Mass spectrometry based proteomics

Question 1

Define proteomics

Answer 1

Study of the proteome, entire set of proteins produced by organism or cell, synonymous with mass spectrometry

Question 2

What can mass spectrometry achieve?

Answer 2

Identify proteins in complex mixture

Identify and localise common protein modifications

Provide relative and absolute quantification

Detect attomolae (10-15) levels of protein, but dynamic range limited to 4 orders of magnitude

Question 3

explain the process of mass spectrometry

Answer 3

1. Sample ionised and accelerated
2. Mass selection:
   - Ionised sample defected by EM field
   - Amount of defection depends on mass/charge ratio
3. Ions detected - mass/charge ratio plotted against signal intensity

Question 4

Two classical theories of mass spectrometry

Answer 4

• Charge particles experience a force when travelling through a magnetic field proportional to their charge (z)

Newton’s second law - Force = mass (m) x acceleration

Making an ion travel at a set non-linear path requires a given acceleration

Solve equation to give mass/charge (m/z) ratio

Experimental variations:
- Ionisation methods
- Different geometries of mass selection (helical, elipsoid etc.)
- Fragmentation chambers

Question 5

Tandem mass spectrometry

Answer 5

Also known as MS/MS or MS^2

1. Standard MS spectrum required
2. m/z of interest selected
3. Ion fragmentation induced
4. Second mass selection event gives fragmentation induced spectrum

Fragmentation provides info about ion identity

Many MS instruments allow sequential fragmentation

Question 6

MALDI-TOF-MS

Answer 6

Matrix assisted laser desorption ionisation- time of flight-mass spectrometry

1. Samples dried in spots on inert solid in a chemical matrix
2. Laser beam causes ionisation with single positive charge
3. Mass calculated by time taken ton travel to the detector

Question 7

Advantages of MALDI-TOF-MS

Answer 7

Ionises peptides, intact peptides, carbohydrates

Question 8

Disadvantages of MALDI-TOF-MS

Answer 8

Sample preparation limits throughput

Question 9

LC-MS

Answer 9

Liquid chromatography coupled to mass spectrometry
- Analysis of peptides as they elute from a separation column
- Electrospray ionisation (ESI)

Question 10

Proteomics

Answer 10

‘Bottoms up’ proteomics

Wholes proteins -> Complex peptide mix -> MS/MS -> forms protein list

Question 11

Sample preparation of proteomics

Answer 11

1. Isolate proteins from biological sample - Tissues, cells, protein extract
2. Denature and reductively alkyate - Breaks up disulphide bonds
3. Digest into peptides - typically trypsin used
4. Fractionatrion to reduce sample complexity - either at protein or peptide level
5. Enrichment or depletion to improve sensitivity

Question 12

Analysis of Proteomics

Answer 12

Standard proteomics: LC-MS/MS analysis

• Nano-flow C18 column with an acetonitrile gradient
  
  • separate peptides on hydrophobicity
• Electrospray ionisation in positive mode
• Data dependent acquisition:
  
  • one MS and 10 MS/MS per ‘duty cycle’
  • dynamically exclude peaks for set time

Orbitrap –- high speed, high mass accuracy and resolution

Question 13

Proteomics – identifying peptides

Answer 13

Peptides fragment in a predictable manner from each end of the peptide

Peptide sequence is read from the MS/MS spectrum
- Each amino acid has a unique mass

Question 14

Protein modifications

Answer 14

Proteins receive co- and post-translational modification in vivo
i.e. Phosphorylation of Ser/Thr/Tyr
N-glycans on Asn
O-glycans on Ser/Thr
Ubiquitinylation of Lys
Often modification occur at low abundance and must be enriched before analysis

Other modifications may be introduced in vitro, either as a deliberate strategy or accidently during sample processing

Modified peptides can be identified by the change in m/z

Increasing the number of modification allowed rapidly increases possible sequence space and CPU time

Question 15

Phosphorylation

Answer 15

Enrichment of phosphopeptides essential:
- Low abundance, ionise poorly

Phosphorylation sites can be localised, and occupancy calculated

Characteristic ions produced:
- pS/T 98 Da neutral loss (H3PO4)
- pY gives immonium ion (m/z 216)

Question 16

N-glycosylation

Answer 16

N-glycosylans have complex branched structures

N-glycosylation sites can be mapped by enzymatic cleavage
EndoH cleaves N-glycan and leave GlcNAc residue

Released glycan structures can identified by GC-MS
Intact glycopeptides can be identified by MSn