MS

Question 1

Why is sequencing proteins relevant? (5)

Answer 1

Knowledge of the sequence of a protein is usually essential to elucidating its mechanism of action (e.g., the catalytic mechanism of an enzyme).

Proteins with novel properties can be generated by varying the sequence of known proteins.

Amino acid sequences determine the three-dimensional structures of proteins.

Sequence determination is a component of molecular pathology, a rapidly growing area of medicine.

The sequence of a protein reveals much about its evolutionary history.

Question 2

What is the first step in protein sequencing?

Answer 2

Proteins need to be digested into peptides (linear): break intermolecular interactions

Question 3

How are proteins digested into peptides?

Answer 3

1. Protein reduction
2. Protein alkylation
3. Protein digestion

Question 4

Why are proteins reduced?

Answer 4

To break disulphide bonds, which stabilise tertiary structure of protein (only strong, covalent interaction involved in this)

Question 5

What chemical is used to reduced disulphide bonds?

Answer 5

Dithiothreitol, used in excess

Question 6

Why is protein alkylation importants in order to digest them into peptides?

Answer 6

Once chains have been reduced, one more step is needed to ensure that the disulphide bonds do not reform

Question 7

What chemical is used for protein alkylation?

Answer 7

Iodoacetate, gives separated carboxymethylated chains on Cys

Question 8

What is the most commonly used enzyme used in protein digestion?

Answer 8

Trypsin, a serine protease

Question 9

What is the function of trypsin and its specificity?

Answer 9

Trypsin is a serine protease found in the digestive system of many vertebrates, where it hydrolyses proteins.

It is produced in the pancreas as the inactive proenzyme trypsinogen.

Trypsin cleaves peptide chains mainly at the carboxyl side of the amino acids lysine or arginine, except when either is followed by proline

Question 10

Why is trypsin so widely used in protein digestion?

Answer 10

highly specific

efficient

generates peptides having an arginine or lysine at the C-terminus (which simplifies sequencing a whole lot)

Question 11

What is the specificity of chymotrypsin?

Answer 11

It cleaves the carboxyl side of the amide bond (the P1 position) is a large hydrophobic amino acid (tyrosine, tryptophan, and phenylalanine)

Question 12

What are the essentials of a MS?

Answer 12

Question 13

What is the best way of ionising a sample for MS or Tandem MS?

Answer 13

ESI

Question 14

How does ESI work?

Answer 14

Protein solutions are passed through a metal needle held at high potential (typically 3-5 kV)

There is a build-up of charge at the needle tip which overcomes the surface tension producing a fine mist of droplets that emerge from the needle tip with a net charge.

A counter current drying gas aids desolvation and droplet evaporation to produce smaller daughter droplets.

This eventually leads to the production of solvent free molecular ions

Question 15

Why are charged particles, with both m and z, needed for MS?

Answer 15

Because they can be handled and their paths controlled thru the use of electric and magnetic fields.

Question 16

Are all ions produced +vely charged?

Answer 16

Generally, yes, but there are important exceptions (NA)

Question 17

Describe the mass spectrum

Answer 17

Ions appear in a spectrum as a result of an electrical current that is generated and amplified when the ion strikes the detector

X-axis is the m/z ratio of the ion
Y- axis is the ion current or more commonly the relative ion abundance

Sometimes base peak is normalised to 100% and all other ions have abundances relative to this peak

Question 18

What is MS/MS?

Answer 18

Means by which to obtain the MS of an MS, by causing ions in the first MS to fragment so that the secondary MS can reveal new information

Question 19

What are the main components of a tandem mass spectrometer and their functions?

Answer 19

Ionisation source: ionises sample so that it can be handled

MS1: separates ions based on m/z value, isolates ion of interest

Collision cell: activates and dissociates ion of interest

MS2: separates and detects fragment ions

Detector: detection of ions, production of spectrum

Question 20

What is CID and what can it be used for?

Answer 20

If an ion collides with a neutral atom (usually a noble gas) or molecule, some of its translational energy is converted into internal energy (bond vibrations) causing the ion to fragment

CID can be used to fragment peptides and therefore obtain their sequence

Question 21

What are the components of the Q-TOF analyser?

Answer 21

Question 22

What is the function of the quadrupole in Q-TOF?

Answer 22

MS mode: guide for ions

MS/MS: can be set to allow only the ion of interest to pass into the collision cell where fragmentation is induced. Fragments are then analysed in detector

Question 23

What does the detector do in Q-TOF?

Answer 23

Converts ion signal from e- to photons and back to e-, it is a photomultiplier tube

Question 24

What are the three different types of bonds that can fragment along the amino acid backbone?

Answer 24

NH-CH , CO-NH, CH-CO

Each bond cleavage gives rise to two species, one neutral and the other one charged. The charged species is the one detected by the mass spectrometer

Question 25

How many possible fragment ions are there for each amino acid residue?

Answer 25

6 (a,b,c,x,y,z)

Question 26

a,b,c ions retain charge on which terminus?

Answer 26

the a, b, and c ions have the charge retained on the N-terminal fragment

Question 27

x,y,z ions retain charge on which terminus?

Answer 27

x, y, and z ions have the charge retained on the C-terminal fragment

Question 28

Which is the most common cleavage site and what ions arise from this?

Answer 28

CO-NH (amide linkage), gives rise to b and y ions

Question 29

What are a ions?

Answer 29

b ions - C=O group

Question 30

What are c ions?

Answer 30

b ions + NH group

Question 31

What does the intensity of the signal in an mass spectrum reflect?

Answer 31

how often that bond breaks

Question 32

Which peak gives the Mr of the ion?

Answer 32

The one with the biggest m/z value, gives [M-H]⁺

Question 33

What is the residue mass for an amino acid?

Answer 33

Residue mass = Amino acid mass – H2O

Question 34

What is the structure of an immonium ion and why are these useful?

Answer 34

Immonium ions appear in the very low m/z range of the MS/MS spectrum but some are of low abundance.

Most amino acid residues yield a diagnostic immonium ion, but. The immonium ions are useful for detecting and confirming many of the amino acid residues in a peptide, although no information regarding the position of the residues in the peptide sequence can be derived from immonium ions.

Question 35

What is the difference in mass between an a and a b ion?

Answer 35

a ions are b ions that have lost a CO group

An a ion has a mass of 28 Da less than the corresponding b ion

Question 36

What are the masses of a,b and y ions?

Answer 36

[N] = mass of the N-terminating group (H = 1)

• [C] = mass of the C-terminating group (OH = 17)
• [M]n = sum of the masses of n amino acid residues (+ any PTMs)

Question 37

How is the probable number of resiudes found?

Answer 37

Divide peak with highest m/z value by 110, which is the average residue mass

Question 38

Identify a,b,c,y,x,z ions in a sequence

Answer 38