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Proteins > Mass Spectrometry > Flashcards

Mass Spectrometry Flashcards

1
Q

What accuracy does MS measure peptide mass?

A

0.01%

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2
Q

What accuracy doe MS detect small organics?

A

5ppm

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3
Q

What molecule can MS be used to sequence?

A

Oligonucelotides and peptides

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4
Q

What are the 2 types of ionisation?

A

Electron Spray

Matrix Associated Laser Desorption

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5
Q

How is a ESI sample administered?

A

Following HPLC

Or by dissolution into volatile solvent

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6
Q

What solvents can be used for ESI?

A

water, methanol, acetonitrile

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7
Q

How are solvents removed from ESI samples?

A

Nitrogen gas

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8
Q

What are ESI protonation additives?

A

Formic acid

triethylamine

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9
Q

What same concentration is used for ESI?

A

1-10pmol/µL

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10
Q

How does ESI work?

A

Sample injected through metal capillary and voltage produces charged droplets

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11
Q

What is the voltage of ESI?

A

1000-4000V

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12
Q

What rate of flow is used for ESI?

A

4-1000µL/min

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13
Q

What rate of flow is used for nano-ESI?

A

30-1000nL/min

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14
Q

How does MALDI work?

A

Small aromatic matrix binds sample
15 minutes solvent evaporation
Laser enters matrix and passes energy/charge to proteins

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15
Q

What MALDI solvents can be used for proteins?

A

3,5-dimethoxy-4-hydroxycinnamic acid

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16
Q

What MALDI solvents can be used for peptides?

A

2,5-dihydroxybenzoic acid

α-cyano-4-hydroxycinnamic acid

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17
Q

What environment does MALDI occur in?

A

High vacuum

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18
Q

How many protons does ESI add to peptides

A

1

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19
Q

How many protons does MALDI add?

A

1

20
Q

How many protons does ESI add to proteins?

A

m/z= (M+nH)/n

21
Q

What does the analyser do?

A

Separate molecules in a high vacuum based on mass/charge ration

22
Q

How does detection occur?

A

Ions hit plate, which disperses electrons down channel

Amplification down channel

23
Q

What type of analysers are there?

A

Quadrupole

Time of Flight

24
Q

What are the components of the detector?

A

Photomultiplier

Microchannel plate detector

25
Q

What can be observed in a MS spectrum?

A

Number of components (purity of sample)
Mr of components
Abundance of components

26
Q

Comparison to database can determine what types of modification on a protein?

A 
Disulphide bonds
Amino acid mutation
Reducing group protonation state
Post translational modification 
Presence of isotopes
27
Q

What does the Right-hand peak tell us?

A

Molecular mass +H

28
Q

Does ionisation only add protons?

A

No, some methods are (M-H)-

29
Q

What is Peptide Mass Fingerprinting?

A

MS spectrum of a whole protein

30
Q

What is tandem mass spectrometry used for?

A

determination of sequence

31
Q

What is the process of a MS-MS?

A

Quadrupole to focus to a single peak/peptide
Argon collision cell generates fragments
ToF analyser detects fragments and difference between peaks is 1 residue

32
Q

When is negative ion mode used?

A

Usually for carboxylic acids/groups that readily lose a proton

33
Q

Which direct is a MS spectrum ladder sequence read?

A

Right to left is N to C

34
Q

Which residues cannot be identified?

A

Isoleucine and leucine have same Mr

35
Q

Why is protease digestion used?

A

To digest protein creating smaller fragments for MS-MS

36
Q

Why is trypsin used?

A

C terminal Arg/Lys are easily protonated

Arg/Lys ideal frequency

37
Q

What is the y1 mass for Arginine fragments?

A

175

38
Q

What is the y1 fragment for lsyine?

A

147

39
Q

What fragments does trypsin digestion produce?

A

neutral and positive

40
Q

What molecular mass does phosphorylation add to a protein?

A

HPO3 adds 80m/z

41
Q

ESI-MS in positive mode shows phosphorylated proteins with what change in mass?

A

H3PO4 lost, -98m/z

42
Q

ESI-MS in negative mode shows phosphorylated proteins with what change in mass?

A

loss of 79m/z for PO3-

43
Q

What effects does pH have on Apo-pseudoazurin?

A

pH7 uses Cu to stabilise

pH2, MeCN destabilises structure to produce more fragments

44
Q

What structural information can be studied by changing conditions?

A

Non-covalent

45
Q

What conditions can be changed to study structure?

A

temperature,
pH
Salt content

46
Q

What can MS be used for in studies of Viral capsids and filaments?

A

Order of assembly of complexes

Proteins (12 decks)

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