Proteomics

Question 1

What are –omics techniques?

Answer 1

Suffix to denote the study of the entire set of something

Question 2

What are genomics?

Answer 2

The study of all genes

Question 3

What are transcriptomics?

Answer 3

The study of all mRNA transcripts

Question 4

What are proteomics?

Answer 4

The study of all proteins –> (large scale) identification and characterisation of proteins and all their properties

Question 5

What is a good research question for proteomics?

Answer 5

Which proteins are present at a certain location or time and how do they look like?

Question 6

Name a challenge in proteomics?

Answer 6

Proteins are more difficult to characterise and work with than for ex. nucleic acids (DNA bases). DNA/RNA has 4 building blocks (ACGT) and are negatively charged. Proteins have 20 building blocks (amino acids) and undergo post-translocational modification. Proteins need to be kept in a functional correctly folded state for most proteomics applification.

Question 7

What is the principle for (Liquid) chromatography

Answer 7

Column chromatography consists of a stationary solid phase that adsorbs and separates the compounds passing through it with the help of a liquid mobile phase. On the basis of their chemical nature, compounds get adsorbed and elution is based on differential adsorption of a substance by the adsorbent

Question 8

Name a few examples of chromatography

Answer 8

• Size exclusion
  -Ion chromatography
• Affinity chromatography
• Reverse/Forward phase

Question 9

What are the steps of chromatography to MS

Answer 9

Sample –> Liquid chromatography –> Collecting fractions –> Trypsin digestion –> Ionisation + Vacuum –> Accelation –> Seperation –> Detection –> Identification

The 1e 3 steps are for seperation, and the rest are for identification

Question 10

What is Massa Spectrometry

Answer 10

Analytical method to measure the molecular weight of molecules (based on charge)

Question 11

Why use Trypsin before MS

Answer 11

The protein is cut into pieces by Trypsin (Fragments). Trypsin cleaves protein behind R (Arginine) or K (Lysine) so it is predictable. Smaller fragments are easier to analyse which leads to larger accuracy

Question 12

What are the 2 techniques for ionization in MS

Answer 12

• Electrospray ionization (ESI)
• MALDI (Matrix Assisted Laser Desorption Ionisation

These are sofi ionizing techniques

Question 13

What are the 3 techniques for analysing MS products

Answer 13

• TOF (Time of flight)
• Ion trap analyzer
• Quadrupole Mass Analyzer

Question 14

What can you see on the graphs made by MS

Answer 14

You can see a lot of peaks. On the X-axis are the m/z ratio displayed and on the Y-axis are the intensity (how much detection) displayed. The mass is in dalton Da and the charge z is per charge
Around the peaks are also isotopes. Isotopes are molecules with the same amount of protons, but a different amount of neutrons. The peaks of isotopes are way smaller than base peak.

Question 15

What is the m/z value of a molecule with a mass of 564 Da and 5 protons charged?

Answer 15

5 PROTONS CHARGED= 5 Da. So 564 + 5= 569 Da total weight.
m/z= 569/5= 113,8

Question 16

How much Da gets added with a proton addition and a sodium addition

Answer 16

Protons adds 1 Da while Sodium adds 23 Da

Question 17

What is a monoisotopic mass?

Answer 17

Sum of masses in a molecule using the principal isotope (most abundant isotope) of each atom in the molecule –> heighest peak

Question 18

What is the average mass?

Answer 18

Sum of the average masses (weighted average of all staple isotopes) of the atoms composing the molecule

Question 19

To divide mass by higher value to obtain the m/z. This is also true for isotopes, but what happens to the distance between isotopes?

Answer 19

Isotopes distance= 1/z

For 1 charge: 1/1=1
For 2 charge: 1/2= 0,5
For 3 charge: 1/3= 0,3
For 5 charge: 1/5= 0,2

Question 20

Is the isotopic distribution of biological molecules (DNA, proteins, lipids) always the same?

Answer 20

NO

Question 21

Is, for a certain molecule produced by a certain species, the isotopic distribution for the molecule always the same?

Answer 21

NO

Question 22

What is the procedure for fragmentation during MS?

Answer 22

You have a sample, then you trypsinize the peptides and use soft ionization. You will have ions now. Then there is a selection/isolation (End of first MS). So now you have +- 1 isolated ion. Then you do fragmentation by adding energy/collision gas. You will end up with fragments. (End of second MS). The procedure for the second MS can be repeated until satisfaction

Question 23

What is the effect of fragmentation

Answer 23

• Analytes will break at specific points
• Usually, multiple fragments for 1 molecule

Question 24

Why do we ionize the samples during MS?

Answer 24

Since otherwise the samples won’t go up to the magnetic field

Question 25

Why is N2 (or any other gas) used during ESI

Answer 25

To let the fluid vaperate (the ions are located there in the fluid)

Question 26

What is the m/z value of a molecule of 564 Da with 5 protons

Answer 26

564 + 5 protons of 1 Da= 569 Da
m/z value: 569/5= 113,8 m/z

Question 27

Imagine you would find signal in mass spectrum: 349,55 m/z with isotopes at 0,25 m/z distance. What is the actual mass

Answer 27

0,25 distance: 1/0,25= 4 charges
m/z= m divided by z so m= m/z multiplied by z
349,55 x 4= 1398,2 Da
minus 4 protons= 1394,55

Question 28

What would be the mass of peptide WTYPR and calculate the isotopes per charge

W= 204,23 Da
T= 119,12 Da
Y= 181,19 Da
P= 115, 13 Da
R= 174, 2 Da

Answer 28

First: add everything together: 793,87 Da. For every peptide bond: substract 18 Da. 4 peptide bonds: 793,87 - ( 4x 18)= 721,87

Charge per proton: +1,2,3 and additional +1 isotopes

Charge 1: 722,87, 723,87, 724,87
Charge 2: 723,87, 724,87, 725,87
Charge 3: 724,87, 725,87, 726,87

Lastly, divide by charge to obtain m/z

Charge 1: 722,87 + distance 1 m/z isotope
Charge 2: 361,94 + 1/2 distance isotope
Charge 3: 241,62 + 1/3 distance isotope

Question 29

We don’t know the amino acid sequence of the performed fragmentation. We obtain the following fragmentation spectrum:

157,10
254,16
440,24
603,30
704,35

From the amino acids:
W= 204,23 Da
T= 119,12 Da
Y= 181,19 Da
P= 115, 13 Da
R= 174, 2 Da

Answer 29

1e: Remove H2O masses then calculate the differences between the fragments

W= 186,23
T= 101,12
Y= 163,19
P= 97,13
R= 156,2

704,35- 603,30= 101,05 T
603,30- 440,24= 163,06 Y
440,24- 254,16= 186,08 W
254,16- 157,10= 97,06 P
157,10- 0= 157,10 R (the 1 difference may be because of proton differences)

Amino acid sequence: RPWYT

Question 30

You find the following mass: 184,55 m/z with isotopes 0,5 distance. What would the composition of the peptides be if you know that it contains a combination of 3 of the following amino acids (increment masses)

Serine: 87,03
Valine: 99,07
Methionine: 131,04

Answer 30

184,55 m/z with 1/0,5 charge: 2 charge
184,55 x 2= 369,1 Da
- 2 = 367,1 Da

The amino acid masses are increment masses which doesn’t include H2O values. We have to add H2O masses according to the peptide bound in the sequence

The only solution will be: 2 x 131,04 (M) with 1x 87,03 (S) = 349,11. There will not be peptide bonds between the same amino acid so M-M binding doesn’t have a peptide bond. There is a peptide bond in M-S, so we only add 18 Da once: 349,11 + 18= 367,11