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mass spec
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- 1what does mass spec doMass spec allows mass of molecule to be measured, can provide info about structure of molecules
Can also be used for quality control, drug testing security and forensics - 2what is the classical and hard mass spec what us it good and bad forelectron ionisation - causes fragmentation, can be good for small molecules bad for macromolecules
- 3how does electron ionisation worksample injected -> vaporised and bombarded with electrons -> generates a positively charged species with a +1 charge
can be coupled with GC as both in a vaccum - 4why must particle analysis be carried out in a vaccumto prevent collision with air particles
- 5what is the mean free path??The mean free path, λ, is the average distance traveled by an ion before it collides with an air molecule, and is given by: λ = 1/N σ
where N is the gas number density, and σ is the collision cross section between the ion and the molecule (typically ~50 Å2 for a small peptide ion). - 6what is the m/z, z, m triangle orderm at top
m/z & z on the bottom - 7how do you calculate ZZ= one peak value – the value of peak to its left
then do 1/value obtained from above - 8how to work out M from m/z and zwork out Z as before then times the z value by the m/z value
an then minus the value of z from (m/z X z) - 9name two soft ionisation techniques• Electro-Spray Ionisation (ESI)
• Matrix-Assisted Laser Desorption Ionisation (MALDI) - 10name 4 advantages to soft ionisation techniques• Ionisation of molecules from solution
- continuous flow
• Ease of coupling with separation techniques
- High-Performance Liquid Chromatography (LC-MS)
- Production of multiply charged ions
• Observed charge envelope characteristic of the number of ionisable sites
- basic sites protonated (positive charge +1 u), acidic sites deprotonated (negative charge -1 u)
• Typically basic sites protonated N-terminal amino group, lysine, arginine and histidine sidechains - 11What does MALDI stand formatrix assisted laser desorption ionisation
- 12how does MALDI work1) analyte is co-deposited with matrix
2)laser excites matrix which trasnfers energy to analyte
3) produces predominately single charged species [M+H](superscript plus )
4) typically used for large biomolecules /polymers
5) MALDI is a high mass/pulsed source so usually combined with TOF
6)Less sensitive to contaminants such as salts and detergents
7)Sensitivity at attomole (10-18) level
8)High throughput analysis (up to ~3000 samples/day)
9) Samples can be re-analysed - 13name the advantages to MALDI-High mol. Weight species can be ionised
-Molecules need not to be volatile
-Good sensitivity ( - 14name MALDI disavantages-MALDI matrix cluster ions obscure low mass range
-Analyte must have low vapour pressure
-Pulsed nature of ion source limits compatibility with many mass analysers
-Analytes that absorb laser light are problematic
- Coupling to LC is difficult - 15name 5 types of mass spectime of flight (TOF, often coupled with MALDI)
Quadrupole (Q)
Ion Trap (IT)
Orbitrap
Ion cyclotron resonance - 16what are some mass analyser considerations• Mass range
• Resolving power
• Mass accuracy and ease of calibration
• Speed
• Scanning or not scanning?
• Ease of interfacing with ion sources
Ease of use for tandem mass spectrometry - 17name time of flights facts-fast, sensitive, simple, cheap
• Electrostatic “mirrors” (Reflectrons) used to lengthen flight time and reduce spread of ion populations → higher resolution
-• Requires pulsed ion packages (MALDI, pulser in ESI-TOF) - 18Quadrupole advantages and dsiadvantagesadvantages: small, light, portable; robust; no high voltage; high dynamic range; cheap
• Disadvantages: low resolution (~2000); mass range m/z <3,000 (can still measure the mass of proteins up to ~50 kDa, by observing highly charged species - 19Ion trap facts-simialr to quadrupole but in 3D
-• Resolution 2,000-10,000 depending on scanning speed
• Mass range <2,000, can only scan down to 1/3 m/z of precursor in tandem MS “1/3 Rule” →loss of low molecular masses. - 20Orbitrap• High mass accuracy (<1 ppm), high resolution (<500k)
• Sensitive and high dynamic range.
• Acts as mass analyzer and detector - 21what are the 3 common detectorsSecondary Electron multipliers (SEM), Channel electron multipliers (CEM) and Microchannel Plates (MCP)
- 22what does tandem mass spec do• Allows analysis of large proteins “soft ionisation” but enables the determination of structural information
- 23in triple quadrupole mass spectrometer what do each of the 'Q's do?• Q1 acts as a mass filter and selects precursor ions “unfragmented”
• Q2 accelerates ions into inert gas molecules (e.g. N2, Ar)
Q3 sorts ion fragments and these are detected - 24name 3 fragmentation techniques• Collision-Induced Dissociation (CID)
• Electron-Transfer Dissociation (ETD)
Higher-energy Collision (C-trap)
Dissociation (HCD) - 25what does bottoms up proteomics doEnables the identification of all proteins in highly complex samples
- 26what are the bottoms up steps1) protein sample is prepared
2) protein sample is digested into peptides using specific proteases
3) peptides resolved by HPLC --> analyses by MS/MS - 27what is a disadvantage to bottoms upcannot make the distinction between multiple proteoforms. E.g. are there long and short isoforms present?, are there mutually exclusive posttranslational modifications?
- 28whats a pro and con to top down proteonomicsindividual species can be more exhaustively characterised in their entirety
con=restricted to simple protein mixtures - 29how does tops down work1. Simple protein mixture or individual protein is prepared
2. Intact proteins are introduced into mass spectrometer
3. Proteins are fragmented (large proteins generate many ions when ESI is employed, MALDI-TOF/TOF is preferred)
3. Peptides analysed by MS/MS
4. MS2 spectra allow protein sequencing - 30name 3 relative quantification techniqueslabel - free
metabolic isotopic labelling (SILAC)
chemical labelling - 31what is absolute qunatificationSynthetic, isotopically labelled peptides are spiked into samples prior to protease digestion
Absolute quantification (AQUA) strategy - 32what does SILAC stand forStable Isotope Labeling by Amino acids in cell Culture
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- Karteikarten am mass spec , erstellt von Zena Dodds am 13/12/2017.
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