Mass Spectroscopy Flashcards
Name 5 key parts of mass spec (simply)
1) sample introduction
2) source (generation of ions)
3) analyser (seperation of ions)
4) detector (detection of ions)
5) acquistion of system
Name 5 methods of sample introduction?
- gas chromatography
- syringe pump
- liquid chromatography
- capillary electrophoresis
- solids
name seven methods of ionisation?
- electron ionisation
- chemical ionisation
- fast atom/ ion bombardment
- secondary ion mass spectrometry
- field desorption
- plasma desorption
- electrospray ionisation
- matrix assisted laser desorption ionisation
what does MALDI stand for?
Matrix Assisted Laser Desorption Ionisation
Give three examples of matrices
α-cyano-4-hydroxycinnamic acid
Sinapinic acid
2-amino benzoic acid
what is the quadrupole analyser?
- mass filter - seperation accomplished using combination of DC and RF electric fields
- ions oscillate through filter towards detector
- only ions with stable trajectory transmitted
- by scanning DC/RF = m/z
Describe LTQ Orbitrap Operation priniciple
- Ions are stored in the Linear Trap
- Ions are axially ejected
- Then trapped in the C-trap
- They are squeezed into a small cloud and injected into the Orbitrap
- Where they are electrostatically trapped, while rotating around the central electrode and performing axial oscillation
what do ions of only one mass generate (in LTQ Orbitrap operation)?
A sine wave signal
Simplify the principles of the Orbitrap Operation
The oscillating ions induce an image current into the two outer halves of the orbitrap, which can be detected using a differential amplifier
what is the axial oscillation frequency formula?
what are the three analysers in the Thermo Fusion Tribrid Orbitrap Mass Spec?
- Quadrupole
- Ion trap
- Orbitrap
Define isotope
Forms of the same element that have same number of protons but a different number of neutrons
Name two radioactive isotopes
C14 and 3H (Tritium)
define a radioactive isotope
an isotope having an unstable nucleus that decomposes spontaneously
name two stable isotopes
Carbon 13 and Deuterium (2 hydrogen?)
what is isotope pattern dependent on ?
the compostition and the number of atoms
Describe the 6 steps before mass spec
1) Sample lysis and protein extraction
2) Complex protein mixture is formed
3) Digested by proteolytic digestion (trypsin)
4) Complex peptide mixture is formed
5) then undergoes seperation and enrichment
6) MS analysis can then be undergone
what does ETD stand for?
Electron Transfer Dissociation
What does CID stand for?
Collisionally Induced Dissociation
Name two types of fragmentation?
ETD and CID
How does CID fragmentation work?
- via several collisions with Helium the precursor ion is internally heated
what sort of cleavage does CID do?
preferences for weak bond cleavages
backbone cleavage is preferred (selected amino acids E, D,P)
How does cleavage work in ETD ?
rapid bond cleavage (no energy dissipation) by random fragmentation of the peptide backbone
whats the best fragment spectra from in CID?
2+ ions
what is the preferrable charge state for ETD?
z > 2
what can native MS provide information on?
- non-covalent protein complexes; oligomeric status/ subunit compostition
- protein-ligand interactions including antibody-drug conjugates
- subunit stochiometry of protein complexes
- topology of protein complexes
- microheterogeneity on proteins present within a protein complex
- metal binding within a protein/ protein complex
what sample solution is used for native denaturing and regular denaturing?
native: aqueous solution - ammonium acetate (pH 6-9)
regular: partial organic solution - water, formic acid, acrtonitrile/ methanol (pH 1-2)
name three examples of regular ESI sample solutions?
- water
- formic acid
- acetonitrile/ methanol
what salt treatment is used for native denaturing and regular denaturing?
native: Off-line desalt
regular: On-line desalt/ RP
what protein concentration is used for native denaturing and regular denaturing?
native: 1 – 10 µM (complex)
regular: < 1 µM (subunit)
what are the benefits of nanoESI?
- less dissociation of oligomer
- symmetrical charge state distribution indicative of a single conformation
- fewer non-specific aggregates
- can use aqueous buffers and ambient temperatures
- Lower sample amounts (flow rate approx 10nL/min vs 5uL/min in ESI)
- Narrower charge states due to fewer adducts
How does collision induced dissociation of protein assemblies occur?
- dissociation is asymmetric with respect to mass
- unfolded, highly charged monomers are removed sequentially
- low subunit flexibility, fewer salt bridges and smaller interfaces tend to dissociate more easilt
