Mass spectrometry

Question 1

What are the fundemental components of a mass spectrometer?

Answer 1

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)

2. seperate ions by mass to charge ratio in analyser. typically under high vaccum.
3. a detector to detect ions
4. an acquisition system to bring all the data together.

Question 2

What are some methods of sample introduction in a mass spec?

Answer 2

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

Question 3

what are methods of ionisation in mass spectrometry?

Answer 3

Electron ionisation

Chemical ionisation

Fast Atom/Ion Bombardment

Secondary Ion Mass Spectrometry

Field desorption

Plasma desorption

Electrospray Ionisation

Matrix Assisted Laser Desorption Ionisation (MALDI)

Question 4

What are soft ionisation techniques?

Answer 4

they dont fragment the molecule so can retain the molecular ion. examples of this are:
- electrospray ionisation and matrix assisted laser desorption ionisation. (MALDI)

Question 5

describe the technique of electrospray ionisation

Answer 5

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

Question 6

Describe the technique of matrix assisted laser desorption ionization (MALDI)

Answer 6

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

Question 7

What is MALDI ionization?

Answer 7

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.

Question 8

What is a matrix in MALDI ionization?

Answer 8

A low molecular weight compound that is mixed with the molecule of interest in MALDI ionization to help ionize the sample.

Question 9

What is the purpose of Time of flight mass spectromertry in MALDI ionization

Answer 9

To separate and analyze the ions that are generated by MALDI ionization based on their mass-to-charge ratio.

Question 10

What is the target plate in MALDI ionization?

Answer 10

The surface onto which the mixture of the molecule of interest and the matrix is spotted and dried in MALDI ionization.

Question 11

What is the role of the UV laser in MALDI ionization?

Answer 11

To provide the specific wavelength of light that the matrix absorbs in order to cause the ionization of the sample.

Question 12

What kind of molecules can be analyzed using MALDI ionization?

Answer 12

Biomolecules such as proteins, peptides, and nucleic acids.

Question 13

How does the time of flight analyser work?

Answer 13

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

Question 14

What is the quadrupole analyzer?

Answer 14

A type of mass filter used in mass spectrometry to separate ions based on their mass-to-charge ratio (m/z).

Question 15

How does the quadrupole analyzer separate ions?

Answer 15

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.

Question 16

What is the role of the DC voltage in the quadrupole analyzer?

Answer 16

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.

Question 17

What kind of information can be obtained from the quadrupole analyzer?

Answer 17

The quadrupole analyzer can provide information about the mass and abundance of specific ions in a sample.

Question 18

What is the hybrid Quadrupole-TOF (Q-TOF) instrument?

Answer 18

A type of mass spectrometer that combines a Quadrupole and Time-of-Flight (TOF) analyzer into a single instrument.

Question 19

How does the Q-TOF instrument work?

Answer 19

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.

Question 20

What is the W-shaped TOF optic used in the Q-TOF instrument?

Answer 20

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.

Question 21

What is the role of the ion mobility separator in the Q-TOF instrument?

Answer 21

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.

Question 22

What is native mass spectrometry (MS)?

Answer 22

Native MS is a technique used to study large biomolecules in their natural state without denaturation or chemical modification.

Question 23

What are the advantages of using native MS?

Answer 23

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.

Question 24

What type of biomolecules can be analyzed using native MS?

Answer 24

Proteins, nucleic acids, and protein complexes can be analyzed using native MS.

Question 25

What information can be obtained from native MS?

Answer 25

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.

Question 26

How does native MS differ from regular (denaturing) MS?

Answer 26

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.

Question 27

what are the benifits of nano electro spray ionisation? (nESI) over normal ESI

Answer 27

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

Question 28

what is collision induced dissociation?

Answer 28

A mass spectrometry technique that involves inducing fragmentation of ions by colliding them with neutral gas molecules, typically helium or nitrogen.

Question 29

what is asymmetric dissociation?

Answer 29

A type of dissociation in which subunits of a protein complex are removed sequentially, and dissociation is not symmetrical with respect to mass.

Question 30

what is a naked protein assembly?

Answer 30

A protein complex that has been stripped of its surrounding detergent molecules during mass spectrometry.

Question 31

what is a detergent micelle and what is its use in CID?

Answer 31

A type of spherical structure formed by detergent molecules in aqueous solution, used to transfer protein samples into the vacuum of a mass spectrometer.

Question 32

What is hydroxyl footprinting?

Answer 32

A structural MS technique that uses hydroxyl radicals to probe the solvent accessibility of protein surface residues.

Question 33

What kind of information can hydroxyl footprinting provide?

Answer 33

Hydroxyl footprinting can provide high-resolution structural information about protein surface residues and their accessibility.

Question 34

How are hydroxyl radicals generated in hydroxyl footprinting?

Answer 34

Hydroxyl radicals are generated by exposing proteins to a source of hydrogen peroxide or other reactive oxygen species.

Question 35

How do hydroxyl radicals react with proteins in hydroxyl footprinting?

Answer 35

Hydroxyl radicals react with solvent-exposed residues in proteins, leading to peptide-level structural changes that can be detected by MS.

Question 36

What is the mass shift observed in hydroxyl footprinting?

Answer 36

Hydroxyl radicals add a mass shift of 16 Da to the modified residue, which can be used to track changes in its accessibility.

Question 37

What are the potential drawbacks of hydroxyl footprinting?

Answer 37

Hydroxyl radicals can generate reactive oxygen species that may damage the protein, and hydroxyl footprinting is limited to the study of solvent-exposed residues.