NPTEL VIDEO COURSE – PROTEOMICS

PROF. SANJEEVA SRIVASTAVA

HANDOUT

LECTURE-01

INTRODUCTI
ON TO
PROTEOMIC
S

I. AN OVERVIEW OF PROTEOMICS

 Proteome describes the protein complement expressed by a genome,

or more precisely, the protein complement of a given cell at a given
time, including the set of all protein isoforms and modifications.
 Study of entire compendium of proteins encoded by a genome is
known as “proteomics”.
 The extent of diversity and complexity due to alternative splicing
and post- translational modification is tremendous, therefore
studying proteins and proteome is very important.
 Steps involved in proteome analysis: protein extraction followed
by their separation, identification and characterization. Protein
extraction from whole
cells, tissue or organisms is first requirement for proteome analysis in
majority of the proteomics experiments. Protein separation and
quantification is achieved by gel-based (e.g. 2-DE) and gel-free
techniques (e.g. iTRAQ) and identification by MS. The functional
characterization of proteins using novel proteomic platforms opens
new horizon for exploration in biology.
 Abundance based proteomics aims to measure the abundance of
protein expression, whereas functional proteomics aims to determine
the role of proteins by assessing protein interaction and biochemical
activities.

Emergence of proteomics
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II. PROTEIN CHEMISTRY TO PROTEOMICS

 Proteomics research originates from classical protein chemistry

and it has embraced new HT techniques to analyze complex
samples. Many of the techniques used under modern proteomics
banner (e.g. 2-DE, MS) have actually originated several years ago.
 The technological advancements in analytical techniques: with
increased sensitivity, resolution and capability to carry out high
throughput studies has led to a transition from protein chemistry to the
new field of proteomics.
 Protein analysis by MS was challenging due to complete degradation of
samples
with available hard ionization techniques. This limitation was
overcome by development of soft ionization techniques, MALDI and
ESI. These techniques greatly improved proteomic studies as they
facilitated MS analysis of protein samples.
 Protein sequencing by Edman degradation is time-consuming and
cumbersome.
Several rounds of sequencing are required for analysis of long
polypeptide chains. However, peptide sequencing by MS is much
faster, and allows large number of samples to be analyzed in short
time.
 Development of IPG strips: facilitated proteome analysis using 2DE:
The pH
gradient in tube gels are established by ampholytes gradients,
which are not always very stable and tend to break down upon
addition of concentrated samples. Analysis of protein mixture by 2-DE
using tube gels often gives a lot of variation across gels.
 The problem of reproducibility was overcome to a large
extent by the development of Immobilized pH gradient strips.
Minimal gel-to-gel variation was observed when samples were run by
2-DE using IPG strips, which made this technique suitable for large-
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 Completion of several genome sequence projects: Genome
sequences of several organisms, including humans, have been
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PROF. SANJEEVA SRIVASTAVA

sequences. Several databases are now readily available which can easily
help in identifying gene sequence of a protein that has been
sequenced by mass spectrometry.

III. GENOMICS TO PROTEOMICS

 Genome represents an important starting point towards

understanding complexity of biological functions. However, proteins,
provide a much more meaningful insight into the mysteries of
essential biological processes.
 To obtain better understanding of cellular processes & regulation, there
has been an increasing interest in studying proteomics.

There are several reasons why one need to study

proteomics? Genomic DNA contains large stretches of

non-coding regions:

 Pre-mRNA is synthesized from genomic DNA by the process of

transcription. mRNA contains both exons, the coding sequences, as
well as introns which are intervening, non-coding sequences.
 By involving series of steps, finally free 3’ hydroxyl group of the first
exon attacks the 5’ end of the second exon such that they are
joined to give the mature mRNA.

Single gene, multiple proteins:

 Alternative splicing is a process by which exons or coding

sequences of pre- mRNA produced by transcription of a gene are
combined in different ways during RNA splicing. Resulting mature
mRNA give rise to different protein products by translation, most of
which are isoforms of one another.
 The diversity of proteins encoded by a genome is greatly
increased due to alternative splicing.
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Post-translational modification of proteins:

 The proteins obtained by translation undergoes folding and various
PTMs such as phosphorylation, alkylation, glycosylation, hydroxylation
etc. to give the final functional protein.
 PTMs generate diversity, complexity and heterogeneity of gene
products and its functional consequences can be modulation in protein
dynamics and alteration of its functional activity.

