Transcript Separation

Introduction to Proteomics
Dr. Ahmed A. Mirza
MLT, FAM, KAU
Objectives
• Protein expression review
• What is proteomics
• Basic methods in protein
biochemistry
• Protein extraction
– Physical force
– enzymatic
• Separation
– Chromotography
•
•
•
•
Size exclusion
Ion exchange
Affinity
hydrophobicity
Analysis
Semi-Quantification
Gel electrophoresis
2D gel electrophoresis
Differential expression
Mass Spectrometry
Separation
Ionization
Identification
Quantification
microarrays
Proteomics
Definition
Application
What is Proteomics??
Proteomics
Proteomics
The proteome is larger than the genome because of
alternative splicing and post-translational protein
modification (e.g., glycosylation)
Important to find out about:
• Protein modification
• Location
• All protein-protein interactions.
• Protein or peptide that have multiple functions
depending on time or place of expression.
• Regulation of protein function.
• Detection and quantification
•
protein concentration may changes by 10 folds.
Gene expression… review
Basic methods in protein
biochemistry
• Protein Extraction:
– Physical force
• Tissue grinder (physical
force)
• Sonication (burst open
cells due to high frequency
sound waves)
– Enzymatic digestion
• Lysozyme (break
open the cells by
poking holes in the
cell membrane)
Protein separation
– More than one level
• Fractionate by organelle (mitochondria, Golgi, etc)
(using centrifugation)
• Fractionate by protein (size, charge, affinity) using
chromatography:
–
–
–
–
Size exclusion
Ion exchange
Affinity
Hydrophobic interaction
Size exclusion
chromatography
Separates proteins
according to their sizes.
Smaller proteins go
through the porous gel
beads, which delays their
movement.
Affinity Chromatography
A Lock and key model that binds STRONGLY proteins with affinity to ligand on the beads
Ion exchange Chromatography
Proteins away from their
isoelectic point are charged.
Accordingly, charged
proteins can bind to beads of
the apposing charges.
Hydrophobic Interaction
Chromatography (HIC)
Analysis
Polyacrylamide Gel Electropharesis
(PAGE)
–
–
–
–
–
Different concentration of acrylamide gels
Proteins are negatively charged
Move toward the positive electrode according to size
Smaller proteins travel furthest
Can be visualized by several types of stains with
varying sensitivity (coomassie blue, silver)
– Can be semi-quantitative
– Can be native or denatured using Sodium Dodecyl
Sulfate (SDS-PAGE) – more common
– The smaller the protein size – the higher acrylamide
concentration used
2D gel Electropharesis
Two steps:
• Isoelectric focusing (IEF)
– This separates proteins based on isoelectric point
– The isoelectric point is the pH at which the protein has no
net charge.
– pH gradients may be large 2-10 or small 6-7
– gel strip (tube) with a pH gradient containing a low
concentration of polyacrylamide.
– Ampholytes are added to create a pH gradient in an
electric field and the proteins are loaded.
– The IEF gel is placed in an electrophoresis system for up to
24 hours
• PAGE
– The second dimension separates the proteins
based on size.
– The IEF gel is soaked in a solution containing
chemical to denature the proteins including
sodium dodecyl sulfate (SDS) a detergent which
gives the proteins a net negative charge..
– The larger proteins are found at the top and the
smaller ones are found at the bottom of the gel.
Differential Expression
Different types and/or levels of proteins are expressed
under different conditions can be easily seen
Excise the spot/band of interest…
Protein Sequencing
two methods:
• Mass Spectrometry
• Edmen degradation
– Can break the one
terminal peptide
bond at a time
without disrupting
the rest
Mass Spectrometry
http://www.youtube.com/watch?v=tOGM2gOHKPc
http://www.youtube.com/watch?v=J-wao0O0_qM
• Mass Spectrometry is a technique for the
detection and resolution of a sample of
ions by their mass-to-charge ratio represented by m/z where m is the mass
in Daltons and z is the charge.
• Samples are isolated, digested or not,
then is analyzed to determine exact AA
sequence MW.
