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CORE Block 3 > Mass Spectrometry and Proteomics > Flashcards

Mass Spectrometry and Proteomics Flashcards

1
Q

What is mass spectometry?

A

Sorting ions based on their mass/charge ratio

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2
Q

What are the four parts of a mass spec machine?

A
  1. front end
  2. ion source
  3. mass analyzer
  4. detector
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3
Q

How does the mass analyzer work?

A

It pulls on ions to different degrees depending on their mass.

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4
Q

When was electron spray ionization developed?

A

1984

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5
Q

When was the matrix assisted laser desorption ionization (MALDI) of proteins developed, and when was the nobel prize?

A

1988, 2002.

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6
Q

What are the applications of mass spec?

A
  • medicine (hospitals)
  • food and agriculture
  • chemistry
  • biology
  • isotope dating (ink in paintings)
  • space exploration
  • explosive detectors (at airports)
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7
Q

What are the front ends?

A

They are a separation from the MS so the sample does not have to be directly loaded.

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8
Q

What are the different types of ionization?

A
  • Hard: EI
  • Soft: ESI (electrospray ionization), MALDI
  • inductively coupled plasma (ICP)
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9
Q

What are the different types of mass selection?

A
  • sector
  • quadrupole
  • ion trap
  • orbitrap
  • fourier transform ion cyclotron resonance
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10
Q

What are the different types of detectors?

A
  • TOF
  • electron multiplier
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11
Q

Describe direct injection into the MS.

A

There are no front ends. Can use any type of molecules

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12
Q

Describe gas chromatography-MS.

A

Might want to look at commercial fish oil to determine how many good oils are actually in it. This uses a front end and can only be done with small, volatile molecules. Gas flows through the column, and molecules separate based on boiling point rather than mass. The higher the bp, the later it will come out of the MS.

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13
Q

Describe LC-MS injection into the MS

A

Liquid chromatography, can be done with all molecules. Uses a front end.

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14
Q

Describe MALDI injection into the MS

A

Can be done with all molecules, injected into the front end.

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15
Q

Describe injection of solids into MS.

A

Does not use front end. Direct injection. Can test any type of molecule (i.e. banana!) using a dipstick.

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16
Q

Descrbie the setup of reverse-phase high performance liquid chromatography.

A

solvent (A and B) -> mixer -> auto sampler -> column -> detector (MS, UV, etc)

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17
Q

What types of column materials can be used for HPLC?

A

in order of increasing polarity:

  • C18
  • C8
  • C4
  • cyano
  • phenyl
  • amino
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18
Q

Difference between HPLC and ultra performance /pressure liquid chromatography?

A

shorter runs because higher pressure in ultra PLC

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19
Q

Describe MALDI

A
  • a metal plate covered in a matrix where samples are spotted onto
  • a laser source hitting the plate, then the plate transferring energy to the analyte
  • molecules then ionized and sucked into the detector
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20
Q

Describe an application of MALDI

A

It can be used in hospitals to look at lipids belonging to bacteria.

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21
Q

What is a downside to MALDI?

A

It can be cumbersome to mix the sample to the matrix on the metal plate.

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22
Q

Describe electrospray ionization (ESI)

A
  • sample injected using LC
  • gas flows through the chamber in order to evaporate solvent
  • sample is sprayed into the chamber and forms a charged parent droplet
  • solvent evaporates leaving a smaller charged droplet

charged droplet -> charged progeny droplets -> naked charged analyte

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23
Q

What are the limitations of ESI?

A
  • only works if the solvent can be evaporated
  • must have a high current
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24
Q

Describe the quadrupole mass analyzer.

A
  • has 4 rods wich interchange in polarity
  • has ion size-dependent filters
  • can select for ion size by changing the strength of the electric field of the quadrupole
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25
Q

Describe the triple quad

A

This is a collision cell quadrupole, or MS/MS

  • Q1: first select for size molecule after ionizatin
  • Q2: molecules collide with gas and fragment
  • Q3: then select for the new size molecule of interest to weed out things you don’t want to analyze
  • molecules then go to detector
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26
Q

Describe the TOF (time of flight) mass analyzer.

A

It relies on long flight tubes and the positioning of mirrors to separate molecules based on size that fly at different speeds. (i.e. larger molecules take longer to reach the detector).

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27
Q

Why is a W shaped flight tube used in a TOF mass analyzer?

A

It makes the path length longer for a better separation of molecules.

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28
Q

Describe the electron multiplier detector.

A

Single ions are amplified in an electron multiplier which gives an electrical signal that is measured.

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29
Q

Describe the orbitrap mass analyzer.

A
  • a trap is within a magnet that has an electric field, causing ions to oscillate inside
  • moving ion rings around the central electrode induce an image current on the outder electrode
  • the wall of the trap is the detector, and the frequency of oscillations is proportional to m/z (i.e. how long the molecule flies around tells us how large it is)
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30
Q

Which bonds are easiest to fragment in MS/MS

A

weaker bonds such as C-N, P-O. Never C-C

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31
Q

What does MS/MS tell us about vitamin B12 content of old and young bacteria species?