IV. CENTRAL DOGMA, OMICS AND SYSTEMS BIOLOGY

 During the last decade we have witnessed the revolution in

biology, as this discipline has fully embraced “omics” tools. The
emergence of genome-wide analyses to understand cellular DNA,
RNA and Protein content by employing genomics, transcriptomics
and proteomics at systems level has revolutionized our
understanding of control networks that mediate cellular processes.
 Genes are the blue-print for life and proteins are the effector
molecules. Due to this fact the central dogma has guided research
at the systems level. After completion of human genome
sequence number of genes ~25,000 are surpassed by an
estimated number of proteins in millions.

Studying large-scale study of protein structure and function, requires a

thorough understanding of protein composition and their various structural
levels by employing HT tools.

 Proteins play an important role in essential characteristic of living

systems, how they function and replicate themselves through
intricate molecular interactions. Amino acids constitute the basic
monomeric units of proteins, which are joined together by peptide
bonds.
 The linear sequence of amino acids constitutes primary structure.
Folding of polypeptide/protein chain into regular structures like a-
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 Three-dimensional compactly folded structure of proteins

makes tertiary structure, which represents overall organization of
secondary structural elements in 3-D space.
 Quaternary structure - refers to interaction between individual protein
subunits in a multi-subunit complex.
 Sickle cell anemia is caused due to single nucleotide substitution
which converts
a glutamic acid residue to valine in beta chain of hemoglobin.
Thalassemia is caused due to abnormalities in hemoglobin synthesis.
 Protein folding is an elegant example of biological self-assembly.
Understanding the mechanisms through which protein folding takes
place remains challenging for scientific community.
 Anfinsen tested the ability of reduced and unfolded Ribonuclease A
protein to
spontaneously fold into its native state. Protein folding is a
cooperative process which arises from simultaneous formation of
multiple interactions within a polypeptide chain. Protein folding
is thermodynamically favorable and spontaneous process.
 Folding efficiency could be limited by processes such as
aggregation. The molecular chaperones are designed to promote
protein refolding.
 From complex proteome it is challenging to purify a protein in
a single chromatographic step. Therefore, sequential pre-
fractionation steps involving
different modes of chromatography becomes necessary.
 Gel filtration chromatography separates protein on basis of
difference in size. When a protein sample is applied to column,
small proteins passes from the pores of the beads while the large
proteins are excluded, therefore this technique is also known as
“molecular exclusion”.
 Ion-exchange chromatography relies on differences between number
of charges and distribution
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condition.
 Affinity chromatography is based on affinity of protein to other
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GENOMICS

 Studying genome of an organism by employing sequencing and

genome mapping is known as “genomics”.
 Several genome sequencing projects that aim to elucidate the
complete genome sequence of organisms have been undertaken by
several research groups all
over the world. From a genomic library clones were isolated and
ordered into a detailed physical map. Further, individual clones were
sequenced by shotgun sequencing to provide the complete genome
sequence.
 Recently next-generation sequencing strategies have dramatically
increased the
pace of sequencing by several order of magnitudes. NGS based on
nanopore structures is known as nanopore sequencing. For NGS
various commercial platforms such as Illumina, Pyro-sequencing,
Helicos, Ion Torrent etc are available.

TRANSCRIPTOMICS

 Study of all the mRNA molecules expressed by a particular cell

type of an individual is known as transcriptomics. The transcriptomic
analysis measures the genes that are being actively expressed at any
given time and varies significantly with external environmental
conditions. Various techniques such as microarrays, Q-PCR etc. have
been widely used for transcriptomics analysis.
 In microarray experiment, mRNA from control and test samples are
extracted
and reverse transcribed into its corresponding cDNA. The cDNA
samples are labeled with Cy5 and Cy3 dyes and mixed cDNA sample is
incubated on printed DNA microarray. This allows hybridization to
occur between the probe oligonucleotides on the array surface
and the labeled cDNA samples of interest. In this manner expression
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simultaneously.
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SYSTEMS BIOLOGY
 In omics era, technological advancements in genomics,
proteomics, and metabolomics have generated large-scale datasets
in all aspects of biology. These large data-sets has motivated the
computational biology and systems approaches with objective of
understanding biological systems as a whole.
 Systems biology and biological network modeling aims to understand
biological
processes as whole system rather than isolated part by synergistic
application of experiment, theory, technology & modeling.
 Systems level studies aim to develop computationally
efficient and reliable models of underlying gene regulatory
network. Quantitative analyses
measures and aims to make models for precise kinetic parameters of
a system network components. It also uses properties of network
connectivity.

V. GEL-BASED PROTEOMICS

 Several techniques used in proteomics typically aim to elucidate the

expression, localization, interaction, and cellular function of proteins.
SDS-PAGE, 2DE and DIGE are commonly used gel-based techniques.

