NAVIGATING THE PROTEOME TOOLS AND STRATEGIES FOR PROTEOME ANALYSIS

1. CCS HARYANA AGRICULTURAL
UNIVERSITY HISAR
@sonulangay
NAVIGATING THE PROTEOME: TOOLS
AND STRATEGIES FOR PROTEOME
ANALYSIS
ANKIT DHILLON
ankitdhill@gmail.com
CCS Haryana Agricultural University, Hisar

2. CCS HARYANA AGRICULTURAL
UNIVERSITY HISAR
@sonulangaya
Introduction
Types of proteomics
Approaches
Steps in proteomic analysis
Proteomics techniques
Case study
Conclusion
CONTENT

3. CCS HARYANA AGRICULTURAL
UNIVERSITY HISAR
@sonulangaya
• Proteome: The set of all expressed proteins in a cell,
tissue or organism.
• Proteomics: The study of the interactions, function,
composition and structures of proteins and their cellular
activities.
• Proteomics provides a better understanding of the
structure and function of the organism than genomics.
(Holman et al., 2003)
INTRODUCTION

4. 4
Expression proteomics
• Large-scale analysis of protein
expression-identify the main
proteins in sample and proteins
differentially expressed in related
samples, such as diseased vs
healthy tissue.
• Represent a useful drug target or
diagnostic marker.
• 2D-PAGE and mass spectrometry
are used.
Structural proteomics
• Large-scale analysis of protein
structure can help to identify the
functions of newly discovered
genes.
• Structural analysis can also show
where drugs bind to proteins and
where proteins interact with each
other.
• X-ray crystallography and mass
spectroscopy.
Interaction proteomics
• Large-scale analysis of protein
interactions
• Protein protein interactions helps to
determine protein functions, how
proteins assemble in larger
complexes.
• Affinity purification, mass
spectrometry and yeast two-hybrid
system are particularly useful.
Types of proteomics

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UNIVERSITY HISAR
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Approaches:

6. 6
Steps in Proteomic Analysis
PURIFICATION
OF PROTEINS
• Extraction of protein samples from whole cell,
tissue or sub cellular organelles.
SEPARATION OF
PROTEINS
• Gel electrophoresis, Spots are detected using
fluorescent dyes or radioactive probes.
IDENTIFICATION
OF PROTEINS
• Separated protein analyzed by mass spectrometry.

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UNIVERSITY HISAR
@sonulangaya
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PROTEIN
MIXTURE
PEPTIDE
MIXTURE
PROTEINS
PEPTIDES
MS ANALYSIS
PROTEIN
IDENTIFICATION
LC BASED
PROTEOMICS
2D GEL BASED
PROTEOMICS
Flowchart of analytical proteomics
DATABASE SEARCH
SEPARATION DIGESTION
DIGESTION SEPARATION

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UNIVERSITY HISAR
@sonulangaya
Proteomics
techniques
Separation
techniques
•1D Slab Gel
Electrophoresis
o2D Gel Electrophoresis
oCapillary Electrophoresis
oChromatography (HPLC,
SEC, IEC, RP, Affinity etc.)
oProtein Chips
Protein
Identification
techniques
Edman sequencing
Microsequencing
Mass spectrometry
Protein Structure
techniques
1.NMR
X-ray
crystallography
Computational
prediction
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UNIVERSITY HISAR
@sonulangaya
• 1D Slab Gel Electrophoresis
• 2D Gel Electrophoresis (SDS-PAGE)
• Capillary Electrophoresis
• Chromatography (HPLC, SEC, IEC, RP, Affinity etc.)
• Protein Chips (Protein microarray)
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Separation techniques

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• 1-DE, can isolate proteins with a molecular weight of
10 kDa to 300 kDa.
• It uses SDS, a detergent that denatures secondary and
non-disulfide-linked tertiary structures, and combines
them with a negative charge proportional to their
volume. This allows the calculation of molecular
weights.
• SDS-PAGE can be used to verify the purity of samples,
test protein purification, and calculate molecular
weights for unknown proteins.
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1D Slab Gel Electrophoresis

