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Proteins
Sugar Chain
2
GENOME
Transcriptome
Nucleus
Nuclear
Proteome
membrae
ER ProteomeMitochondria
Golgi
an
br
m
Vesicles
Me
e
Chloroplasts Vacuole
mRNA
Re
2D electrophoresis
g
•
ula
Protein sequencing
ion
• Translational fusional
Proteins (Proteome) • Immunodetection
• Enzyme activities
Metabolites
(Metabolome) • Chromatography
• Mass spectrometry
• NMR
09.01.2003
Snapshot of the human genome
•Only 1.1% to 1.4% of
human genome DNA
actually encodes
proteins.
6
Protein functions
GFP consists of 238 amino acids, linked together in a long chain. folds up into the
shape of a beer can. Inside the beer can structure the amino acids 65, 66 and 67
form the chemical group that absorbs UV and blue light, and fluoresces green.
(Credit: Image courtesy of Nobel Foundation)
Classification of proteins by posttranslational modification
After protein translation some are subjected to posttranslational
modification.
12
Classification of Amino Acids by Polarity
pK2 ~ 9
+1 0 -1
Isoelectric point
Juang RH (2004) BCbasics
Amino Acids Have Buffering Effect
pH 12
★ pK2
9
NH2 H+
6 H-C-R Isoelectric point = pI
COO- pK1 + pK2
3 ★ 2
pK1
0
[OH] →
Juang RH (2004) BCbasics
H first Aspartic acid
HOOC-CH2-C-COOH +1
NH3+ Isoelectric point is the average
pK1 = 2.1 of the two pKa flanking the
zero net-charged form
second H
2.1 + 3.9
HOOC-CH2-C-COO- 0 2
= 3.0
NH3+
Isoelectric point
pK2 = 3.9
H -2
-
OOC-CH2-C-COO- -1 pK3
NH3+ third -1
pK2
pK3 = 9.8 0
H pK1
+1
-
OOC-CH2-C-COO- -2 [OH]
NH2
Juang RH (2004) BCbasics
pKa of Amino Acid Residues
Pengikatan
substrat dan
koenzim
Terjadi
katalisis
Lactate
dehydrogenase
Phospho
fructokinase
Three Dimensional Structure
3-D structure very important in understanding function
of protein
2 major methods:
› X-ray crystallography
can be used for any size protein or even a huge
macromolecular complex the size of a ribosomal
subunit (many proteins + RNA molecules) IF the
molecule or complex can be crystallized
(combination of scientific skill, art, and luck!)
› NMR (currently only useable for total structure
determination for small proteins, < ~25 kilodaltons)
more than 10,000 structures have been determined,
most in the last decade as new, more powerful
instruments have become available
X-ray Crystallography
• Electrons in crystal scatter x-rays to produce an image.
• Fourier transformation is used to convert raw data into 3-D
structure.
Two protein with similar structure but different functions
Lysozyme Alfa lactalbumin
pMAL™
(Protein
Fusion &
Purification
(pMAL™)
System)
MBP
Maltose
Binding
Protein
- separation of proteins by size in polyacrylamide gel
Zymogram Analysis of Protease Derrived
from Random DNA from the Soil
94.0 kD
74 kD
67.0 kD
56 kD
43.0 kD 45 kD
30.0 kD
20.1 kD
14.4 kD
Detection of selected protein on Western blot
Protein electrophoresis (SDS-PAGE)
Detection by antibodies
(primary = interacts with the protein
secondary = interacts with primary antibodies
from certain species
W. blot
Vizualization by enzymatic reaction
(color precipitate, chemiluminiscence)
- 200 kD
SDS-PAGE
+ 20 kD
Yarmush &
Generalized proteomics scheme
Jayaraman
, 2002
65
Staining of Polyacrylamide Gels
66
Clinical applications of 2-D electrophoresis
Body fluids Solid tissue
› Blood cell › Heart
› Plasma and serum › Brain
› Urine › Thyroid
› Cerebrospinal fluid › Muscle
› Amniotic fluid Malignant diseases
› Synovial fluid Tissue culture
› Saliva Malignant cells
› Sweat Bacterial proteins
› Tears
› Semen
Protease
69
Determination of primary structure
chemical methods
› amino acid composition
› amino terminal residue determination
› Edman degradation
› fragmentation and determination of overlapping
fragment sequences
› mass spectrometry (useful in many other ways,
too, e.g. for identifying proteins, even in complex
mixtures)
EDMAN DEGRADATION
Edman degradation can be used to sequence
proteins rapidly, and with little starting
material.
The process is easily automated, and
sequencers can operate sometimes with as
little as a few picomoles of sample.
The machine performs the coupling, cleavage,
conversion and identification steps for each
amino acid residue, and researchers often
choose to identify 10-15 residues from the
protein.
The protein may then be identified by
searching a database to match the sequence
and properties of the protein.
PROTEIN SEQUENCING STRATEGY
8 STEPS
Sample Analyzer
input Ionization Detector
85
The basic components of a mass spectrometry system
Sample Input:
Gas Chromatography (GC), Liquid Chromatography
(LC), Capillary Electrophoresis (CE), Solid crystal
etc.
Ionization:
Electrospray, Matrix-assisted Laser Desorption /
Ionization (MALDI) etc
Analysis:
Quadrupole, Time of Flight (TOF), ion trap etc.
Detection:
Electron multiplier, Scintillation counter
87
Two important methods are used to create protein ions:
89
MALDI-TOF
Laser
displaces
sample into
ionization
chamber.
Ions travel
through
electrical
field.
Heavier
ions travel
more slowly.
Protein identification by „peptide fingerprinting”
Protein digestion (by trypsine)
protein I (12 kD)
DNA (i EST), protein database
2 fragments (4 kD, 8 kD)
MALDI ToF
protein I
protein a: 3, 5, 9, 12 kD
protein b: 2, 7, 9 kD
4000 8000 m/z protein c: 4, 8 kD
protein d: 3, 9, 12 kD
protein e: 2, 6, 8 kD
protein II
Trypsin *
*
*
* * *
*
92
94
MALDI-TOF mass spectrum of the in-gel trypsin digest of a 2D gel spot that is identified
as 130 KDA LEUCINE-RICH protein.
After accurate determination of the peptide masses, databases are searched for
identification of the original proteins. Identification requires a minimum of four peptide
matches and the highest probability ranking in at least two of the search programs
95
employed.
Preferential fragmentation in peptide bond
MALDITOF-TOF spectrum of human β-endorphin (m/z 3465.4 selected) obtained
under optimized experimental conditions. The y- and b-ions detected are listed The
peak donated by an asterisk is the b12 ion, the complementary of the y19 ion
Typical mass spectrometry scheme
peptide mass fingerprint & tandem mass spectrometry
100
A tandem mass spectrometer sequences peptides by
generating gas-collided fragments derived from the
peptides,
101
Serial dichromatic detection of glycosylated
and un-glycosylated proteins
Pro-Q Emerald 488 glycoprotein stain kit SYPRO Ruby protein gel stain
103