Protein extraction

Protein extraction is the first step for proteomic analysis. The protein
extraction methods aim that most, if not all the proteins in a cell or its
organelles are extracted by the procedure and the presence of interfering
compounds are minimized.

 Different biological samples pose different challenges. E.g. serum

proteome analysis shown here, illustrates that proteins in biological
systems such as serum may have difference of several order
of magnitudes.
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lower
concentrations. It is therefore preferred to remove these high
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PROF. SANJEEVA SRIVASTAVA

 In bacterial protein sample preparation sonication is an important step

to disrupt the bacterial membrane. Sonication breaks open the
cellular membranes to release the intracellular contents. Protein
extraction can be performed using different methods and protein
pellets are reconstituted in lysis buffer for proteomic analysis.

Protein quantification

 Protein quantification is sensitive to detergents or certain ions

therefore it is crucial to select the correct quantification method.
 In Bradford color reagent transfer of electrons converts the dye to its
blue form thereby giving the solution a blue color. Absorbance of
standard and unknown protein samples can be measured at 595 nm
and protein concentration can be determined from the standard plot of
the absorbance values.

Gel-based proteomics

 In gel-based proteomics, proteins are commonly analysed using SDS-

PAGE and two- dimensional (2D) gel electrophoresis. Separation in
SDS-PAGE occurs almost exclusively on the basis of molecular
weight and in 2DE the complex mixtures are resolved first by
isoelectric point and then by size on a polyacrylamide gel.
Some of the limitation of 2DE can be overcome by Difference gel
electrophoresis (DIGE) technique.
 2DE in combination with mass spectrometry has been the standard
technique for proteome analysis.
 Two-dimensional electrophoresis involves protein separation on a pH
gradient
based on their isoelectric point (pI) using isoelectric focusing (IEF)
followed by separation in the second dimension using SDS-PAGE
where the proteins are resolved according to their molecular weight.
 To perform 2DE, add the reconstituted protein sample to the
rehydration tray and
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place the IPG strip for rehydration. Isoelectric focusing involves the
application of an electric field, which causes the proteins to migrate to
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PROF. SANJEEVA SRIVASTAVA

gradient that matches the pI of a specific protein after which it does not
move in the electric field owing to the lack of charge. The proteins
migrate along the strip and come to rest at a point where their net
charge becomes zero known as their isoelectric point.
 Prior to second dimension separation, an equilibration step is
required. In equilibration, Dithiothreitol brings about cleavage of the
protein disulphide bonds while iodoacetamide prevents reformation
of these bonds by binding to free sulphydryl groups.
 On SDS-PAGE gel, proteins get separated on the basis of their
molecular weight with the low molecular weight proteins having high
mobility and migrating further through the gel and the high molecular
weight proteins remaining close to the point of application.
 Gels can be visualized by different dyes such as Coomassie blue
staining, Silver staining, Cyanine dyes etc.

Data Analysis

 The gel analysis involves, images processing, detection of spots,

making match- set, landmarking, viewing histograms etc.
 Various information regarding the spots such as their area, volume,
intensity and statistical parameters such as standard deviation, can
also be calculated.
 2DE has high resolving power but it has several limitations such as
staining and reproducibility.

2D DIGE

 Fluorescence two-dimensional difference in-gel electrophoresis (2-D

DIGE) is an advanced 2DE technique that allows for accurate
quantification with statistical confidence, while controlling non-
biological variation.
 In DIGE, proteins extracted from different types of cells or tissue
samples are labeled with different fluorescent reagents (Cy2, Cy3 and
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separated by 2 DE on a single gel. The proteins are detected separately

using Cy2, Cy3 and Cy5.
 The commercial software such as DeCyder facilitate the automated
analysis of DIGE gels and provide differential expression analysis,
principal component
analysis, pattern and discriminant analysis.
 2DE, DIGE followed by mass spectrometry technique has been applied
for many applications.

VI. MASS SPECTROMETRY

 Mass spectrometry is technique for protein identification & analysis by

production of charged molecular species in vacuum, & separation by
magnetic and electric fields based on m/z ratio.
 MS has become the method of choice for analysis of complex protein
samples in proteomics studies due to its ability to identify thousands of
proteins.
 The gel-based techniques typically resolve only products of a few
hundred genes at best, had low throughput and low dynamic range.
 To overcome such issues, Mass Spectrometry has become an
important analytical tool in proteomics, and in biology in general. It
offers high-throughput, sensitive and specific analysis for many
applications.
 The basic components of MS involve “Sample inlet” to transfer
sample into the
ion source. “Ionization source” which converts neutral sample
molecules into the gas-phase ions, A “mass analyzer” to separate
and analyze mass of ionic species. Detector, which measures and
amplifies ion current of mass-resolved ions and data system to
process and analyze data.
 Soft ionization techniques such as matrix-assisted laser
desorption/ionization (MALDI) and electrospray ionization (ESI), are
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 In MALDI protein is mixed with matrix and laser beam ionizes matrix
molecules. It
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PROF. SANJEEVA SRIVASTAVA

