Lecture 8 - protein modification

Question 1

What is the most important regulator of expressed protein activity?

Answer 1

Phosphorylation of proteins

Via. Kinases, Phosphatases and secondary messengers

Question 2

How are phosphorylated proteins studied?

Answer 2

1) Purification of phosohorylated proteins/peptides
2) Radiolabelled-P 2D SDS PAGE
3) IMAC purification
4) Immunoaffinity

OR

1) MS detection only of phophopeptides
2) Derivatisation reaction with MS
3) Precursor ion discovery (PID)
4) Constant neutral Loss (CNL)

Question 3

Outline 2D-Gel with radiolabelled P

Answer 3

Add either 32P or 32P-labelled ATP
MS cant use radioactive atoms

N-term sequencing

Separation of radiolabelled peptides

Run a second non-radiolabelled gel for comparison

Radiolabelled P must compete with unlabelled P

Question 4

What is IMAC?

Answer 4

Immobilised metal affinity chromatography

This relies on the interaction between phosphate group and an immobilised metal such as iron

1) Photolabelled protein is digested
2) Immobilized metal affinity resin
3) Non photolabelled peptides are eluted
4) Wash resin with salt and urea
5) Non specifically bound peptides are eluted
6) Wash with phosphate buffer
7) Photolabelled peptides are eluted
8) Further purification by HPLC
9) Sequence determination

Question 5

What is immunoaffinity?

Answer 5

Antibodies raised against phospho STY which are immobilised on a column or used for Western blotting 2D PAGE gels

Question 6

How is derivatisation reaction achieved for MS?

Answer 6

1) Reduce
2) Alkylate or oxidize with performic acid
3) O-deglycosylation
4) Proteolytic digestion
5) Base catalyzed reaction of P-Ser/P-Thr peptides

6) Ethanethiol > MS
6) Dithioreitol (DTT) > SH > Solid phase affinity > MS
6) Ethanedithiol > Biotinylation > (malemide,nonmalemid,photosensitive linker) > Solid phase affinity > MS

Question 7

How MS detects phosphopeptides?

Answer 7

Relies upon CNL or PID
Detection of a characteristic product ion during MSMS

e.g CNL analysis: loss detected is the loss of the phosphate (98kDa)

BUT only aplicable to peptides with phosphorylation on the tyrosine residue

Question 8

What is the m/z of a phosphopeptide and its precursor?

Answer 8

Phosphopeptide m/z = peptide + 80

Precursor m/z = -98

Question 9

What is the m/z of immonium ion (NH=CHR)

Answer 9

m/z = 216

Question 10

Compare the difference between CNL, PID for phophate analysis.

Answer 10

CNL - detects all phosphopeptides

PID - detects phosphotyrosine

Phosphoserine and threonine have too unstable immonium ions

CNL is the most commonly applied technique

Question 11

What % of the human body proteins can be glycosylated?

Answer 11

50-60%

Account for 25% of the currently available cancer biomarkers

Question 12

Why is protein glycosylation problematic?

Answer 12

Heterogenous and diverse nature of glycans and the complex nature of this modification.

This is fixed through enrichment methods:
Lectin affinity purification (most used)

Concanavalin A binds to N-linked glycans

Less specific lectins = Glyco-catch approach

Question 13

What is the derivatisation enrichment methods : O-Gly-Nac?

Answer 13

1) Metabolic labelling using galactose transferase GalT
2) Aminooxy biotin
3) Western blot using horse radish peroxidase conjugated streptavidin
4) Protease digestion
5) Cation exchange; Avidin agarose

Question 14

What are the MS sample preparation methods?

Answer 14

Add fractionated protein sample to MALDI plate
-Obtain protein profile

Treat with glycopeptidase

• Release glycans
• Done on plate

See which protein masses have changed.
Change in mass = protein modification occured

Question 15

How are glycans studied using the sample preparation method?

Answer 15

Mass shift in protein corresponds to mass of glycan
Protein ID unknown and sequence of glycan maybe unsure.

This is fixed using PID or CNL to characterise the products

Question 16

What do ubiquinated proteins partake in?

Answer 16

Proteasomal degradation
Protein sorting
DNA repair
Translation control
Kinase activation

Ubiquination can be mono or poly

Question 17

What do farnesylated proteins partake in?

Answer 17

Targeting proteins to membrane

-Chemotherapy against cancer