Introduction to

Proteomics
Dr. Sharda Bharti
Syllabus

UNIT-3: FUNDAMENTALS OF PROTEOMICS

• Concept and components of proteome;
• importance of proteomics in biological functions;
• protein-protein interactions and methods to study it:
• protein arrays, cross linking methods, affinity methods, yeast hybrid systems.

UNIT-4: MASS SPECTROSCOPY ANALYSIS OF PROTEIN

Mass Spectrometry (MS)- Peptide mass finger printing, Mass accuracy,
Resolution, Sensitivity; Ion sources: Electrospray ionization, Matrix
assisted laser desorption and ionization; Mass analyzers: Quadrupole,
Ion-trap, Time-of-flight, Orbitrap, Fourier-transform ion cyclotron
resonance, Hybrid analyzers; Detectors; MS-MS; LC-MS.
 1.Proteomics: from protein
sequence to Function by
S.R. Pennington and M.J.
dunn. Viva Books. Private
Reference Limited (2001)
Books 2.Introduction to Proteomics
by Daniel C. Liebler.
Humana Press
 Introduction

• Genome function can be studied at the translation level as well as

the transcription level.
• The entire collection of proteins that an organism produces is called
its proteome.
• Thus, proteomics is the study of the proteome or the array of
proteins an organism can produce.
• Proteomics provides information about genome function that mRNA
studies cannot because a direct correlation between mRNA and the
pool of cellular proteins does not always exist. Much of the research
in this area is referred to as functional proteomics.
• It is focused on determining the function of different cellular
proteins, how they interact with one another, and the ways in which
they are regulated.
Easy to remember

Proteins
• molecules of amino acids that perform much of life’s function

Proteome
• set of all proteins in a cell

Proteomics
• study of protein’s structure & function

5
 Genome – sum total of all the genes in an
organism.

Genomics – study of sequence of all genes in an

organism.

Transcriptome – sum total of all the RNA

transcripts an organism can make in its lifetime.

Transcriptomics – study of levels of RNA

Nomenclatures produced from many genes in a cell at a given
time.
Proteome – properties and activities of all the
proteins that an organism makes in its lifetime.

PROTEin complement to a genOME .

Proteomics - the qualitative and quantitative

comparison of proteomes under different
conditions to unravel biological processes.
Biochemical context
 • The terms “proteomics” and “proteome”
were coined by Marc Wilkins and
colleagues in the early 1990s and mirror the
terms “genomics” and “genome,” which
describe the entire collection of genes in an
organism.
• Proteomics is the study of the proteome, the
protein complement of the genome.
• Definition: Proteomics is a scientific discipline
Proteomics concerned with systematic analysis of proteins
present in cells at a given time under given
conditions.
• Proteomics includes the identification,
characterization and quantitation of the entire
complement of proteins in cells, tissues or whole
organisms with a view to understanding their
function in relation to the life of the cell.
 Genomics vs Proteomics
Genomics Proteomics
Genomics is the study of the entire set of Proteomics is the study of proteins that
genes or the genome of a cell or organisms are expressed by functional genes
It is the study of genome structure and It is the study of proteins structure,
expression expression and activity
Every cell contains the same set of genes. Proteome varies in different types of
Genome is fixed in an organism cells according to function, internal and
external factors
Mere presence of a gene does not tell about Study of proteins is more conclusive as it
its functionality. Gene expression is regulated is the functional product of a gene, and
at the transcription and translation level tells about the actual condition of a cell
or an organism
It involves sequencing, mapping and It involves the study of 3D structure,
expression analysis of a genome function and interaction of proteins
Technologies used are DNA sequencing and Technologies involved are immunoassay,
bioinformatics mass spectrometry, etc.
The human genome project is a part of It takes the help of genomic projects
genomics
 The proteome is larger than the genome due to
alternative splicing and protein modification.
All protein-protein interactions. E.g involved in plant
defense reactions.
One protein or peptide may have multiple functions
depending on context.
Regulation of protein function.
Proteomics
Modification: to map protein modification to
includes determine the difference b/w wild type and a GMO.
Location

The goal of proteomics is to Detection and quantitation

understand how the structure
and function of proteins allow
The concentration of protein changes by 10 orders
them to do what they do, what of magnitude within the cell.
they interact with, and how they
contribute to life processes.
Different subdivisions of proteomics:
• Structural proteomics -- in-depth analysis of protein structure, X-ray crystallography and NMR
spectroscopy
• Expression proteomics -- analysis of expression and differential expression of proteins
• Interaction proteomics -- analysis of interactions between proteins to characterize complexes
and determine function