 ESI requires sample of interest to be in solution and produces gas-

phase ions from solution. The distinguishing feature of ESI is its
ability to produce multiply charged ions.
 Mass analyzer disperses all ions based on their (m/z) ratio and focuses
all mass- resolved ions at a single focal point and maximizes their
transmission.
 Time of flight - measures m/z ratios of ions based on time it takes for
ions to fly in
analyzer & strike the detector
 Ion Trap - traps ions using electrical fields and measures mass by
selectively ejecting them to a detector.
 Quadrupole consists of four parallel metal rods and mass
separation is
accomplished by the stable vibratory motion of ions in high-frequency
oscillating electric field.
 Many advancement in MS during the last decade have provided new
ways for protein analysis and facilitated proteomic analysis of various
biological systems.
 The Q-TOF LC/MS system performs MS/MS analysis using a
quadrupole, hexacollision cell and time of flight (TOF) mass analyzer.
 Quadrupole selects precursor ions, which are further fragmented in
collision cell. The product ions move to detector and spectrum is
generated.
 Protein labeling with stable isotopes are effective methods for
quantitative proteome profiling using MS. Stable isotope labeling by
amino acids in cell
culture SILAC, which is a metabolic-labeling strategy to encode
whole cellular proteome, is widely used methods for quantitative
proteomics.

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Quantitative proteomics
 In SILAC two groups of cells are cultured in media that are
identical in all respects except that one contains a heavy, isotopic
analog of an essential amino acid while the other contains the normal
light amino acid. After a number of cell divisions, the grown cells are
combined and digested using trypsin. The complex mixture is further
separated by SDS-PAGE to simplify the analysis. Further purification
is carried out by liquid chromatography and purified peptide
fragments are analyzed by MS/MS.
 iTRAQ, it is a MS based technique for relative and absolute
quantification of proteins. iTRAQ reagents are a set of four
isobaric amine-specific labeling
reagents (114, 115, 116, or 117). An iTRAQ reagent consists of a
reporter group, a balance group, and a peptide reactive group.
 Pooled samples are purified on a strong cation exchange column to
remove excess unbound reagent. These isobaric labels are detected
upon fragmentation
and release in MS.
 The data obtained from mass spectrometry can be analyzed by
using search engines such as Mascot. The analysis requires inputs
regarding the experimental parameters such as enzyme cleavage,
modifications, instrument used, peptide
tolerance etc. The data file generated from MS is uploaded and
the search carried out by employing databases such as NCBI, MSDB
and SwissProt.

VII. INTERACTOMICS

 Biology has evolved several mechanisms that regulate interactions,

including a variety of PTMs and the presence or absence of an
activator or inhibitor molecule. A detailed understanding of
protein interactions provides an opportunity to understand the
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 Inspired from the success of gene microarrays various protein
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 Some of the widely recognized technologies that have been used to

map protein- protein interactions at large scale, include yeast-
two-hybrid, IP with mass spectrometry and different types of
microarray platforms.

Yeast two hybrid

 In Y2H, binding domain is fused with the bait protein while the
activation domain is fused with the prey protein. Binding of either one
of the fusion proteins to the promoter is insufficient to bring about
transcription of the gene. When the bait protein bound with the
binding domain interacts with the prey protein fused with the
activation domain, there will be expression of the reporter gene
which can easily be detected.

Protein microarrays

 There are two types of protein arrays that are commonly used. In
forward phase arrays, immobilized antibody is probed by the test
lysate. In reverse phase arrays, cellular lysate is immobilized on the
array surface and then probed using detection antibodies specific to
the target of interest.
 The gene coding for the protein of interest is expressed in a
suitable heterologous host system such as E. coli. Protein
purification can be done by chromatographic procedures to obtain the
pure target protein. Tags like His6 are
often fused with the protein of interest to facilitate the purification
process due to its specific affinity towards nickel. Protein purity is
tested on SDS-PAGE gels.
 The array surface can be functionalized with suitable chemical
reagents such as Aldehyde and silane derivatizations that will react
with groups present on the
protein surface. Protein is printed on to the array surface in
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Protein detection
 In protein detection using direct labeling, all the target proteins are
labelled with a fluorescent tag. In sandwich assay, however, a
fluorescent tagged secondary antibody that recognizes a different
epitope on the target antigen binds to it and is detected by means of
the fluorescence.
 Protein purification is a laborious and time-consuming procedure
which posses several technical challenges eg. protein purity, protein
folding and functionality during the purification and immobilization
steps.