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UNIVERSITY HISAR
@sonulangaya
• 2-DE differentiates proteins better than 1-DE due to the variation
in molecular weight and isoelectric point of protein molecules.
• It also has a better resolution than 1-DE because the protein is
separated into two different dimensions. In first dimension, the
protein is separated based on net charge, in second dimension,
protein separation is based on the molecular mass and isoelectric
point. Thus, this method can detect different forms of proteins
such as PTMs and phosphorylation.
• There are many applications of 2-DE, including protein
expression profiling and cell map proteomics. Protein expression
profiling can be used for comparing normal and diseased tissues.
• However, 2-DE cannot detect proteins at a low molecular weight
and the limits of separation by isoelectric point and size.
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2D Gel Electrophoresis (SDS-PAGE/IEF)

12. CCS HARYANA AGRICULTURAL
UNIVERSITY HISAR
@sonulangaya
• Chromatography of affinity, size exclusion chromatography
(SEC) and ion-exchange chromatography (IEC) techniques can
be used to purify protein-based chromatography. In addition,
western blotting and the enzyme-linked immunosorbent assay
are used to identify selective proteins.
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Chromatography

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UNIVERSITY HISAR
@sonulangaya
• Protein microarrays also known as protein chips are the emerging class of proteomics
techniques capable of high-throughput detection from small amount of sample.
Protein microarrays can be classified into three categories;
• Analytical protein microarray: Antibody microarray is the most representative class
of analytical protein microarray.
• Functional protein microarray: Functional protein microarray is constructed by
means of purified protein, thus permits the study of various interactions including
protein–DNA, protein–RNA and protein–protein, protein–drug, protein–lipid,
enzyme–substrate relationship.
• Reverse-phase protein micro: Cell lysates obtained from different cell states are
arrayed on nitrocellulose slide that are probed with antibodies against target proteins.
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Protein microarrays

14. • Edman sequencing
• Microsequencing
• Mass spectrometry
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Protein Identification techniques

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UNIVERSITY HISAR
@sonulangaya
• Edman sequencing has been used to detect the sequence of amino acids in
peptides or proteins.
• This technique includes the reaction of chemicals, which remove and
determine amino acid residues present at the N-terminus of the
polypeptide chain.
• Mechanism: The peptide to be sequenced (50 aa) is adsorbed onto a solid
surface (glass fibre) coated with a cationic polymer.
• The Edman reagent, Phenylisothiocyanate (PITC), is added to the
adsorbed peptide, together with a mildly basic buffer solution. This reacts
with the amine group of the N-terminal amino acid.
• The terminal amino acid can then be selectively detached by the addition
of anhydrous acid. It can be washed off and identified by
chromatography, and the cycle can be repeated.
Limitations:
• It will not work if the N-terminal amino acid has been chemically
modified.
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Edman sequencing

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UNIVERSITY HISAR
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• Automated process of Edman reaction
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Microsequencing
(A Beckman-Coulter Porton
LF3000G protein sequencer)

17. CCS HARYANA AGRICULTURAL
UNIVERSITY HISAR
@sonulangaya
• MS is the best analytical tool for rapidly facilitating the sequencing of proteins. It can also be used to detect the
molecular weight of proteins. In this technique, protein molecules are ionized, and their mass is calculated
according to mass-to-charge ratios.
• The mass spectrometer has three main components: an analyzer, an ion source and a detector. The methods
used for ionization are ESI and MALDI.
• In Matrix Assisted Laser Desorption/Ionization (MALDI), a chemical matrix is mixed with the peptides and
spotted onto a metal plate to make a crystal lattice. The matrix chemicals pass the energy to the samples after
absorbing it. Then peptide ions are detected by a mass analyzer. MALDI creates mostly singly charged ions that
help to determine the m/z value.
• In electrospray ionization (ESI), the power is activated in the protein sample to create charged droplets that
increase gaseous ion production, which then are analyzed with a mass analyzer.
• Advantages of ESI are its high reproducibility and high elasticity to combine many categories of MS.
• Disadvantages of ESI is that it requires a large quantity of samples and multiple peaks are produced due to the
many charged ions that result in the complexity of MS/MS spectra.
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MASS SPECTROMETRY

