Introduction to Proteomics University of Missouri-Columbia Jay Thelen Ph.D., Assistant Professor Department of Biochemistry

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Introduction to ProteomicsUniversity of
Missouri-ColumbiaJay Thelen Ph.D., Assistant
ProfessorDepartment of Biochemistry
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Outline

• Introduction to proteomics and protein separation
• Contemporary mass spectrometry as applied to
  proteomics

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Why study proteins?

• DNA ? mRNA ? protein ? phenotype

• - Genomics provide indirect evidence for the
  existence and expression of proteins
• Protein stability, turnover, post-translation
  modifications all influence protein expression
  and activitythus predictions from DNA/RNA
  research are frequently invalid

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What is Proteomics?
..the large-scale study of proteinswhile it is
often viewed as the next step, proteomics is
much more complicated than genomics. while the
genome is a rather constant entity, the proteome
differs from cell to cell and is constantly
changing through its biochemical interactions
with the genome and the environment. One
organism will have radically different protein
expression in different parts of its body, in
different stages of its life cycle and in
different environmental conditions.
Wikipedia, http//en.wikipedia.org
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ANIMALS
PLANTS
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What is Proteomics?

• Multidisciplinary field of research
• biology (research question)
• protein biochemistry (protein isolation,separation
  ,quantification)
• analytical chemistry (protein identification
  using mass spectrometry)

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Challenges associated with proteomics

• Dynamic range of protein expression in a cell is
  1 x 109
• Alternative splicing and post-translational
  modification increases the number of protein
  species 5-6-fold
• - 30,000 genes in Arabidopsis, gt 150,000
  proteins species
• 3) Certain classes of proteins are
  under-represented in proteomic investigations
  using conventional techniques
• - Membrane, highly basic/acidic and low MW
  proteins

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Tools to study proteins

• Protein separation
• one-dimensional polyacrylamide gel
  electrophoresis
• two-dimensional (2-D) gel electrophoresis
• liquid chromatography (HPLC, capillary, FPLC)
• Protein identification
• Edman degradation
• mass spectrometry

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Approaches for protein separation

• One-dimensional polyacrylamide gel
  electrophoresis (SDS-PAGE)

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• One-dimensional polyacrylamide gel
  electrophoresis (SDS-PAGE)

MW high low
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• Two-dimensional gel electrophoresis

1st dimension - isoelectric focusing 2nd
dimension - SDS-PAGE
Most widely used protein separation technique in
proteomics Capable of resolving thousands of
proteins from a complex sample (i.e. blood,
organs, tissue)
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Two-dimensional gel electrophoresis

• 1st dimension - isoelectric focusing
• -separation of proteins according to native
  charge.

native charge
isoelectric point -pH at which net charge is zero
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Two-dimensional gel electrophoresis
pI
3
10
6.5
kDa
100
75
mass
50
25
plant leaves
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New variation of 2-D gelsDifference Gel
Electrophoresis (DIGE)

• Differential Gel Electrophoresis (DIGE)
• - samples resolved in the same gel
• - simplifies spot matching

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Difference Gel Electrophoresis (DIGE)
Cy5
Cy3y3
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Techniques for identifying proteins using mass
spectrometry
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What is mass spectrometry ?
Mass (molecular weight) determination of sample
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(No Transcript)
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Basics of mass spectrometry

• determination of mass to charge ratio (m/z)
• components of MS
• - ionization and entrance to gas
• phase
• - mass analysis

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Ionization - giving an electric charge to a
neutral species
M H MH M Na MNa M H M-H-
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Soft-ionization methodsfor proteomics

• MALDI
• electrospray/nanospray

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MALDI Matrix AssistedLaser Desorption
Ionization

• analyte is dried with matrix on target
• matrix CHCA, DHB, sinapinic acid
• generates predominantly singly-charged ions
• fragmentation dependent on laser power, choice of
  matrix, modification of peptide

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MALDI Matrix AssistedLaser Desorption
Ionization
laser
M
A
M
A
M
M
A
M
M
M
M
A
M
M
A
M
A
M
M
M
M
A
A
A
M
M
M
A
A
M
M
M
M
M
M
A
M
A
M
(ions and neutrals)
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Mass Analyzers Time of Flight (TOF)
Ion Source
Detector
Flight tube
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MALDI-TOF mass spectrometers
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How does mass spectrometry assist Proteomics
research ?
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Peptide mass fingerprinting
intact protein
defined protease digestion (e.g. trypsin
cleaves after K,R residues)
peptides
mass spectrometry
mass
952.0984 1895.9057 1345.6342
899.8743 2794.9761
intensity
m/z
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2319.1430
1.6E4
100
1950.1408
1108.6889
90
80
70
60
Intensity
50
1142.7207
1637.9620
40
2457.2961
3313.7365
634.3435
2301.1896
1768.9952
30
999.6277
2096.2165
20
1713.9404
1654.9660
1019.6834
3117.5553
2332.2139
4325.9686
1558.9500
10
2008.1049
1525.9309
1076.6995
2642.5282
3328.7509
606.3184
1993.1114
2808.5225
1621.8725
0
0
499.0
1599.4
2699.8
3800.2
4900.6
6001.0
Mass (m/z)
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Prospector or Mascot
2-D gel
candidate protein list
Voyager DE-PRO MALDI-TOF
96-well plate
MALDI plate
In-gel digestion