Types
Scope of
Proteomics
Application of Proteomics
 1. growth of gene, expressed sequence tag
(EST), and protein-sequence databases
Three during the 1990s
developments
2. introduction of user-friendly, browser-based
formed bioinformatics tools to extract information
the foundation from these databases.
of the new
biology: 3. oligonucleotide microarray: contains a
series of gene-specific oligonucleotides or
cDNA sequences on a slide or a chip.
• By applying a mixture of fluorescently labeled DNAs
from a sample of interest to the array, one can probe
the expression of thousands of genes at once.
 Level of transcription of a gene ≠
Level of expression of the gene
mRNA - degraded rapidly -
translated inefficiently
Why do we Post-translational Modifications /
need Translocations
Proteomics?
One gene / transcript > many
proteins
One protein > many processes
 • Proteomics represents the
effort to establish the
Not so identities, quantities,
structures, and biochemical
easy to and cellular functions of all
remember proteins in an organism,
organ, or organelle, and how
these properties vary in space,
time, or physiological state.
Proteomics
A true multidiscipline science

• Protein chemistry
• Mass spectrometry
• Genomics
• Bioinformatics
• Computer science
• Separation science

17
From Genome to
Proteome

18
From cell to gene
The human genome is located right
in the heart of the cells, in the
nucleus.

The genes, parts of the DNA

(double helix), are the functional
units of the genome.

They hold all the information

necessary to create the proteins you
need.

Genes are located along thread-like

structures called chromosomes.

19
Protein Synthesis

20

Protein synthesis is the transcription and translation

of specific parts of DNA to form proteins.
Central Dogma of Biology

genome transcriptome proteome

21
The building polypeptide chain

22
Amino acids

23
Amino acid

• The human body needs 20 amino acids to be able to

make (or synthesize) its thousands of proteins.

24
Aminoacid structure
 What are Proteins?
Proteins are strings of amino acids and are the active elements of cells.

Where we find proteins?

Cells and body tissues, hormones, antibodies, and enzymes.

Protein functions?
• structure providing proteins (hair and fingernails).

• help in digestion (stomach enzymes), detoxify poisons, or help fight disease.

•cell membranes proteins and are important in controlling how substances pass
through these membranes.

• enzymes proteins are catalysts for biochemical reactions.

• antibodies proteins react with foreign substances to defend the body.

 Source of Proteins

• Grow cells and blow them up

(lyses)
• Dissect tissue sample,
homogenize and lyse
• Synthesize

27
Source of Proteins ( cell fractionation)
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 Protein Immunoprecipitation

Immunoprecipitation (IP) is the technique of precipitating

a protein antigen out of solution using an antibody that
specifically binds to that particular protein.

29
Biological fractionation
• Necessary!! To decrease complexity before analytical
fractionation
– Abundant protein depletion
– Membrane fraction
– Soluble fraction
– Organellar fractionation
– Affinity chromatography
– Etc…

30
Separation techniques versus protein
characteristics
• Charge
Ion exchange chromatography
Electrophoresis
Isoelectric focusing

• Polarity
Adsorption chromatography Paper
chromatography
RP chromatography
Hydrophobic interaction chromatography

• Size
Dialysis and ultrafiltration
Gel electrophoresis
Gel filtration chromatography (size exclusion chromatography)
Ultracentrifugation

• Specificity
Affinity chromatography
PI Protocol
• Lyse cells and prepare sample for immunoprecipitation.
• Pre-clear the sample by passing the sample over beads that are not coated with
antibody to soak up any proteins that non-specifically bind to the beads.
• Incubate solution with antibody against the protein of interest. Antibody can be
attached to solid support before this step (direct method) or after this step (indirect
method). Continue the incubation to allow antibody-antigen complexes to form.
• Precipitate the complex of interest, removing it from bulk solution.
• Wash precipitated complex several times. Spin each time between washes or
place tube on magnet when using superparamagnetic beads and then remove
supernatant. After final wash, remove as much supernatant as possible.
• Elute proteins from solid support (using low-pH or SDS sample loading buffer).
• Analyze complexes or antigens of interest. This can be done in a variety of ways:
– SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis)
followed by gel staining.
– SDS-PAGE followed by: staining the gel, cutting out individual stained protein
bands, and sequencing the proteins in the bands by MALDI-Mass
Spectrometry
– Transfer and Western Blot using another antibody for proteins that were
interacting with the antigen followed by chemiluminesent visualization.