A

It tells us that the older ones have a type of B12 that is different and not usable by humans.

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32
Q

What does it mean to fragment a parent peak?

A

It means to zoom in to look at high energy, and more peaks

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33
Q

What is the problem with the ‘omics’?

A

They are all separated and not integrated, though the instruments we all use are the same!

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34
Q

Why would we study proteomics?

A
  • functional macromolecules of the cell
  • concentration is different than that of mRNA
  • post-translational modifications are diverse
  • alternative splicing gives different protein isoforms
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35
Q

What is proteomics?

A

the study of the full set of proteins involved in structural, metabolic and regulatory functions of a cell or organism under a given set of conditions

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36
Q

What can the study of proteins be useful for?

A
  • disease: expression may go up or down
  • diagnostics: protein biomarker
  • basic science (i.e. localization of proteins, protein interactions)
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37
Q

What is the typical workflow of studying proteiomics?

A
  1. obtain protein sample
  2. separate proteins
  3. identify/quantify proteins (digest, fractionate, identify)
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38
Q

Why do we have to cut proteins into peptides, fractionate, and do MS/MS to sequence proteins?

A

Because mass spec can not tell the sequence, just the mass.

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39
Q

By what methods can proteins be seaparated?

A
  • electrophoresis
  • pull down
  • sucrose gradient centrifugation
  • microarray
  • LC (FPLC, UPLC/HPLC)
40
Q

What are the pros and cons of 1D electrophoresis?

A
  • simple and cheap
  • trouble resolving bands
41
Q

What are the pros and cons of 2D electrophoresis?

A
  • more complex
  • labor intensive
  • but, we get an extra dimension of separation
42
Q

How does 2D electrophoresis work?

A
  • move down gel in decreasing pI
  • then rotate gel and move according to size
43
Q

What are the pros and cons of capillary electrophoresis?

A
  • it is expensive equipment
  • but, it is automated and shows results as a chromatogram
44
Q

describe the different types of gel stains

A
  • calorimetrics: commassie (cheap!)
  • fluorescent
  • silver
  • tagged (fusion protein, fusion tag, specific functionality)
45
Q

Describe difference gel electrophoresis (DiGE)

A
  • dye proteome 1 a color and proteome 2 a different color
  • mix them and run 2 D gel, looking for differences and/or overlap
  • can look for differences between diseased and healthy proteomes
46
Q

What are ways to pull down proteins?

A
  • affinity
  • covalent
  • immunoprecipitation (antibodies)
47
Q

What are the limitations of sucrose gradient centrifugation?

A

Need an ultracentrifuge to reach very high speeds

48
Q

How does sucrose gradient centrifugation separate proteins?

A

Based on size

49
Q

What is sucrose gradient centrifugation used for these days

A

Separating the mitochondria or ER from the rest of the cell.

50
Q

Describe how microarray can be used to separate proteins.

A

A chip has different antibodies or antigens spotted at different points on the chip. Other proteins and peptides can be used to look at protein interactions. These things bound to the chip are called probes. Then flow the protein sample over the chip and observe differences in binding.

51
Q

How is microarray used with MS?

A

You can perform MALDI ionization directly from the microarray chip.

52
Q

What is the most common chromatography separation method for proteins?

A

Fast-paced liquid chromatography because it is not high pressure, the proteins are not degraded

(ion exchance, size exclusion, affinity resins)

53
Q

How can proteins be seaparated based on affinity?

A
  • tags (His)
  • probes (biotin)
  • antibodies
54
Q

Describe solid phase extraction (SPE) to separate proteins.

A

uses gravity to move samples through the column (can take a while)

separates large from small proteins

normally uses C18 resin

55
Q

What are the pros and cons of fast protein LC.

A
  • slower because low pressure
  • but, keeps proteins in native state
56
Q

When is HPLC or UPLC used to separate proteins?

A

For analytical purposes, not purification, because the protein will no longer be in its native state due to high pressure.

57
Q

Why are smaller columns used to separate smaller proteins or peptides?

A

Because the smaller the diameter, the more resolution.

58
Q

Describe 2D-LC.

A

Also called nano-LC. Uses tiny column used to separate peptides.

59
Q

What are the methods of detecting proteins?

A
  • western
  • edman sequencing
  • mass spec
60
Q

Where do antibodies for westerns come from?

A
  • polyclonal: inject antigen in animal, collect antiserum, affinity purify
  • monoclonal: fusion short-lived antibody expression spleen cells and immortal myeloma cells, screen and select.
61
Q

Describe Edman sequencing to detect proteins.

A

It chews the protein down one amino acid at a time, sequencing each as it goes. Very slow, so we now use mass spec instead.

62
Q

When might we use mass spec versus western blotting for protein detection?

A

Western for larger protein, mass spec for smaller peptides

63
Q

How is MALDI-TOF used in proteiomics?

A

With 2D gels and microarrays (basically need a chip)

64
Q

In what type of protein study would we use LC-triple quad?

A

for a targeted study in which we know the sequence of the protein already. identification

65
Q

In what type of protein study would we use LC-QTOF or LC-Orbitrap?

