Proteomics. Sarfaraz Hussain Nasri. GCU-Faisalabad

1. SARFARAZ HUSSAIN
Department of Bioinformatics & Biotechnology
GCU-Faisalabad.

2. Introduction
Techniques
Techniques
Applications
Databases
Limitations

3. Proteomics is the branch of molecular biology
concerned with the study of proteome.
Proteomics is a quite recent field. The term
proteomics was introduced in 1994.

4.  to study the structure and function of protein
 To study the 3D structure of protein
 Study of qualitative and quantitative analysis
of proteins.

5.  Structural proteomics
Helps to identify newly discovered genes and
drug interaction
 Expression proteomics
Helps to identify the main gene in a particular
sample

6.  Interaction proteomics
It the pathway in which proteins combined in
large complexes
Advantages of study of proteomics
 Shows that genetic alterations are not the reason
for all types of diseases
 Helps in determining the proper treatment of
diseases

7.  With the help of three dimensional analysis of
proteins we have found that HIV protease is
the enzyme which is responsible for AIDS.
 One of the most important use of proteomics
in diagnosis is the identification of
biomarkers. The study of drugs in proteomics
is called pharmacoproteomics.

8.  The study of proteins is very complex
because the concentration of protein is
different in each organism and in each
cell of the organism.

10.  Proteomes of interest, such as the human
proteome have such complexity that no
single technique is adequate for complete
analysis of the constituents.
 There are so many techniques for
identification and analysis of the proteins in
proteomics such as
Matrix-assisted Laser Ionization etc.

11.  It is a form of electrophoresis which is
commonly used to analyze proteins.
 In this technique proteins are extracted from
chosen specimen .
 Software packages include BioNumerics2D,
Delta2D, Progenesis and PDQuest for
quantification and detection of particular
protein resolved on a gel.

12.  PROTOMAP stands for “Protein Topography and Migration Analysis
Platform”.
 Protomap a proteomic technique used for characterizing protolytic
events using mass spectrometry.
 Protomap was invented and developed by Ben Cravatt and
colleagues at The Scripps Research Institute.

13.  To perform a protomap analysis proteins are
separated via 1D-SDS-PAGE.
 Protomap is a recently developed proteomic
technique for identifying changes to proteins that
manifest in altered migration by one- dimension
SDS-PAGE.
 It is similar conceptually to two- dimension gel
electrophoresis.

14.  Protomap is performed by resolving control and experimental
samples in separates lanes of a 1D-SDS-PAGE gel.
 Each lane is cut into evenly spaced band (usually 15-30 bands).
 Information from all of these bands are bioinformatically integrated
into a visual format called a peptograph.

15.  Which plots gel migration in the vertical dimension (high
to low molecular weight, top to bottom)
 Sequence coverage in the horizontal dimension (N- t0 C-
terminus, left to right).

17.  A peptograph is generated for each protein in the sample.
 This data format enables rapid identification of proteins undergoing
proteolytic cleavage.

18. Introduction
 MS/MS plays important role in
protein identification (fast and
sensitive)
 Derivation of peptide sequence an
important task in proteomics
 Derivation without help from a
protein database (“de novo
sequencing”), especially important
in identification of unknown protein

19. • Sours ionized the
sample
• Analyzer separate the
ions on m/z ratio
• Detector sees the
ions and
analyzed the result

20. • Isolate cell or other protein source
• Lyses cells and isolate proteins
•Break up proteins into smaller (but still
relatively large) amino acid chains
• Separate chains (2D gel, gas or liquid
chromatography)
•Analyze separated protein parts by mass
spectrometry

21.  Proteins consist of 20 different
types of a. a. with different
masses (except for one pair
Leu and Ile)
 Different peptides produce
different spectra
 Use the spectrum of a peptide
to determine its sequence

22. 1.Peptide mass fingerprinting
 Protein is cleave in smaller peptides
 Masses measured with MS
 These masses are then compared to
known protein
 Computer programs translate the known
genome of the organism into proteins
 Cut the proteins into peptides and
calculate the masses of peptides
 Compare the known and unknown protein

23. • Two Mass Specs, (MS1, MS2)
• A specific peak (corresponding to a specific
peptide chain is identified and fragmented to
form ions
• The ions are analyzed by MS2, and identified
as amino acids
• This way each selected piece of the whole
protein can be broken up and analyzed

25. Bottom up
approach
 In this method
by using mass
spectrometer,
entire peptides
of protein is
determined.
 Advantage
Small masses
are easier to be
handling.
Top down
approach
 By using mass
spectrometer, entire
protein is
determined without
solution digestion.
 Advantage
it provide the
complete covering
of protein.

