Mass Spectrometry

Question 1

What accuracy does MS measure peptide mass?

Answer 1

0.01%

Question 2

What accuracy doe MS detect small organics?

Answer 2

5ppm

Question 3

What molecule can MS be used to sequence?

Answer 3

Oligonucelotides and peptides

Question 4

What are the 2 types of ionisation?

Answer 4

Electron Spray

Matrix Associated Laser Desorption

Question 5

How is a ESI sample administered?

Answer 5

Following HPLC

Or by dissolution into volatile solvent

Question 6

What solvents can be used for ESI?

Answer 6

water, methanol, acetonitrile

Question 7

How are solvents removed from ESI samples?

Answer 7

Nitrogen gas

Question 8

What are ESI protonation additives?

Answer 8

Formic acid

triethylamine

Question 9

What same concentration is used for ESI?

Answer 9

1-10pmol/µL

Question 10

How does ESI work?

Answer 10

Sample injected through metal capillary and voltage produces charged droplets

Question 11

What is the voltage of ESI?

Answer 11

1000-4000V

Question 12

What rate of flow is used for ESI?

Answer 12

4-1000µL/min

Question 13

What rate of flow is used for nano-ESI?

Answer 13

30-1000nL/min

Question 14

How does MALDI work?

Answer 14

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

Question 15

What MALDI solvents can be used for proteins?

Answer 15

3,5-dimethoxy-4-hydroxycinnamic acid

Question 16

What MALDI solvents can be used for peptides?

Answer 16

2,5-dihydroxybenzoic acid

α-cyano-4-hydroxycinnamic acid

Question 17

What environment does MALDI occur in?

Answer 17

High vacuum

Question 18

How many protons does ESI add to peptides

Answer 18

1

Question 19

How many protons does MALDI add?

Answer 19

1

Question 20

How many protons does ESI add to proteins?

Answer 20

m/z= (M+nH)/n

Question 21

What does the analyser do?

Answer 21

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

Question 22

How does detection occur?

Answer 22

Ions hit plate, which disperses electrons down channel

Amplification down channel

Question 23

What type of analysers are there?

Answer 23

Quadrupole

Time of Flight

Question 24

What are the components of the detector?

Answer 24

Photomultiplier

Microchannel plate detector

Question 25

What can be observed in a MS spectrum?

Answer 25

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

Question 26

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

Answer 26

``` Disulphide bonds Amino acid mutation Reducing group protonation state Post translational modification Presence of isotopes ```

Question 27

What does the Right-hand peak tell us?

Answer 27

Molecular mass +H

Question 28

Does ionisation only add protons?

Answer 28

No, some methods are (M-H)-

Question 29

What is Peptide Mass Fingerprinting?

Answer 29

MS spectrum of a whole protein

Question 30

What is tandem mass spectrometry used for?

Answer 30

determination of sequence

Question 31

What is the process of a MS-MS?

Answer 31

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

Question 32

When is negative ion mode used?

Answer 32

Usually for carboxylic acids/groups that readily lose a proton

Question 33

Which direct is a MS spectrum ladder sequence read?

Answer 33

Right to left is N to C

Question 34

Which residues cannot be identified?

Answer 34

Isoleucine and leucine have same Mr

Question 35

Why is protease digestion used?

Answer 35

To digest protein creating smaller fragments for MS-MS

Question 36

Why is trypsin used?

Answer 36

C terminal Arg/Lys are easily protonated | Arg/Lys ideal frequency

Question 37

What is the y1 mass for Arginine fragments?

Answer 37

175

Question 38

What is the y1 fragment for lsyine?

Answer 38

147

Question 39

What fragments does trypsin digestion produce?

Answer 39

neutral and positive

Question 40

What molecular mass does phosphorylation add to a protein?

Answer 40

HPO3 adds 80m/z

Question 41

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

Answer 41

H3PO4 lost, -98m/z

Question 42

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

Answer 42

loss of 79m/z for PO3-

Question 43

What effects does pH have on Apo-pseudoazurin?

Answer 43

pH7 uses Cu to stabilise | pH2, MeCN destabilises structure to produce more fragments

Question 44

What structural information can be studied by changing conditions?

Answer 44

Non-covalent

Question 45

What conditions can be changed to study structure?

Answer 45

temperature, pH Salt content

Question 46

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

Answer 46

Order of assembly of complexes