Lecture 4

Question 1

What are proteomics

Answer 1

Study of entire set of proteins produced by an organism

Question 2

Problem of proteomics

Answer 2

• Genome of each cell in an organism is identical
• Proteome differs
• Protein abundance varies >6 orders of magnitude
• Co/post-translational modifications widespread

Question 3

How does MS solve protein

Answer 3

• Identifies protein in complex mixture
• Identify/localize protein modifications
• Relative/absolute quantitation
• Detect attomolar levels of proteins
• Limitation - dynamic range (detects 1 in 1000)

Question 4

Overview of MS

Answer 4

• Several interconnected chambers
• Under high vacuum

1. Sample ionized/accelerated
2. Mass selection - ionized sample defected by EM field (depends on m/z ratio)
3. Ions detected - mass/charge ratio plotted against signal intensity

Question 5

theory surrounding MS

Answer 5

• Charged particles experience force proportionate to charge
• Ions travelling non-linear path requires given acceleration

Experimental variations:
- Ionisation methods
- Different geometries
- Fragmentation chambers

Question 6

Tandem mass spectrometry

Answer 6

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

• Provides identity of ion
• MS instruments allow sequential fragmentation

Question 7

MALDI-TOF-MS

Answer 7

Matrix-assisted laser desorption ionization-time of flight

• Sample dried on inset solid in chemical matrix
• Laser beam cause ionisation with single +ve charge
• Mass calculated by time taken to travel to detector

Advantages: Ionise peptides, intact proteins, carbs

Disadvantages: Sample prep limits throughtput

Question 8

Liquid chromatography-mass spectrometry

Answer 8

• Analysis of eluted peptides
• Electrospray ionisation

Question 9

Overview of proteomics

Answer 9

Whole protein -> Complex peptide mixture -> MS/MS -> Protein list

Question 10

Proteomics sample prep

Answer 10

• Isolate proteins from biological sample e.g. tissues/cells
• Denature and reductively alkylate - break disulphide bonds
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• Digest into peptides - use trypsin
• Fractionation used to reduce sample complexity
• Enrichment or depletion improves sensitivity

Question 11

Analysis of proteomics

Answer 11

• Nano-flow C18 column with acetlonitrile gradient
• Positive electrospray ionization
• Data dependent acquisition: 1 MS scan and 10 MS/MS per duty cycle
• Dynamically exclude peaks for set time

Question 12

Identifying peptides using proteomics

Answer 12

• peptides fragment in predicable manner from each end of peptide
• Peptide sequence read from MS/MS spectrum

Question 13

protein modifications

Answer 13

Phosphorylation of Ser/Tyr/Thr

N-glycans on Asn

O-glycans on Ser/Thr

Ubiquitination of Lys

• Modifications occur at low abundance
• Introduced in vitro as deliberate strategy or accidently during sample processing
• Modified peptides identified by m/z
• Increasing modifications increases sequence space/CPU time

Question 14

phosphorylation

Answer 14

• Enrichment of phosphopeptides essential
• Sites localised and occupancy calculated

Tryptic digest -> Phosphopeptide enrichment -> IMAC/Anti-pY IgG/TiO2/SCX -> LC-MS/MS

Question 15

N-glycosylation

Answer 15

Complex branched structures

Mapped by ezymatic cleavage e.g. EndoH cleaves N-glycan and leaves GlcNAc residue

Identified by GC-MS

Inact glycopeptides identified by MS^n

Question 16

Ubiquitinylation

Answer 16

• Ubiquitin attached to lysine side chain
• Contains multiple lysines allows polyubiquitinylation

Mapped by tryptic digest which digests protein and Ubq modification

Question 17

SILAC quantification

Answer 17

• Stable isotope labelling by amino acids in cell culture
• Metabolic labelling technique

Use 2H, 13C, 15N labelled amino acids

• Labelled L-Arginine and L-lysine used

Light - C12-Arg label

Medium - 13C6-Arg or 13C4-Lys used

Heavy - 13C6/15N4-Arg or 13C4/15N4-Lys used

Question 18

Proteomics data processing

Answer 18

• Peptides and theoretical fragments calculated from sequence/allowed modifications
• Theoretical fragments matched to peaks
• proteins assembled from peptide data
• False discovery rate estimated using decoy sequences

Question 19

iTRAQ quantification

Answer 19

• Isobaric tagging for relative/absolute quantification

Chemically reactive multiplexed tag:
- All tags have same mass in MS
- Each tag produces distinct mass in MS/MS

• Up to 10 conditions can be simultaneously labelled

Question 20

Affinity purification MS

Answer 20

Isolates biologically relevant complexes

Distinguishing contaminant from real components is major problem

Question 21

AP-MS

Answer 21

qMS can identify non-specific contaminants

Stoichiometry can be obtained

Complex of interest is bound by a GFP affinity tag

Anti-GFP antibodies bind GFP where they have a non-specific contaminant

GFP tagging produces greater intensity than untagged complexes

Unspecific background binders same across untagged and tagged complexes

Question 22

Cross-linking MS

Answer 22

• Cross linking agents gain distance information
• CHemically reactive cross-linkers form covalent bonds
• Reveal weak interactions

Process:
Protein complex crosslinked

Proteolysis forms peptide mixture

Peptide mixture undergoes LC-MS/MS

Map of cross-links and set of distance restraints formed

Selection of structural models