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UNIVERSITY HISAR
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• NMR (Nuclear magnetic resonance spectroscopy)
• X-ray crystallography
• Computational prediction
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Protein Structure techniques

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UNIVERSITY HISAR
@sonulangaya
• The NMR is a leading tool for the
investigation of molecular structure,
folding and behavior of proteins.
• Nuclear magnetic resonance (NMR)
spectroscopy is the study of molecules
by recording the interaction of
radiofrequency (Rf) electromagnetic
radiations with the nuclei of molecules
placed in a strong magnetic field.
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NMR (Nuclear magnetic resonance spectroscopy)

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UNIVERSITY HISAR
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• X-ray crystallography is the most preferred technique for three dimensional structure
determination of proteins.
• The highly purified crystallized samples are exposed to X-rays and the subsequent
diffraction patterns are processed to produce information about the size of the
repeating unit that forms the crystal and crystal packing symmetry.
• X-ray crystallography has an extensive range of applications to study the virus
system, protein–nucleic acid complexes and immune complexes.
• Further, the three-dimensional protein structure provides detailed information about
the elucidation of enzyme mechanism, drug designing, site-directed mutagenesis and
protein– ligand interaction
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X-ray crystallography

22. CCS HARYANA AGRICULTURAL
UNIVERSITY HISAR
@sonulangaya
• In theory, a protein structure can solved computationally as it folds into a 3D structure
to minimizes its potential energy.
• ab initio/de novo folding methods: not practical (yet) due to its high computational
complexity. These procedures tend to require vast computational resources, and have
thus only been carried out for tiny proteins.
• Comparative modeling methods: uses previously solved structures as starting
points, or templates.
a. Protein threading — make structure prediction through identification of “good”
sequence-structure fit.
b. Homology modeling — identification of homologous proteins through sequence
alignment; structure prediction through placing residues into “corresponding” positions
of homologous structure models.
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Computational prediction

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UNIVERSITY HISAR
@sonulangaya
25

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UNIVERSITY HISAR
@sonulangaya
26
CASE STUDY 1

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UNIVERSITY HISAR
@sonulangaya
• This study identified differences between maize and sorghum grown in
water deficit conditions and identified proteins associated with drought
tolerance in these plant species.
• Label-free proteomics analysis was carried out to identify differences in
protein expression in the two species in response to water deficit.
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INTRODUCTION

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UNIVERSITY HISAR
@sonulangaya
• The Liquid Chromatography–Tandem Mass Spectrometry (LC–MS/MS) analysis
yielded 3154 distinct peptides, including 2752 entries for maize and 2794 for
sorghum.
• There were no matches for 718 (23%) peptides in both plant species for which an
orthologous had been named.
• 4 proteins (phenylalanine/tyrosine ammonia-lyases, indole-3-acetaldehyde oxidase,
sucrose synthase and phenol/catechol oxidase) were differentially expressed between
maize and sorghum in response to drought.
28

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UNIVERSITY HISAR
@sonulangaya
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Protein orthologs with differential expression between sorghum and maize in response to water deficit stress