Shotgun (bottom-up) proteomics
LNDLEEALQQAKEDLAR
NKLNDLEEALQQAK
NVQDAIADAEQR
SKEEAEALYHSK
SLVGLGGTK
TAAENDFVTLK
TAAENDFVTLKK
TSQNSELNNMQDLVEDYK
TSQNSELNNMQDLVEDYKK
VDLLNQEIEFLK
YEELQVTVGR
YLDGLTAER
ADLEMQIESLTEELAYLK
ADLEMQIESLTEELAYLKK
AETECQNTEYQQLLDIK
LNDL
EEAL
QQAC
EDLA
R
N
KLND
LEEAL
QQAK
Proteins
Separation Digestion Peptides Separation
SDS-PAGE
SCX
Antibody-based
High pH RP LC
approaches
Low pH RP LC
Analysis
MS-MS/
Tandem MS
Peptide IDs
+ Quantitation
Most modern proteomic MS is ‘bottom-up’
IPI:IPI00000073.2
IPI:IPI00217963.3
IPI:IPI00031065.1
IPI:IPI00376379.4
IPI:IPI00397801.4
IPI:IPI00009950.1
IPI:IPI00395488.2
IPI:IPI00295414.7
IPI:IPI00554711.3
IPI:IPI00009867.3
IPI:IPI00019449.1
IPI:IPI00016915.1
IPI:IPI00060800.5
IPI:IPI00013885.1
IPI:IPI00221224.6
Observed Proteins
+ Quantitation
Amino Acid Masses
Amino acid
G
Mass(avg)
57.0520
Amino acid
D
Mass(avg)
115.0886
A
71.0788
Q
128.1308
S
P
V
T
C
I
L
N
87.0782
97.1167
99.1326
101.1051
103.1448
113.1595
113.1595
114.1039
K
E
M
H
F
R
Y
W
128.1742
129.1155
131.1986
137.1412
147.1766
156.1876
163.1760
186.2133
Peptide Fragmentation
88
S
1166
145
G
1080
292
F
1022
405
L
875
534
E
762
663
E
633
778
D
504
907
E
389
1020
L
260
1166
K
147
b ions
y ions
% Intensity
100
0
250
500
750
1000
m/z
Peptide mass fingerprinting
• is an analytical technique for protein
identification in which the unknown protein of
interest .
• The peptide masses are compared to either a
database containing known protein sequences
or even the genome.
• The results are statistically analyzed to find
the best match.
Microarrays
a)
b)
c)
d)
e)
f)
antigen-antibody interactions
sandwich immunoassays
protein-protein interaction
Nucleic Acid-protein interaction
receptor-ligand interaction
enzyme-subrates specificity
•
Analytical microarrays:
• Various molecules of interest (Ag, ab, DNA, RNA, …)
are arrayed on a chip.
• Unknown protein samples testing two different
biological states are labeled with two colors and
incubated with the chip and bind to a specific pattern
accordingly.
•
Functional microarrays:
• individual proteins are purified and arrayed on a chip
• Other proteins can be incubated with the chip to
determine protein-protein interactions and posttranslational modifications among other uses.
Why should we care?
Proteomics Application
Assignment
• Write one full page description of two clinical
uses of Proteomics.
References
All resources and links below were last accessed on 12/09/2014
• Mass Spectroscopy, Bhavana Vedantam.
http://www.slideshare.net/mnmnmknmjjk/mass-spectroscopy-28595894?related=2
• Proteomics, Dimitri Raptis. http://www.slideshare.net/dimitriraptis/proteomics6887316
• Proteomics 101, http://www.slideshare.net/many87/proteomics-101?related=1
• Application of Proteomics Workflows to Biomedical Research. Karen Jonscher.
http://www.ucdenver.edu/academics/colleges/medicalschool/programs/bioanalytics
/education/Documents/Lecture%201.pdf
• Proteomics, Ruchi Yadav.
http://www.slideshare.net/ruchibioinfo/proteomics?related=2
• Proteomics Processes and Applications. Khalid Hakeem.
http://www.slideshare.net/khalidhakeem/proteomics-processes-andapplications?related=3
• Introduction to Proteomics. Phil Charles.
http://www.well.ox.ac.uk/dtc/bioinformatics_course/IntroductionToProteo
mics.pptx
•
Proteomics PPT.
http://www.ndsu.edu/pubweb/~mcclean/plsc411/proteomics%20lecture%202b.ppt