26. •In this method high electric field is applied
to the tip of capillary, from which solution
will pass through and get the ions of
interest.
•Ions said to be multiple ions
• Multiple charged ions measure the high
mass biopolymer.

27. By pulses of laser light on the sample, ions of
interest formed. Large bio molecules can be
determined and synthetic polymer greater than
200,000 Dalton.
Advantage
•High speed
•Relative immunity to contaminants

28.  MALD with MS is called time of flight or
TOF. This enables accurate and fast molar
masses determination along with
determined impurities and sequencing
repeated units.
 It is a method of mass spectrometry in
which an ion’s mass-to-charge ratio is
determined via a time measurement.

30.  Most important and promising
applications to come forward is the
manufacture and production of
potential new drugs for the
treatment of perticular diaease.
 This depends upon genome and
information in the form of protein
expression.
 Which computer sofwares can use
as target for new drug discoveries.

31.  Example: if a protein is implicated in a
perticular disease its 3D structure
provides information for drug
discoveries.
 A competitive molecule that
inactivates the enzyme .
 This forms the basis for new drug
dicovery tools.
 Aim: Manufacture drugs to inactivate
proteins involved in a disease.

32.  Due to the presence of
genetic differences among
individuals therefore
scientists and researchers are
trying to develop personalised
drugs that can be more
effective for an individual.

33.  Most of biotech companies now have a proteomic
oriented biotech partner.
 Common applications in drug discoveries include
target identification and validation.
 Involves identifying proteins whose expression
level or expression changes in diseased state.
 Proteins may serve as potential theraptic targets
.

34.  Biomarkers are used to access
the effects and mechanism of
action of specific drugs.
 And in screening compounds
in pre-clinical studies.

35.  Functional genomics and proteomics have provided a
huge amount of drug targets.
 Most drugs acts on proteins.
 For example: Protease Inhibitor is design to disable
protease enzyme that allows perticular virus to
reproduce.
 Shape and structure of both compound and protein is
important.

36.  Majority of drug target are proteins.
 Proteins are key component in
pathways involved in disease and
therefore rich source of new drug
targets.
 Pattern of protein changes after drug
application will give information about
mechanism of action either for
therapeutic or toxicological effects.
 Various drugs are grouped according
to their signals in metabolic pathways.

37.  The correlation of dynamic
expression of protein and
physiologic changes related to
health or distressed conditions
can help to :
a) Support and understand disease
management.
b) Design new drug discoveries
and validation.
c) Disease models.
d) Find new diagnostic markers.
e) Identify potential therapeutic
targets.
f) Characterize drugs.

38.  A characteristic that is objectively measured and
evaluated as an indicator of normal biological process
pathogenic process , pharmacological response to a
therapeutic intervention is called a biomarker .
 It measures characteristic which may lead as an
indicator of biological state.
 In medicine biomarker can be a traceable substance that
is introduced into an organism as a mean to examine
organ function and aspects of health .

39.  A biomarker indicates a change in
expression or state of a protein that
correlates with the risk or progression of a
disease .
a) There are many techniques of proteomics
that allow tests for proteins produced in a
particular disease helping to diagnose
disease quickly , these include:
b) West blot.
c) Immunohistochemical staining.
d) Enzyme linked immune sorbent assay
e) Mass spectrometery.