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UNIVERSITY HISAR
@sonulangaya
• Proteomic analyses identified proteins differing in expression patterns between the
two species, revealing key metabolic pathways that explain the better drought
tolerance of sorghum than maize.
• This study highlights the importance of phenylpropanoids, sucrose, melanin-related
metabolites and indole acetic acid (auxin) as determinants of the differences in
drought stress tolerance between maize and sorghum.
• The selection of maize and sorghum genotypes with enhanced expression of the genes
encoding these differentially expressed proteins, or genetically engineering maize and
sorghum to increase the expression of such genes, can be used as strategies for the
production of maize and sorghum varieties with improved drought tolerance.
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29. CCS HARYANA AGRICULTURAL
UNIVERSITY HISAR
@sonulangaya
31
Proposed mechanism through which proteins differentially regulated between maize and sorghum
under water deficit stress lead to drought tolerance

30. CCS HARYANA AGRICULTURAL
UNIVERSITY HISAR
@sonulangaya
• Drought-induced phenylalanine and tyrosine metabolism leading to phenolic acid
biosynthesis via phenylalanine/tyrosine ammonia lyase (PAL/PTAL, i.e., PTAL)
enhances antioxidant activity.
• When activation of indole acetic acid oxidase occurs, auxin biosynthesis is enhanced
and can act coordinately with the biosynthesis of abscisic acid and jasmonic acid to
regulate plant responses to water deficit, leading to drought tolerance.
• Furthermore, improved drought tolerance can be achieved by regulation of sucrose
synthase to enhance osmotic adjustment through sucrose and D-fructose metabolism,
and through catechol oxidase-mediated detoxification of ROS that can be coupled to
biosynthesis of melanin-related and other phenolic compounds
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UNIVERSITY HISAR
@sonulangaya
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CASE STUDY 2

32. CCS HARYANA AGRICULTURAL
UNIVERSITY HISAR
@sonulangaya
• In this study proteomic analyses was done to understand the molecular
mechanisms regulating CMS in soybean using 2 CMS lines (W931A and W931B)
• Compared to W931B, which had healthy, oval pollen grains, W931A showed
shrunken or degraded pollen grains with an irregularly thickened endothelium and
decreased starch accumulation.
• A total of 630 DEPs were identified in W931A compared to W931B at the
uninucleate microspore (UM) stage, with 305 up-regulated and 325 down-
regulated.
• At the binucleate pollen (BP) stage, 242 proteins were up-regulated in W931A
compared to W931B, whereas 384 were down-regulated.
• Proteomic analysis revealed 343 differentially expressed proteins (DEPs), which
were mainly involved in carbon metabolism, glycolysis/gluconeogenesis, and
nitrogen metabolism.

33. CCS HARYANA AGRICULTURAL
UNIVERSITY HISAR
@sonulangaya
• Four genes with demonstrated roles in pollen development were differentially
expressed, including AGAMOUS-LIKE 104, PROTEIN-TYROSINE-
PHOSPHATASE 1 and PHOSPHOLIPASE A2.
• A total of 53 genes and the corresponding proteins were differentially expressed in
W931A at both the uninucleate microspore (UM) and binucleate pollen (BP) stages
and many of these were pectinesterases, polygalacturonases, peroxidases and ATPases
• Soybean pollen abortion in W931A was likely regulated by multiple pathways
involved in pollen development, pectin catabolism, responses to oxidative stress,
carbon metabolism, the TCA cycle and oxidative phosphorylation.
• These results provide novel insights and candidate genes for further studies into the
mechanism underlying CMS in soybean; this will ultimately promote the application
of W931A in soybean heterosis and breeding.
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34. CCS HARYANA AGRICULTURAL
UNIVERSITY HISAR
@sonulangaya
• Conclusively, Proteomics is a fast, sensitive technology that provides
high proteome coverage.
• All fields related to biological sciences have been benefited with
increasing use of proteomics techniques.
• Overall, the application of proteomics in plant breeding holds immense
promise for unravelling novel biomarkers and advancing the development
of resilient crop varieties, although ongoing efforts are imperative to
optimize the reproducibility and efficacy of proteomic tools in this
context.
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CONCLUSION

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UNIVERSITY HISAR
@sonulangaya
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