5. Proteomics Research
•Basic research:
To understand the molecular mechanisms
underlying life.
•Applied research:
Clinical testing for proteins associated with
pathological states (e.g. cancer).
7. For example: Hemoglobin
Picks up oxygen in the lungs, travels through
the blood, and delivers it to the cells.
O2
hemoglobin
Hbβ Hbα
Hbβ
Hbα
8. ATG GTG CAC CTG ACT CCT GAG GAG … ATG GTG CAC CTG ACT CCT GTG GAG …
E
E
M V H L T P … E
V
M V H L T P …
Normal Hbβ Mutated Hbβ
Sickle cell disease is caused
by a single amino acid change.
9. Summary – what is proteomics?
•Involves the study of proteins
•Proteomics is multidisciplinary
•Proteomics is being applied to both basic and clinical
research
13. Proteins are comprised of amino
acid building blocks
R
O
OH
C
N H
H
Acid
Base
Variable
CH
+
H2O
Dipeptide
Peptide Bond
Amino acid 1 Amino acid 2
R1
O
C
C
N O
H
H2 H
R2
H
C
C
N O
O
H
H H
R1
O
C
C
R2
O
C C
H
H
N
H2
N
H OH
14. Summary – why study proteins?
•Biological workhorses that carry out most of the
functions within the cell
•Serve diverse functional and structural roles
•Composed of amino acids that are covalently
linked by peptide bonds
•Synthesized during the translation process
•Must fold correctly to perform their functions
18. How are proteins isolated?
• Mechanical Methods
– grinding – break open cell
– centrifugation – remove insoluble debris
• Chemical Methods
– detergent – breaks open cell compartments
– reducing agent – breaks specific protein
bonds
– heat – break peptide bonds to “linearize”
protein
19. Protein isolation procedure
Find a sample
Pick it
Grind sample in buffer
Transfer to tube
Heat the sample
Centrifuge to remove
insoluble material
“pure” protein
solution
Recover supernatant Keep solution for gel analysis
21. Summary – protein isolation
•Proteins can be isolated from a variety of samples
•Proteomics includes the use of both mechanical and
chemical methods to isolate proteins
•Opening cell or cellular compartments
•Breaking bonds and “linearizing” proteins
•Removal cell debris
23. Why separate proteins?
“PURE” Protein Solution
Tube 1
Decreased Protein ID
Increased Complexity
Tube 2
Increased Protein ID
Decreased Complexity
24. How to separate proteins?
Separating intact proteins is to take
advantage of their diversity in
physical properties, especially
isoelectric point and molecular weight
25. Methods of Protein Separation
• Sodium Dodecyl Sulfate –
Polyacrylamide Gel Electrophoresis
(SDS-PAGE)
• Isoelectric Focusing (IEF)
27. SDS-PAGE separates only by
molecular weight
• Molecular weight is mass one molecule
• Dalton (Da) is a small unit of mass
used to express atomic and molecular
masses.
28. PAGE is widely used in
• Proteomics
• Biochemistry
• Forensics
• Genetics
• Molecular biology
29. Polyacrylamide gels separate
proteins and small pieces of DNA
• Major components of polyacrylamide gels
• Acrylamide – matrix material/ NEUROTOXIN
• Bis-acrylamide - cross-linking agent/ NEUROTOXINS
• TEMED - catalyst
• Ammonium persulfate - free radical initiator
34. During SDS-PAGE proteins separate
according to their molecular weight
Bromophenol
Blue dye front
Cathode (-)
Anode (+)
Standard Sample1 Sample2
20 kDa
100 kDa
75 kDa
50 kDa
37 kDa
25 kDa
150 kDa
36. Separation of Protein X
Bromophenol
Blue dye front
Cathode (-)
Anode (+)
Standard Sample1 Sample2
20 kDa
100 kDa
75 kDa
50 kDa
37 kDa
25 kDa
150 kDa
Protein X
11 kDa
25 kDa
37. Two-dimensional gel
electrophoresis (2-DGE)
Most widely used protein separation technique in
proteomics
Capable of resolving thousands of proteins from a
complex sample (i.e. blood, organs, tissue…)
1st dimension - isoelectric focusing
2nd dimension - SDS-PAGE
38. Isoelectric focusing (IEF) is separation of
proteins according to native charge.
isoelectric point -pH at which net charge is zero
1st Dimension-Isoelectric
Focusing
42. 1-DGE vs. 2-DGE
1-DGE (SDS-PAGE)
• High reproduciblity
• Quick/Easy
• Separates solely based
on size
• Modest resolution,
dependent on complexity
of sample
2-DGE
• Modest reproducibility
• Slow/Demanding
• Separates based on pI and
size
• High resolution, not
dependent on complexity
of sample
43. Summary – protein separation
•Protein separation takes advantage physical
properties such as isoelectric point and molecular
weight
•SDS-PAGE is a widely used technique to separate
proteins
•1-DGE is a quick and easy method to separate protein
by size only
•2-DGE combines isoeletric focusing (IEF) and SDS-
PAGE to separate proteins by pI and size
45. Peptide mass
fingerprinting
intact protein x
protein digestion
mass spectrometry
m/z
intensity
952.0984
1895.9057
1345.6342
899.8743
2794.9761
mass
Protein ID
Make proteolytic peptide
fragments - Digest the
protein into peptides (using
trypsin)
Measure peptide masses -
“Weigh” the peptides in a
mass spectrometer
Match peptide masses to
protein or nucleotide
sequence database - Compare
the data to known proteins
and look for a match
46. Protein digestion
We use the enzyme TRYPSIN to digest (cut) proteins
into peptides – trypsin cuts after Lysine (K) and
Arginine (R)
????????K?????R????????
????????K?????R????????
????????K?????R????????
Protein X
????????K?????R????????
????????K?????R????????
????????K?????R????????
47. How does mass spectrometry
identify unknown proteins?
48. Basics of mass spectrometry
• determination of mass to charge ratio
(m/z)
• Mass spectrometer = very accurate
weighing scales
– third or fourth decimal place
55. The unknown peptides have been
identified
?????R = 692.31 Da
????????K = 1106.55 Da
???????? = 1002.37Da
WEGETMILK
ASTEER
MANYCQWS
56. Protein X has been identified
????????K?????R????????
????????K?????R????????
????????K?????R????????
WEGETMILK AFTEER MANYCQWS
57. Summary – tools to study proteins?
•Proteins are digested into peptides
•Peptides are analyzed with a mass spectrometer
•Match observed peptide masses to theoretical
masses of all peptides in database
•Assemble those peptide matches into a protein
identification
58. Concluding points about Proteomics
-Proteomics is the analysis of all proteins
-Interdisciplinary research
-Essential to both basic and clinical research
-Protein are the workhorses of the cell
- Discovery research – drugs and diseases
-Proteomics tools allow identification of proteins
