Proteomics

Question 1

Define the proteome

Answer 1

A proteome is the complete set of proteins expressed by an organism // mechanistic expression of the gene expression. It differs from cell to cell and changes over time.

Question 2

How many amino acids have the potential to make a protein?

Answer 2

20 potential amino acids that can be
combine together to create a protein

Question 3

Explain the different levels of protein organisations (4)

Answer 3

Primary structure – sequence

Secondary structure – alpha helix or pleated sheets

Tertiary structure – how sheets or helixes fold together

Quaternary structure - multiple tertiary structures

Question 4

when interpreting mass spectra, which isotope should be used in calculations?

Answer 4

Use the lightest and most abundant isotope of the element when doing calculation – don’t use the average molecular mass!

Question 5

Explain the role of mass resolution within mass spectrometry?

Answer 5

• the ability to resolve closely related adjacent mass peak
• the bigger the number the higher the resolution
• higher resolution is better but more expensive

Question 6

Define Scan Speed?

Answer 6

How fast can the mass analyser scan the entire range of the mass spectrum (again the higher the number, the faster it can scan)

Question 7

what are the different types of MS acquisition modes?

Answer 7

MRM, PRM, DDA, DIA

Question 8

what is MRM?

Answer 8

Multiple reaction monitoring.
- targeted acquisition mode that is used to detect and quantitate specific, known compounds in a sample
- uses two mass spectrometer scans
- one to select a specific precursor ion, and a second to detect the corresponding product ions after fragmentation

Question 9

what is PRM?

Answer 9

Parallel Reaction Monitoring.
- similar to MRM but it allows to monitor multiple transitions
- useful in cases where multiple target compounds are present in a sample and need to be quantified simultaneously

Question 10

what is DDA?

Answer 10

Data-Dependent Acquisition.
Looks for things in DISCOVERY.
- selects and analyzes a series of precursor ions from a sample based on their relative abundance
- the most intense ions are selected and fragmented, and the resulting product ions are analyzed

Question 11

what is DIA?

Answer 11

Data-Independent Acquisition.
- DIA does not rely on the relative abundance
- instead splitting chamber and indistcriminatley fragmenting of precursor ions, thus it allows for detection of low-abundance precursors

Question 12

Explain how a Quadrupole works

Answer 12

• a quadrupole is a type of ion trap
• consists of four parallel metal rods arranged in the shape of a square
• by applying different voltages to the rods, an electric field is created that can trap and manipulate the ions
• quadrupole can be tuned to allow only ions of a specific mass-to-charge ratio to pass through; others are trapped or deflected
• used in mass spec. to separate and analyze different ions based on their mass-to-charge ratio

Question 13

Explain how a A triple quadrupole (QT) functions

Answer 13

• a triple quadrupole (QT) is a type of mass spectrometer
• utilizes three quadrupoles
• performs MRM
• first quadrupole acts as a mass filter, selecting ions of a specific mass-to-charge ratio
• second quadrupole, also called the collision quadrupole, is used to perform collision-induced dissociation (CID) on the selected ions - FRAGMENTS them
• third quadrupole, known as the detection quadrupole, is used to analyze the fragment ions generated in the second quadrupole

Question 14

Describe an Orbritrap

Answer 14

The Orbitrap is a type of mass spectrometer that utilizes ion trapping.

• ions are first generated and then introduced into the instrument
• ions are then trapped and oscillate in an electric field within the Orbitrap analyzer
• oscillating motion of the ions generates a current, which is measured and used to determine the mass-to-charge ratio of the ions

Question 15

explain how a Quadrupole Time of flight (TOF) functions

Answer 15

A type of mass spectrometer that combines the capabilities of a quadrupole mass filter and a time-of-flight (TOF) detector.

• uses a quadrupole mass filter to selectively pass ions of a specific mass-to-charge ratio
• then uses a TOF detector to measure the mass of the ions based on the time it takes them to travel a fixed distance

Question 16

Explain how Time-of-flight spectrometry is measured

Answer 16

• a measurement technique used to determine the mass-to-charge ratio (m/z) of ions in a mass spectrometer
• ions are first generated and accelerated into a flight tube
• ions are then allowed to travel a fixed distance through the flight tube
• they are detected by a detector at the end of the tube
• time it takes for the ions to travel the fixed distance is measured, and this time is used to calculate the mass-to-charge ratio of the ions

Question 17

Explain the role of MALDI and how it works

Answer 17

MALDI is a soft ionization method used in mass spectrometry to analyze large biomolecules such as proteins.

• sample is mixed with a small molecule called a “matrix” and applied to a metal plate
• matrix is typically a weak acid or a compound that absorbs at the wavelength of the laser
• a laser beam is then directed at the sample, and the energy from the laser causes the matrix to desorb and ionize the sample molecules
• resulting ions are then analyzed by a mass spectrometer

Question 18

ADV/DISADV of MALDI

Answer 18

+ Useful for intact proteins
- Multiple charges states due to large proteins so hard to derive the original mass.
- not suitable for small ionised molecules

Question 19

Explain the diffrence between MS1 and MS2

Answer 19

MS1 is used to determine the mass-to-charge ratio (m/z) and the relative abundance of each precursor ion are determined.
MS2 selects a specific precursor ion, and it fragments the precursor ion using collision-induced dissociation (CID) or other fragmentation methods. Analysis shows info about the structure of the precursor ion.

MS2 is useful for compounds
that have exact same chemical
formula but different chemical
structure

Question 20

What are the current limitations of MS based proteomics?

