron frequencies, causing rotation in phase
- As ion packets pass electrodes, induce image current
- Current (superposition of sine waves) is converted to mass spectrum using Fourier transform
How does Orbitrap mass analyser work?
Ions orbit around central spindle-like electrode while oscillating in axial dimension
- Axial oscillation detected as transient signal by surrounding electrodes
- Transient = frequency of ion oscillation, unique for each m/z
- Fourier transform transforms transient into mass spectrum
What needs to be considered during data analysis?
- One peak could indicate several biomolecular isomers (same chemical formula, different structures/properties)
- 2 mols may have similar m/z that cannot be distinguished by mass spectrometer
- Isotopes can show expression at multiple m/z values
Describe features of mass spectrum and what it shows?
Ions appear in as result of electrical current that is generated and amplified when ion strikes detector
X-axis: m/z ratio
Y-axis: relative ion abundance/ ion current
What areas of biology can mass spectrometry be used for?
Identification
- eg. metabolomics, proteomics
Structural
What are extreme conditions?
Alkaline, acidic, unsually hot/cold, without water/oxygen
Dangerous for humans so analysis of environments is difficult