40.  Tumor metastasis is a dominant cause of death in
cancer patients.
 The identification of protein molecules with their
expression correlated to metastatic process will help to
understand the metastatic mechanism and thus
facilitates the development of strategies for therapeutic
intervention and management of cancer

41.  In neurology many applications of proteomics have
involved neurotoxicology and neurometabolism as
well as in determination of several proteomis aspects of
individual brain areas and body fluids in
neurodegeneration.
 Investigation of brain protein group in
neurodegeneration , such as enzymes, cytoskeleton
proteins, chaperones, synaptosomal proteins and
antioxidant proteins, is in progress as phenotype related
proteomics.

42.  The concomitant detection of several hundred proteins
on a gel provides sufficiently comprehensive data to
determine a pathophysiological protein network and its
peripheral representative.

44.  Correct identification and alignment of spots
- hence very slow.
 The processing power available with today’s
PCs means that automated analysis.
 One vendor claims a throughput of 4 gel
pairs per hour can be compared and
annotated by an experienced user.

45.  Gel matching, or “registration”, is the process of aligning two images
to compensate for warp.
 Some packages still require the user to identify corresponding spots
to help with gel matching.
 The Z3 program from has a fully-automated gel matching algorithm:
◦ define set of small, unique rectangles.
◦ compute optimal local transformations for rectangles.
◦ Interpolate to make smooth global transformation.
 This makes use of spot shape, streaks, smears and background
structure, which other programs discard.

46.  Once the gel
images have been
matched, the
program
automatically
detects spots.
Algorithms are
generally based on
Gaussian statistics.

47.  The positions of detected spots are calibrated
to give a pI / mW pair for each protein.
 A value for the expression level of the protein
can be calculated from the overall spot
intensity.
 Some programs do not quantitate each gel
separately, but calculate relative intensity.

48.  The user can set
threshold values for the
detection of differential
expression. This helps
reduce the amount of
information displayed at
once.
 In this example, a protein
expressed only in the
second sample is circled
in red. The yellow circles
show proteins which are
differentially expressed.

49.  Some systems allow semi-automatic
annotation of spots, based on a database.
 Proteins of interest can also be excised from
the gel and sent on to mass spectrometry for
definitive identification. The Proteome Works
system offers such an integrated solution for
2D-PAGE.

50.  One useful feature of modern programs is the
ability to collate data.
 Spots which only appear in one gel are likely
to be artifacts, and are removed from the
analysis.
 This is an excellent way to reduce noise and
enhance weak signals

51.  With the advent of many 2-D PAGE databases
there are a number of protein spots that are
already "identified" in a few cell lines.
Combined with the aims of the experiment,
these databases may give one the
opportunity to guess at the identity of a
particular protein spot and confirm or deny
this by immuno-blotting

52. • A number of 2-D Gel databases exist.
• Quantitative databases: S.cervisiae .
• Annotative databases: E.coli and
human keratinocytes.
• An annual issue of the journal
“Electrophoresis”-Major database for
these databases!!!…
• A best one would obviously the
database which is regularly
updated.(e.g : Swiss 2D page).

53.  One can find an extensive list of such
databases by following these links.
 We would discuss a few “Interesting ones”.
• World 2-D PAGE
• NCIFCRF
• DEAMBULUM-Protein Databases
• Ludwig Institute of Cancer Research

54. • Basically a link to various 2-D Page databases.
• Has a useful tool called 2-D Hunt.
• Indexed as databases for multi species, mammals, yeast, plant ,
bacteria , viruses and parasites, cell lines.

55. • It gives cross reference to Medline and a few
other databases.
• In addition to this textual data, SWISS-
2DPAGE provides several 2-D PAGE images
showing the experimentally determined
location of the protein, as well as a
theoretical region computed from the
sequence protein, indicating where the
protein might be found in the gel.

56.  There have been some recent additions to the
database.
 SDS and 2-D Page of nuclear proteins from
Human HeLa cells have been added to the
growing list of reference maps.It is still an
ongoing project.Information about known
proteins found within that gel stretch has been
mapped(see below: right-SDS, left-PAGE)

57.  From SWISS-PROT, the user can select a link to
SWISS-3DIMAGE to see the three-dimensional
structure of the protein, if it is known, or to
submit the sequence to the SWISS-MODEL
three-dimensional modelling tool or view the
domain structure .
 - Also, from SWISS-PROT, the user can select
links to pertinent information from DNA
sequence databases (EMBL),chromosomal and
genomic maps (GDB Genome Database),
bibliographic references and abstracts
(Medline), and databases on the association of
human proteins with diseases (OMIM Online
Mendalian Inheritance in Man).

