Mass spectrometry Flashcards
What are the fundemental components of a mass spectrometer?
1.Generate ions from liquid or solid phase into gas phase. can only measure charaged molecules so have to have a way to generate charge. (typically happens at source)
- seperate ions by mass to charge ratio in analyser. typically under high vaccum.
- a detector to detect ions
- an acquisition system to bring all the data together.
What are some methods of sample introduction in a mass spec?
Gas chromatography (volatile ions) - not going to focus on this
Syringe pump - fill syringe with protein and solvent and pump into mass spec
Liquid chromatography - level of operation to reduce complexity of sample before it enters mass spec
Capillary electrophoresis - take capillary and fill it with a buffer e.g. phosphoric acid along with protein of interest, apply an electric field and the protein will migrate within the capillary
Solids - going from Solid phase into gas phase
what are methods of ionisation in mass spectrometry?
Electron ionisation
Chemical ionisation
Fast Atom/Ion Bombardment
Secondary Ion Mass Spectrometry
Field desorption
Plasma desorption
Electrospray Ionisation
Matrix Assisted Laser Desorption Ionisation (MALDI)
What are soft ionisation techniques?
they dont fragment the molecule so can retain the molecular ion. examples of this are:
- electrospray ionisation and matrix assisted laser desorption ionisation. (MALDI)
describe the technique of electrospray ionisation
Go from liquid phase to gas phase ions
Metal capillary containing a liquid
Apply a high potential (around 3000 V) to get an electrophoretic effect and forms a Taylor cone
A spray of charged droplets forms
These undergo desalvation - basically evapiratio. Where droplets get smaller and smaller until gas ions are left to put into the spectrometer
Describe the technique of matrix assisted laser desorption ionization (MALDI)
Take molecule of interest mix with a matrix (low molecular weight absorbing compound)
Spot this onto target plate
And dry it onto surface
Now have sample embedded into a crystalline matrix e.g. α-cyano-4-hydroxycinnamic acid, Sinapinic acid, 2-amino benzoic acid
The matrix absorbs UV light in a certain wavelength
So use laser at this wavelength, matrix flakes/comes off surface and part of the energy is imparted onto the sample and hence that becomes ionises and comes off the surface and becomes charged
Now ions are generated, need a way to separate them
Use Time of flight mass spectrometry
What is MALDI ionization?
A method of ionizing molecules for analysis in mass spectrometry by mixing the molecule of interest with a low molecular weight matrix and then firing a UV laser at the mixture.
What is a matrix in MALDI ionization?
A low molecular weight compound that is mixed with the molecule of interest in MALDI ionization to help ionize the sample.
What is the purpose of Time of flight mass spectromertry in MALDI ionization
To separate and analyze the ions that are generated by MALDI ionization based on their mass-to-charge ratio.
What is the target plate in MALDI ionization?
The surface onto which the mixture of the molecule of interest and the matrix is spotted and dried in MALDI ionization.
What is the role of the UV laser in MALDI ionization?
To provide the specific wavelength of light that the matrix absorbs in order to cause the ionization of the sample.
What kind of molecules can be analyzed using MALDI ionization?
Biomolecules such as proteins, peptides, and nucleic acids.
How does the time of flight analyser work?
In vacuum - measure the time it takes for the ion to travel to the detector
Fire UV laser at sample, generate ions
Start timing
Stop when they reach detector
The lighter the ions the faster they move
Equations are worked through to give value that is m/z
What is the quadrupole analyzer?
A type of mass filter used in mass spectrometry to separate ions based on their mass-to-charge ratio (m/z).
How does the quadrupole analyzer separate ions?
By applying a combination of DC and RF electric fields to four metal rods arranged in a square pattern, causing ions to oscillate towards a detector. Only ions with a stable trajectory will make it through the filter, allowing specific m/z values to be isolated.
What is the role of the DC voltage in the quadrupole analyzer?
By changing the DC voltage applied to the rods, the quadrupole analyzer can selectively allow certain m/z values to pass through while filtering out others.
