L13 : Mass Spec and Biosignatures Search Flashcards
Why use 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 basic components 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
Compare the two methods of sample induction?
Direct infusion:
- Simple samples (small mol, single protein)
- Top down analysis (whole protein)
- Faster but less identified
Chromatography:
- Complex mixture )whole cell)
- Bottom up analysis (digested)
- Slower but more identified
What are hard and soft ionisation techniques?
Hard:
- Electron ionisation
- Chemical ionisation
Soft:
- Electrospray ionisation
- MALDI
Explain how electrospray ionisation works?
Liquid phase ionisation technique
1. Proteins passed through metal needle at high potential (typically 3-5 kV)
2. Build up charge at needle tip overcomes surface tension
3. Produces fine mist of droplets emerging with net charge
4. Counter current drying gases aid desolvation and droplet evaporation
5. Production of solvent free molecular ions
Explain how matrix assisted laser/desorption ionisation (MALDI) works?
Solid phase sample mixed with matrix
1. Sample (analyte of interest) diluted with large excess of matrix solution
2. Deposited on metal sample plates to dry
3. Ionisation takes place from solid state
4. 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 and what determines their performance?
Separate ions with different mass to charge ratios (m/z)
- 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
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 in metabolomics, proteomics etc.
Structural
What are extreme conditions?
Alkaline, acidic, unsually hot/cold, without water/oxygen
Dangerous for humans so analysis of environments is difficult
What are the advantages of mass spec analysis in extreme conditions?
Mass specs are portable and don’t need to be manned
Can face extreme conditions without risk to life
How can mass spectrometry be used in space?
Every space mission has MS on board
- Analysing rocks and soil on surface of comet/planet
- Analysis of gases in atmosphere
- Could be used to identify life
How can mass spectrometry be used for volcanoes?
- Measure geothermal gas data
- Use data to understand volcanic activity (increase in levels of H2S, CO2, SO2 in volcanic plumes)
How can mass spectrometry be used on sea floor?
Use MS in robotic submarine
- Study metabolic rates of deep sea microbes
- Analyse seabed, water, thermal vents
How can mass spectrometry be used at North Pole?
- Analysis of trace metals in snow surface provides insight into air pollution (use ICP-MS as elemental analysis technique)
Proteomics of extremophiles?
Want to identify proteins involved in growth and survival
- Proteomics can provide qualitative and quantitative
Due to extremophilic nature, proteins may be thermostable and resistant to chemical denaturants/ digestive enzymes
- Careful experimental planning required