58. Here we click on
this spot in
reference map
of the Colorectal
epithelia cell
Throws a screen
showing
the pictures of
different image
maps with respect
to that protein

59. The red
rectangle is the
expected region
of the protein
on the gel.
Spots are the
proteins
identified
Dotted lines are extensions of
the possible regions if the
protein is acetylated,
phosphorylated or glycosylated.
Protein
identification
on chosen reference map

60.  Human 2-D PAGE Database:
 The keratinocyte 2D PAGE database constructed using carrier
ampholytes, is the largest of its kind and currently list 3625
cellular (2313 isoelectric focusing, IEF; 954 non equilibrium pH
gradient electrophoresis, NEPHGE), and externalised
polypeptides (358, IEF) of which 1285 have been identified
using a combination of techniques including immunoblotting
[32], Edman degradation of internal peptides [33, 34], and mass
spectrometry [35]. (Might be outdated!!!!)

61.  By clicking on each of the available reference
gels,we can get information(links to
medline,swissprot,PDB,cellular
location,Knockout,method used) on the
available proteins(checked spots) on the gel.
Databases for study of skin biology
HK-NEPHGE d’base
KP present in medium
IEF Database
HK-IEF d’base
865 IP 372 IP
59 IP

62. Database for study of Bladder Cancer
TCC-NEPHGE d’base Urine-IEF d’base
TCC-IEF database BSCC-IEF d’base
449 IP
144 IP
309 IP
197 IP

63. Other 2D Page Databases
Human MRC-5-
Fibroblasts-NEPHGE
D’base
Human MRC-5-
Fibroblasts-IEF
D’base
84 IP262 IP

64.  Search Options:
 Seacrh by protein name, keyword, sample spot
number,
 Relative Molecular mass, pI, organelle /component.
 Other options relating listing of proteins,views of the
gels are quite self explanatory.
 Other utilities of the Database:
 Has links to
 -NCBI’S Human-Mouse Homology maps through its
Mouse 2D-PAGE Databases.

65. 2D Protein Gel Databases
FlickerWebGel dbEngine
Maintained by
Image Processing
Section
Maintain the
gel analysis
software-
GELLAB II

66. WebGel:
WebGel is an Internet-based, interactive, qualitative and
quantitative gel
database analysis system.
A WebGel database contains previously quantified gel data
generated from a
stand-alone quantitataive gel analysis system.
wbdemoDB
demonstration
database
of serum
proteins in a
fetal alcohol
syndrome study.
melanie2DB
demonstration
database
of E.coli gelsfrom the
Melanie 2.3
demonstration
database.
fasDB
database
of serum proteins
in a fetal alcohol
syndrome study

67.  “Flicker is a method for comparing
images from different Internet
sources on your Web browser. In
the case of 2D protein
electrophoretic gel images, maps
identifying proteins in these gels
are becoming increasingly
available.’’

68.  It is a simple database search engine
which may be used to quickly create
a searchable database on a World
Wide Web (WWW) server. Data may be
prepared from spreadsheet programs
(such as Excel, etc.) or from tables
exported from relational database
systems. This Common Gateway
Interface (CGI-BIN) program is used
with a WWW server such as available
commercially, or from NCSA or CERN.

69. Broad study of cellular proteins
Detailed analysis are required
Various technologies that helps to figure
out complete functionalities of every
protein
There are some limitations also

70. The level of transcription gives a rough
estimation of translation.
Proteins experience post transcriptional
modifications.
Many transcripts give rise to more than
one protein.

71. Proteins degradation plays an important role.
Reproductibility
 Many proteins may form complexes with
other proteins.
 e.g, Peng at al identified 1504 yeast
proteins of which 858 were from previous
study

72. It is a manual and prolonged procedure
Do not permit analysis at single cell level.