What kind of information can be obtained from the quadrupole analyzer?
The quadrupole analyzer can provide information about the mass and abundance of specific ions in a sample.
What is the hybrid Quadrupole-TOF (Q-TOF) instrument?
A type of mass spectrometer that combines a Quadrupole and Time-of-Flight (TOF) analyzer into a single instrument.
How does the Q-TOF instrument work?
The Quadrupole analyzer is used to select and filter ions based on their mass-to-charge ratio (m/z) before they enter the TOF analyzer. The TOF analyzer separates ions based on their flight time through a vacuum tube.
What is the W-shaped TOF optic used in the Q-TOF instrument?
The W-shaped TOF optic provides high resolution mass spectrometry by slowing down ions and separating them in a more controlled manner, resulting in higher resolution and greater accuracy than a traditional linear TOF analyzer.
What is the role of the ion mobility separator in the Q-TOF instrument?
The ion mobility separator slows down ions by filling the area with helium and passing them through, adding another dimension of separation based on the ion’s surface area and providing additional information about the structure of the ions.
What is native mass spectrometry (MS)?
Native MS is a technique used to study large biomolecules in their natural state without denaturation or chemical modification.
What are the advantages of using native MS?
Native MS allows for the analysis of protein complexes and other biomolecules in their native state, which provides valuable information about their composition, subunit stoichiometry, topology, and interactions.
What type of biomolecules can be analyzed using native MS?
Proteins, nucleic acids, and protein complexes can be analyzed using native MS.
What information can be obtained from native MS?
Native MS can provide information on the oligomeric status and subunit composition of protein complexes, subunit stoichiometry and topology, microheterogeneity such as protein glycosylation, metal binding, and protein-ligand interactions.
How does native MS differ from regular (denaturing) MS?
Regular MS involves denaturation and/or chemical modification of the sample prior to ionization and analysis, whereas native MS does not disrupt native interactions and allows for analysis of biomolecules in their natural state.
what are the benifits of nano electro spray ionisation? (nESI) over normal ESI
Lower sample amounts
Can use aqueous buffers and ambient temperatures
Narrower charge states due to fewer adducts
Less dissociation of oligomer (just actual protein complex)
Symmetrical charge state distribution indicative of a single conformation
Fewer non-specific aggregates
what is collision induced dissociation?
A mass spectrometry technique that involves inducing fragmentation of ions by colliding them with neutral gas molecules, typically helium or nitrogen.
what is asymmetric dissociation?
A type of dissociation in which subunits of a protein complex are removed sequentially, and dissociation is not symmetrical with respect to mass.
what is a naked protein assembly?
A protein complex that has been stripped of its surrounding detergent molecules during mass spectrometry.
what is a detergent micelle and what is its use in CID?
A type of spherical structure formed by detergent molecules in aqueous solution, used to transfer protein samples into the vacuum of a mass spectrometer.
What is hydroxyl footprinting?
A structural MS technique that uses hydroxyl radicals to probe the solvent accessibility of protein surface residues.
What kind of information can hydroxyl footprinting provide?
Hydroxyl footprinting can provide high-resolution structural information about protein surface residues and their accessibility.
How are hydroxyl radicals generated in hydroxyl footprinting?
Hydroxyl radicals are generated by exposing proteins to a source of hydrogen peroxide or other reactive oxygen species.
How do hydroxyl radicals react with proteins in hydroxyl footprinting?
Hydroxyl radicals react with solvent-exposed residues in proteins, leading to peptide-level structural changes that can be detected by MS.
What is the mass shift observed in hydroxyl footprinting?
Hydroxyl radicals add a mass shift of 16 Da to the modified residue, which can be used to track changes in its accessibility.
What are the potential drawbacks of hydroxyl footprinting?
Hydroxyl radicals can generate reactive oxygen species that may damage the protein, and hydroxyl footprinting is limited to the study of solvent-exposed residues.
