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Mass Spectrometry > Post-translational modification > Flashcards

Post-translational modification Flashcards

1
Q

What is biological complexity linked to?

A

Gene products

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2
Q

PTM

A

Chemical modifications of a protein after its translation

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3
Q

How many different types of PTMs are there

A

200

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4
Q

How do PTMs alter proteins

A

Alter localisation, turnover, activity state and interaction with other proteins

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5
Q

Mod form

A

Modified form of the protein (2 to the power of the number of PTM sites)

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6
Q

2 most abundant forms of PTM

A

Phosphorylation and glycosylation

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7
Q

What AAs can be phosphorylated

A

Ser, thr, tyr

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8
Q

BK channel

A

Large conductance voltage- and Ca2+-gated K+ channel

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9
Q

What is the structure of the BK channel

A

Pore forming alpha subunits and modulatory beta subunits (4 alpha subunits assemble to make functional channel)

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10
Q

What is BK channel involved in

A

Neuronal firing and neuropeptide release

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11
Q

How is the BK channel activated

A

Alcohol activates - increases K+ channel - causes reduced neuronal excitability and neuropeptide release - leads to depression of central neurons, motor incoordination, diuresis

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12
Q

What happens to BK channel when repeated exposure to alcohol

A

Phosphorylation of T107 by CAMKII kinase - switches channel alcohol response from activation to inhibition - molecular dimmer switch - mediate alcohol tolerance

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13
Q

How to detect phosphorylation

A

Phosphospecific stain, Pro-Q Diamond, 32P or 33P labelling and autoradiography), western blotting with phospho-T, S, Y antibodies

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14
Q

Phosphoproteomics enrichment

A

Immunoprecipitation, affinity chromatography, chemical derivitisation

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15
Q

Immunoprecipitation

A
  1. Immobilised pS, pT, pY antibodies 2. Immobilised antibodies against kinase consensus sites
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16
Q

Affinity chromatography

A
  1. Immobilised IMAC (immobilised metal ion affinity chromatography) beads 2. Immobilised TiO2 beads 3. SIMAC - peptide mix + IMAC beads
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17
Q

Chemical derivitisation

A
  1. Beta-elimination 2. PAC - phosphoramidate chemistry
18
Q

Why are sugars important?

A

Act as recognition molecules

19
Q

What is the name of the sugar coat that all cells have

A

Glycocalyx

20
Q

What is key for cell-to-cell communication

A

Glycan-lectin recognition

21
Q

How is there variation in glycosidic bond formation

A

Orientation of planes (alpha or beta)

22
Q

What enzyme aids glycosidic bond formation

A

Glycosyltransferase (link to hydrolysis of phosphate bond)

23
Q

What are the 2 forms of glycosylation

A

N- and O-linked

24
Q

N-linked glycosylation

A

Sugar linked to amide nitrogen in the side chain of asparagine

25
Q

O-linked glycosylation

A

Sugar linked to oxygen in the side chain of serine or threonine

26
Q

N-glycosylation consensus sequence

A

Asn-X-Ser/Thr (X = any AA except proline)

27
Q

What type of modification is N-linked glycosylation

A

Co-translational modification

28
Q

How is N-linked glycosylation initiated

A

In ER by en bloc transfer of pre-formed lipid anchored conserved glycan

29
Q

What are the two sections of the pre-formed lipid anchored conserved glycan?

A

Dolichol (lipid), core, antennae

30
Q

N-glycan biosynthesis

A
  1. Synthesis of lipid linked precursor oligosaccharide 2. En bloc transfer to protein 3. Processing
31
Q

Types of N-linked glycans

A
  1. High mannose N glycans 2. Hybrid N-glycans 3. Complex N-glycans
32
Q

What sugars are antennae capped with and why are they capped

A

Fuc or NeuAc to form ligands for specific recognition by lectins

33
Q

O-glycosylation consensus sequence

A

No consensus sequence but some rules e.g. nearby proline, tandem repeats of Ser/ Thr

34
Q

How is O-glycosylation initiated

A

In golgi by addition of single sugar (usually GalNAc)

35
Q

How are O-glycans classified

A

By their core structure (8 known)

36
Q

What are the most common O-glycan core structures

A

Cores 1 and 2

37
Q

What O-glycans are most common on mucins

A

Cores 1 - 4

38
Q

What are mucins

A

Cell surface and excreted glycoproteins - protect mucus membranes by keeping them hydrated, acting as lubricants and prevent microorganism invasion

39
Q

What are O-GlcNAc proteins

A

Type of glycosylation localised in cytoplasm or nucleus - they are phosphoproteins - dynamic

40
Q

What are proteoglycans

A

Special type of O-glycoprotein found in ECM and structural tissues (e.g. cartilage) which form massive molecular aggregates (act as shock absorbers)

41
Q

What is a GPI-anchor

A

Glycosylphosphatidylinosotol-anchors. Protein anchored to membrane via diacylglycerol glycolipid.

Mass Spectrometry (11 decks)

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