A

discovery, not targeted

66
Q

Describe “top-down” protein study.

A

Protein not digested. Take the pattern of the MS spectra, and deconvolute the charges and the masses. Use excel to determine where a charged protein might be based on what you have.

67
Q

Why is a “top-down” protein study done?

A

When you have no information about the protein sequence, but still want to know if you have made the protein or have the protein you want (must know mass)

68
Q

If we want to determine whether a protein is phorphorylated using MS, how would we do it?

A

If we know the mass of the protein, can look for the mass + that of a phosphate. Can fragment using MS/MS then if we want to determine which residue is being phosphorylated.

69
Q

How can we use MS to determine the location of disulfide bonds in a protein?

A

There will be different retention times in the MS depending on the location of the disulfide bond. Would have to already know the sequence of the protein.

70
Q

Describe the shotgun approach to proteomics. What type of experiment is it?

A

It is bottom up, meaning that the protein is digested. First do proteolytic digestion of the protein (shotgun). Then do MS and compare sequence to other known sequences on databases and can do comparisons and statistics.

71
Q

What is the most common type of protease used for shotgun protein studies?

A

Trypsin, it is the least specific.

72
Q

How is a peptide sequence read on a mass spectra using MS/MS?

A

From left to right, as the smaller peptides (smaller sized polypeptides) will elute first

73
Q

What are some problems to proteomics and their solutions?

A

problems:

-many, many peptides

  • complex mixtures
  • difficult to quantify
  • difficult to compare
  • low abundant proteins (especially when comparing proteomes of healthy and sick individuals)

solutions:

  • SRM
  • Mudpit
  • itraq
  • Silac

15N

74
Q

What are SRM and MRM?

A
  • SRM: selective reaction monitoring (looks specifically at one protein)
  • MRM: multiple reaction monitoring (looks at multiple protein)
75
Q

How does SRM and MRM work?

A

Just like MS/MS triple-quad, only instead of detecting all fragmented ion, only those selected for will be detected.

76
Q

What does MudPit do?

A

Uses 2D chromatography coupled to MS/MS

77
Q

What are the cons to using itraq?

A

The dyes are very expensive

78
Q

What is ITRAQ

A

isobaric tag for relative and absolute quantitation. Used to compare quantities of proteins in different samples

79
Q

How does iTRAQ work?

A
  • Proteins are extracted from a sample and digested
  • protein of interest is dyed iTRAQ tags of slightly different masses
  • Can mix all samples and run LC-MS, will know the sizes of the tags and proteins to determine relative quantities.
80
Q

What is the benefit of using iTRAQ compared to SILAC?

A

The samples can be dead

81
Q

What is the benefit of using SILAC compared to iTRAQ?

A

It is cheaper, just using 15N media rather than buying expensive dyes

82
Q

Describe how SILAC works.

A
  • One sample is cultured in 15N heavy amino acid media, the other is cultured in 14N light media.
  • proteins are pulled out
  • MS reveals ratio of light to heavy protein
83
Q

What does SILAC stand for?

A

Stable isotope labelling with amino acids in cell culture

84
Q

What is SILAC used for?

A

Relative quantification of protein. Can be used to determine differences in protein expression in diseased and healthy samples

85
Q

What type of ionization is used for imaging mass spec?

A

MALDI

86
Q

What can imaging MS be used for?

A
  • cancer grading
  • drug mapping
  • tumor diagnostics
  • chemical fingerprint (i.e. cocaine)
  • analysis of ink (i.e. paintings)
87
Q

How does imaging MS work?

A
  • small amount of liquid sprayed onto the sample so it can be ionized
  • sample stage moves so that the area of the sample is studied and you can get an image of the distribution of something like a drug or layering of ink
88
Q

What can be used to study the chemical warfare between bacteria species?

A

Imaging mass spec

89
Q

What is a drawback of imaging MS?

A
  • it is tedious, lots of scanning
  • direct injection, so the sample is dirty
  • resolution is not down to the single cell level
90
Q

What is iKnife?

A

It is a clinical use of direct MS injection in which part of a tissue is burned away and the vapors enter a MS. Can get real time MS of margins of something being cut. No longer need pathology lab. Can shorten the length and number of surgeries

91
Q

What are the limitations of MS?

A

Cannot determine molecular structure

Can’t measure tiny concentrations

92
Q

How can molecular structure of a protein be determined?

A

NMR

X-ray crystallography

cryo EM

93
Q

What are the drawbacks to X-ray crystalography?

A
  • strong beams are expensive
  • hard to do in house
  • optimization of conditions is time-consuming
  • some proteins are difficult to crystallize due to location or highly dynamic
94
Q

When is NMR useful?

A

When small molecules cannot be crystallized.

95
Q

How does X-ray crystallography work?

A
  • crystal made in tray
  • beam shot at crystal (stronger beam for larger proteins)
  • electron density map given to model the protein using software
96
Q

What are the disadvantages of NMR?

A

The protein needs labelled with an isotope by enriching the media which can be pricey

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