Cell-free expression based microarrays

 The limitations of protein purification have motivated the advent

of cell-free expression based microarrays, which carry out in
situ transcription and translation, and eliminate the drawbacks of
traditional cell-based methods.
 Nucleic Acid Programmable Protein Array (NAPPA) replaces complex
process of
spotting purified proteins with simple process of spotting plasmid DNA.
By using recombinational cloning, & cell-free expression system,
proteins are produced in vitro, and captured on array.
 In DNA Array to Protein Array (DAPA) slides bearing the DNA template
and the
protein tag-capturing agent are assembled face-to-face with a lysate
containing permeable membrane placed in between. The
expressed protein slowly penetrates the membrane and gets
immobilized on the slide surface through its capture agent.
 Multiple Spotting Technique (MIST) involves addition of template DNA
on to the solid array support. and second spotting step involves the
addition of the cell-free lysate directly on top of the first spot.
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 In HaloTag method slide is activated with the HaloTag ligand which
captures the expressed protein through firm covalent interactions
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 The protein microarray experiment involves blocking the slides

with milk or SuperBlock. Application of primary AB, and washing
with milk followed by incubation of secondary AB and signal
detection.
 Although microarray experiments are simple but data analysis
is very
challenging. Biological research has witnessed a paradigm shift from
focused reductionist approaches to a greater dependence on data
provided by large “Omics” techniques to provide insight into biological
systems and organization of physiological networks.
 The microarray scanning and data analysis will be discussed with
application expert of Spinco for Molecular Devices.
 Single or multiple slides can be scanned by using scanner. Using
defined scanning parameters, robotic arms can select slides and
position it for scanning.
The laser power wheel can adjust the laser strength. Fluorescence
signal is collected from the photomultiplier tubes. Each channel is
scanned sequentially and tiff images are saved.

Microarray data analysis

 It becomes crucial to make sense out of massive amount of data.

Software tools can help but they can’t answers all the questions
related to functional genomics and proteomics.
 It is more important to have a good understanding of both the
biology involved and the analytical techniques rather than relying only
on software. Challenges of microarray data analysis will be
discussed with Prof. Sudesh from Tulane University USA.
 Protein microarrays have found wide applications for discovery and
functional proteomic studies. Microarrays are used for analyzing
both antigens and antibodies in blood samples and other biological
fluids for biomarker discovery. Some of the representative applications
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VIII. Label-free detection technique

 Several conventional label-based detection approaches such as
fluorescence, chemiluminscence and radioactive isotopes are in
practice but researchers are exploring methods for label-free analysis
to get rid of the interference due to the tagging molecules and reduce
the complexity and assay time.
 Label-free techniques rely on measurement of inherent properties of
the query
molecules such as mass and dielectric property, and allow direct,
real-time detection of biomolecules in a HT manner eliminating
the requirement of secondary reactants.
 Many label-free techniques such as SPR, SPRi, Ellipsometry,
Interference and
nano-technique based approaches are emerging rapidly as a
potential complement to labeling methods.
 SPR-based biosensors provide label-free, real-time detection of
interactions. SPR sensorgram describes the changes in SPR signal
versus time.
 The SPR biosensors have played an important role in research into
biomolecules and their interactions and now they are increasingly
being used for detection and identification of chemical and biological
substances.
 Performing good SPR experiment and accurate interpretation of
binding
reactions from biosensors are always very challenging. Performing
good SPR experiments, data collection and processing can eliminate
artifacts and provide good quality data.
 The success of SPR experiments depends on the kinetic
measurement in real
time, monitoring adsorption of unlabeled analyte molecules to the
surface and, ability to monitor weakly bound interactions due to high
surface sensitivity.
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 Several nanotechniques
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quantum dots, gold nanoparticles are increasingly being used for
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phenomenon of Diffraction and Interference and use of diffraction-

based biosensors is another .
 These techniques holds great promise to become a technically robust
and user- friendly platform for clinical and diagnostic studies.

This century is considered as century of biology, in which life science

research is undergoing a profound transformation by employing various
omics technologies. Unraveling structural and functional details of
proteins at the proteome scale is very daunting task. However, Proteomics
has come to mean virtually everything in protein research and it has
quickly evolved to become an integral aspect of human biology and
medicine.

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