Proteomics Steven Meinhardt Lectures Lecture 1 Introduction - - PDF document

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Proteomics

Steven Meinhardt

Lectures

• Lecture 1

Introduction – review of proteins

• Lecture 2

Gel Electrophoresis

• Lecture 3

Mass Spectrometry

• Lecture 4

Programs for MS analysis

• Lecture 5

MIAPE (Minimum Information About a Proteomics Experiment)

• Lecture 6

Protein-Protein Interaction

• Lecture 7

Proteome experiments

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Review of Proteins

• Composition and Structure

– Amino Acids

• 20 common amino acids
• Other amino acids

– Other components

• Cofactors

– Metals Hemes vitamins (cobalmin) – Others

– Modifications

Common Amino Acids

There are 20 common amino acids Name Three Single Name Three Single Letter Letter Letter Letter Code Code Code Code Alanine Ala A Methionine Met M Cysteine Cys C Asparagine Asn N Aspartate Asp D Proline Pro P Glutamate Glu E Glutamine Gln Q Phenylalanine Phe F Arginine Arg R Glycine Gly G Serine Ser S Histidine His H Threonine Thr T Isoleucine Ile I Valine Val V Lysine Lys K Tryptophan Trp W Leucine Leu L Tyrosine Tyr Y Asp or Asn B Glu or Gln X

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Protein Modification

• Modified amino acids (15)

– (A, C, D, E, H, K, L, M, N, P, R, S, T, V, W, Y) – Hydroxylation (C, D, I, K, L, N, P, V, W) – Glycosylation (S, T, N, (OH)P) – Sulfination (S, T, Y) – Phosphorylation (S, T, Y) – Oxidation Methionine sulfoxide, sulfone (C) – Selenocysteine (U)

• Browse by modification
• http://dbptm.mbc.nctu.edu.tw/browse.php

Proteomics volume 4 Issue 6 , Pages 1525 - 1853 (June 2004) http://www.ebi.ac.uk/RESID/ 532 different amino acid modifications. (2010)

Amino Acid Modification

–Acetylation (n-terminal and K or C)

• Cyclization of N-terminal glutamine to make

pyroglutamate

–Fatty acid addition (K, C, N- and C-terminus) –ADP-Ribosylation (H, N, R, and K) –Disulfide bond formation (C) –Methylation (A, C, E, F, H, K, M, N, P, Q, R)

• N-terminal phenylalanine and C-terminal C

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Protein Modification

• Ubiquitin or SUMO addition

– Ubiquitin signals degradation

• Ubiquitin (76aa, 8.5 kDa) is transferred to lysine in target

protein

• Degradation occurs in proteosome

– SUMO signals retention (Small Ubiquitin like Modifier

• SUMO (~12 kDa) attaches to lysine in target
• Helps control trafficking
• consensus motif Ψ-K-x-D/E

(Ψ is hydrophobic)

• Proteolytic processing

– SUMO (activated by removal of last 4 amino acids) – Insulin

Cofactors

• Metals

– Fe, Cu, Mo, Zn, Mn, Mg, Co, W, Se – Other molecules S, I

• “Small” Organic Molecules

– AMP ADP ATP cyclic-AMP – GMP GDP GTP cyclic-GTP – Coenzyme A Coenzyme Q Vit K RNA – FAD+ or FADH2 NAD+ or NADH – Porphyrins (Hemes and chlorophyll) – FeS clusters – Cobalamin Thiamine pyridoxine folic acid

• Others

Allosteric regulatory molecules

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Protein Structure

• Some portions of proteins are cleaved.

– Signal sequence

• Excretion, mitochondria, chloroplast, golgi,

nucleus

– Prosequence – Internal sequence

• Mature protein – final protein after

cleavages and or modification, usually functional

Protein Structure

• Primary (amino acid sequence)
• Secondary

– Helix, beta turn, parallel and antiparallel beta sheet. – Triple Helix

• Tertiary

– Proteins fold into common structures

• All  and the four helix bundle
• -barrel

-clamshell

• helix-turn-helix
• E-F Hand
• Quarternary

– Multisubunit structures

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Examples of structures Common  helical structures

4 helix bundle Coiled-coil EF Hand

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Function

• What do proteins do?
• Things that affect that function.

– Location

• Proteins move
• Function changes with location
• Tissue specificity
• Soluble vs membrane bound

– Modifications – Association with other proteins – Expression (temporal, spatial, amount)

Protein Regulation

• Transcription
• Translation
• Proteolysis

– Activiation – Ubiquitin – Small Ubiqitin-like Modifier (SUMO)

• Phosphorylation
• Dissociation
• Binding of signal, substrate, product,

etc.

• Concentration

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Basic Proteomics Information

• Composition
• Structure
• Function
• Regulation
• Location
• Interactions
• Quantity