Answer 20

• detection limited as it can be difficult to detect low-abundance proteins
• can be limited by the complexity of the sample

Question 21

What preparation do MS1 & 2 samples require?

Answer 21

to be trypsinized and turned
into peptide mixture

Question 22

what are the methods for protein estimation?

Answer 22

• Bradford assay
• Lowry method
• Bicinchoninic acid assay
• Fluorescent based assa

Question 23

Explain how protein estimation works

Answer 23

• A calibration is created using a set of known concentration standards (such as BSA) and a minimum of 6 measurements are taken
• This will generate equation of the line (Y = Ax+B)
• Concentration and the response recorded, using the equation is it then
  back calculate to estimate the protein content (solve for x)

Question 24

why protein estimation is crucial for the trypsinization step?

Answer 24

trypsinization is a stichometrical reaction meaning we need a very specific ratio to fragment proteins correctly.

Question 25

How would you solve for X within a calibration curve for protein estimation using (Y = Ax+B). Additinally the sample was in 10ul solution and underwent a 50 fold dilution?

Answer 25

1. Subtract B (Y = Ax+B) = (Y-B = Ax)
2. Divide by A (Y-B = Ax) = (Y-B)/A=x
3. Divide by solution volume %10 = xuL = units
4. Multiply Dilution x50

Question 26

Explain Gel base method for protein fractionation
SDS-Page

Answer 26

• based on the principle that proteins will migrate through a gel matrix under an applied electric field
• proteins are first denatured by the addition of a detergent, sodium dodecyl sulfate (SDS), which coats the proteins and gives them a uniform negative charge
• allows for proteins to be separated based on their size, rather than charge
• proteins are then applied to a polyacrylamide gel, which acts as the matrix
• proteins will migrate under an applied electric field

Question 27

what is DiGE?

Answer 27

Difference gel electrophoresis.
Allows for detection of changes in protein modification.

Changes in any type of modification can potentially be detected, however we still need to identify the nature of that modification.

Question 28

Describe the steps of Shot gun (Bottom up) proteomic analysis and the aim.

Answer 28

(1) Extraction of protein content from the biological samples

(2) Subsequent determination of approximate protein (Bradford
assays)

(3) Fractionation (SDS gels) and trypsin digestion

(4) Clean up step and lyophilization

5) Chemical analysis (DIA or DDA)

(6) Database matching and peak table generations

(7) Data analysis and interpretation

Question 29

Explain Trypsin digestion

Answer 29

• trypsin is a serine protease
• it cleaves peptide bonds by hydrolyzing the peptide bond adjacent to the carboxyl group of the lysine and arginine residues
• therefore generates peptides of varying lengths

optimum conditions: pH of 8.3, 37°C

Question 30

List all the stages of protein analysis

Answer 30

1.Protein collection/ estimation techniques
2. Gel electrophoresis
3. tripsinisation + urea + wash to end reaction
4. Ionisation source - Electrospray ionization
Convert liquid samples to
gas phase ions
5 mass analyzer MS1
6. fragmentation
7. MS2 fragment analysing to tell structural component
8. Database matching (Mascot, Global proteome machine (GPM)
* Peakatable (Common proteins detected, ID, relative abundance)
* Quality of data, reproducibility, QCs

Question 31

Describe the function and mechanisms of Liquid chromatography system

Answer 31

Main purpose of LC block is to separate the complex peptide mixture into simpler ones. It should be reproducible.

how? :
- Analytical LC proteomic column and solvent gradient profile to separate complex mixture in both time and space

Question 32

Explain Electrospray ionization (ESI)

Answer 32

• electrospray ionization (ESI) is a method used to generate gas-phase ions from liquid samples
• involves the application of a high voltage to a liquid solution, which causes the liquid to form a spray of small droplets
• these droplets are then vaporized and ionized, resulting in the formation of gas-phase ions

Question 33

Explain the difference between top-down and bottom-up proteomics

Answer 33

Bottom-up
digest and separate into multiple peptides and build the protein structure e.g Shotgun proteomics

Top down
MALDI analysis of the whole protein with a high resolution mass spectrometer

Question 34

What are the benefits of top-down proteomics?

Answer 34

Intact protein analysis

Identification of protein isoforms

100% protein sequence coverage is possible

Question 35

What are the negatives of top-down proteomics?

Answer 35

Lower sensitivity and throughput

High sample purity is required (expensive)

Expensive instrumentation

Complex data and information rich

Question 36

What are the benefits of Bottom up proteomics?

Answer 36

More complex mixture can be analysed (thousands
of proteins can be measured)

Higher throughput

MS2 level information, ID and database matching

Question 37

What are the negatives of Bottom up proteomics?

Answer 37

High rate of false positive

Complex system, maintenance, calibrations, system performance assessment

Question 38

Explain Post-translational modification (PTM)

Answer 38

• PTM are a set of chemical modifications that plays a important role in the proteins functions
• can occur at any step in the life cycle of proteins
• regulate protein activity,
  localisation and interactions with other proteins, nucleic acids, lipids and co-factors
• changes in response to stimuli

Question 39

give examples of PTM events.

Answer 39

• Glycosylation
• Ubiquitination
• S-nitrosylation
• Methylation
• Acetylation
• Lipidation
• Hyroxylation

Question 40

What are Quality control (QCs) and why are they important?

Answer 40

• pool of the all the samples involved in the study
• is the most reproducible data points within the experiment
• used to assess for instrumental drift/analytical reproducibility over the course of the profiling experiment
• it is embedded into the experiment at regular intervals