32
Peptide bond formation
Two amino acids can undergo a condensation reaction to form a dipeptide.
Further condensation reactions result in a polypeptide.
R1-COOH + R2-NH2 R1-CO-NH-R2 + H2O

Amino acids are linked with the peptide bond, amide bond
33
 The aliphatic amino acids

Glycine, Gly, G Alanine, Ala, A Valine, Val, V Leucine, Leu, L

Isoleucine, Ile, I Proline, Pro, P

34
 The aromatic amino acids

Phenylalanine, Phe, F Tyrosine, Tyr, Y Tryptophan, Trp, W

35
The sulfur containing amino acids

Cysteine, Cys, C Methionine, Met, M

36
S-S bond

2Cysteines/oxidation=disulfide
bond (only in extracellular and not
intracellular proteins)

Disulfide bonds stabilize protein

structure by providing crosslink

37
The hydroxyl amino acids

Serine, Ser, S Threonine, Thr, T

38
The acidic amino acids

Aspartate, Asp, D Glutamate, Glu, E

39
The amide amino acids

Asparagine, Asn, N Glutamine, Gln, Q

40
The basic amino acids

Lysine, Lys, K Arginine, Arg, R

41
The imidazole amino acid

Histidine, His, H

42
Amino acids that accept a positive
charge

Histidine Lysine Arginine

H K R
His Lys Arg

43
Aminoacids clasification

Side chains of aminoacids

Responsible for many of the

uniques properties of proteins

charged or polar groups provide

interesting catalytic groups

nonpolar amino acids - a protein

folding issue

44
 Amino acid 3LC SLC Average Monoisotop
ic
Glycine Gly G 57.0519 57.02146
Monoisotopic Alanine Ala A 71.0788 71.03711
and Average Serine Ser S 87.0782 87.02303
Mass Proline Pro P 97.1167 97.05276
Valine Val V 99.1326 99.06841
Threonine Thr T 101.1051 101.04768
Cysteine Cys C 103.1388 103.00919
Leucine Leu L 113.1594 113.08406
Isoleucine Ile I 113.1594 113.08406
Asparagine Asn N 114.1038 114.04293
Aspartic Asp D 115.0886 115.02694
acid
Glutamine Gln Q 128.1307 128.05858
Lysine Lys K 128.1741 128.09496
Glutamic Glu E 129.1155 129.04259
acid
Methionine Met M 131.1926 131.04049
Histidine His H 137.1411 137.05891
Phenyalani Phe F 147.1766 147.06841
ne
Arginine Arg R 156.1875 156.10111
Tyrosine Tyr Y 163.1760 163.06333
Tryptophan Trp W 186.2132 186.07931
What Proteomics
can do?

46
 • Protein separation: In order identify each protein in the mixture.

• Protein identification
• Mass spectrometry
• Antibody based assays can also be
used, but are unique to one sequence
motif. Edman degradation used to
confirm sequence when MS
unavailable.
Types of • Protein quantification
Experiments; • Gel-based methods, differential staining of gels with
fluorescent dyes (difference gel electrophoresis). Gel-
key free, various tagging or chemical modification methods,
label free methods.
questions of • Protein sequence analysis : Searching databases.
Proteomics • Structural proteomics
• High-throughput determination of protein structures in three-
dimensional space. Methods are x-ray crystallography and NMR
spectroscopy.

• Interaction proteomics
• The investigation of protein interactions on the atomic, molecular and
cellular levels.

• Protein modification: Phosphoproteomics and glycoproteomics.

• Cellular proteomics
• The goal is to map location of proteins and protein-protein
interactions during key cell events. Uses techniques such as X-ray
Tomography and optical fluorescence microscopy.
Methods for protein analysis
High resolution mass spectrometry methods
Top down methods
Intact proteins

Bottom up methods
Digested peptides

De Novo Sequencing

Gel based methods

One/Two-dimensional gel electrophoresis

48
 Mass spectrometer based proteomics
• This area is most commonly associated with
proteomics and is a method to determine which
proteins are expressed and the amounts of those
proteins.
What areas Array based proteomics
are being • This method uses various arrays to try and define
the function of the proteins, regulation levels and
studied in interacting partners within the cell.
Proteomics? Informatics
• This is trying to define what information will be
needed, stored, accessed and how it can be used
to study the proteome of the cell.

Clinical

Orthagonalomics
 • Principles
MASS • Mass spectrometry is based on the fact that
SPECTROMETER ions of differing charge and mass will move
BASED differently in a magnetic field. In proteomics
the proteins are first separated by some
PROTEOMICS means and then analyzed with a mass
spectrometer
Separating
the
The protein genome is separated by several
Proteome different methods.

Many researchers are first separating

portions of the genome, such as isolating
organelles, and then analyzing that portion.

This is because often proteins of interest,

regulatory proteins are in low abundance.

The most commonly used method is 2-

dimensional gel electrophoresis.

• Consists of using isoelectric focusing with SDS

polyacrylamide gel electrophoresis
Isoelectric focusing

• 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
• Typically this is done with an immobilized pH gradient gel strip or with
a tube gel 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
and the proteins form tight bands at their isoelectric point.
• The IEFgels are now ready for the second method.
Figure is from “Principles of Biochemistry” Lehninger, Fourth Edition
SDS Polyacrylamide Gel
Electrophoresis

• The second dimension separates the proteins based on size.

• There are two parts, the stacking gel which concentrates the
sample and the running gel that is used to separate the proteins.
• The IEF gel is soaked in a solution containing chemical to
denature the proteins including sodium dodecyl sulfate a
detergent which gives the proteins a net negative charge. This
means that all proteins will move in one direction.
• The IEF gel is then put in the one long well in the stacking gel,
sealed in place with agarose, and the proteins subjected to an
electric field to separate.
• The larger proteins are found at the top and the smaller ones
are found at the bottom of the gel.
 In a 2D gel the proteins appear as spots on the
gel rather than bands. These spots can then
be further processed or used for mass
spectrometry directly.

Further processing usually includes spot

excision, trypsin digestion, and mass
2-Dimensional spectromety

Gel
Electrophoresis Analysis may also include differential 2D gel
electrophoresis

• In this case a control and sample are separately labeled

with a fluorescent molecule.
• The samples are mixed and electrophoresed in the same
gels.
• A laser scanner is used to identify each spot and a
program puts the two images together.
2-D Gel
Electrophoresis
2-D Gel (denaturing)
 2-D Gel (non-denaturing)

pH 4 pH 10
 The first dimension is run in larger agarose
tube gels with ampholytes.

• This has less resolution than polyacrylamide gels. The

tubes are sliced and the proteins are allowed to
diffuse out.

Alternate Gel regions are cut, proteins eluted and the

proteins are then separated by capillary
Separation electrophoresis.

Methods Capillary electrophoresis has a much greater

resolution for the proteins mass.

Proteins are eluted from the capillary in the

process and can be collected. They are
readily available for mass spectrometry.
 Whole proteome is analyzed at once.

Proteome is digested with protease

(trypsin)

Alternative Digested proteome is injected to HPLC

Separation with 2 columns in series (mixed bed
ion exchange and reverse phase)
Methods Peptides are eluted from ion exchange
onto reverse phase and then separated
on reverse phase column.
Peptides then enter ESI-MS-MS
Mass Spectrometery

• Separates ions based on mass to charge ratio.

• Charges are placed on the protein or the peptide by ionization.
• Two most common types of ionization are:
• Matrix-Assisted Laser Desorption Ionization.
• MALDI causes fragmentation of the protein during ionization.
Can be used to get more information about the fragments.
Easier to do than ESI.
• Electrospray ionization (ESI)
• ESI can give whole protein masses as well as complex masses.
If the proteins is first separated by reverse phase HPLC
before injection only the subunits masses will be known.
 MALDI causes fragmentation of the
protein during ionization. Can be
used to get more information about
the fragments. Easier to do than ESI.
Matrix-Assisted
Laser Requires sample to be placed in
Desorption matrix that absorbs appropriate
Ionization wavelength light.
(MALDI)
Matrix generates heat and forms ions
of matrix and what is around it.
Electrospray Ionization
Mass Analyzers

• Important parameters
• Sensitivity
• How few ions can be detected.
• Resolution
• How well different masses can be determined.
• mass accuracy
• How reproducible and correct are the masses.
 Quadrapole
• High Sensitivity, acceptable
mass accuracy and resolution
• Easily coupled to
Mass chromatography

Analyzers Time of Flight

• High Sensitivity, high mass
(MS) accuracy, high resolution
• Limited to small m/z ratios
• Not easily coupled to
chromatography
• Easily coupled to MALDI
 Ion Trap

• High Sensitivity
• Low mass accuracy and
Mass resolution
Analyzers Fourier Transform ion
(MS) cyclotron
• High sensitivity, mass accuracy,
resolution, dynamic range
• Expensive, difficult to operate,
low fragmentation efficiency
Amino Acid Masses

Amino Amino
Mass(avg) Mass(avg)
acid acid
G 57.0520 D 115.0886
A 71.0788 Q 128.1308
S 87.0782 K 128.1742
P 97.1167 E 129.1155
V 99.1326 M 131.1986
T 101.1051 H 137.1412
C 103.1448 F 147.1766
I 113.1595 R 156.1876
L 113.1595 Y 163.1760
N 114.1039 W 186.2133
Peptide Fragmentation

88 145 292 405 534 663 778 907 1020 1166 b ions
S G F L E E D E L K
1166 1080 1022 875 762 633 504 389 260 147 y ions

100
% Intensity

0 m/z
250 500 750 1000
 Quadrapole
• Sensitive, acceptable mass accuracy and
resolution
• Easily coupled to chromatography.

Time of Flight

Mass • Sensitive, high mass accuracy, high resolution

• Limited to small m/z ratios

Analyzers • Not easily coupled to chromatography

Ion Trap
(MS) • Sensitive
• Low mass accuracy

Fourier Transform ion cyclotron

• High sensitivity, mass accuracy, resolution,
dynamic range
• Expensive, difficult to operate, low
fragmentation efficiency