Proteomics

Gurbachan S. Miglani
Visiting Professor

School of Agricultural Biotechnology

Punjab Agricultural University
Ludhiana
 Why Study Protein Expression?
Nucleus Cytoplasm
Inactive mRNA
RNA
Degradation
control

Primary
DNA RNA mRNA mRNA
transcript
Translation control
RNA
protein Modified
RNA Transport
protein
Transcriptional Processing control
control control
Post-translational
control

Gygi et al. 1990. Mol. Cell. Biol. 2: 1720-1730

Relation
ship • Genomics focuses on an organism's genetic makeup,
while proteomics focuses on gene products
betwee • Both are interrelated
n • Molecular genetic analysis would be incomplete
Genomi without protein analysis
• Proteomics checks out the functional relevance of the
cs and protein and how different proteins work together
Proteo
mics
Proteome

• Term “proteome” introduced by Mark Wilkins (1994) in a

symposium on "2D Electrophoresis: from protein maps to
genomes"
• Total protein complement of a given cell which
may change
(a) from one cell type to another,
(b) under different environmental conditions, or
(c) during development
• Proteome of a cell is more complex than
transcriptome under given conditions
Complexity of proteome compared to transcriptome and genome

http://www.piercenet.com/method/overview-post-translational-modification
Techniques involved in Proteomics
1. Protein Extraction
Phenols/Ammonium acetate/Methanol/Acetone

2. Protein Separation
Gel-based and Gel-free methods

3. Protein Analysis
Edman degradation N-termini sequencing

Peptide mass fingerprinting

Mass spectrometry
Tandem mass
spectrometry
 What does proteomic analysis involve?

1. Protein distribution, characterization and identification

2. Protein-protein interactions
3. Differential display proteomics
1. Protein Distribution, Characterization and Identification

• Proteomics combines
o two-dimensional polyacrylamide gel electrophoresis (2D-
PAGE) that separates, maps and quantifies proteins
with
o mass spectroscopy (MS)-based sequencing techniques
identifying both the amino acid sequences of proteins and
their post-translation appendages
• Differences between the reference (standard) and altered
states are measured by quantifying spot intensities
• Protein spot are excised and incubated with proteases (e.g.,
trypsin) which reduce the proteins to peptides that are
more easily analyzed by Mass Spectroscopy
• The masses of the peptides are measured and protein is
identified
2. Protein-Protein Interactions

• Protein-protein interactions are studied by yeast-two hybrid (Y-

2H) system developed by Fields and Song (1989)
• Y-2H uses two fusion proteins to activate the transcription of a
reporter gene in yeast
o The first fusion protein contains a DNA-binding (DB) domain
fused to a bait protein of interest
o The second is transcriptional activation domain (AD)fused to a
second protein of interest
• Specific interaction between the two chimeric proteins leads to
transcriptional activation of the reporter genes which in easily
scored by either colour-based assay or by auxotrophic
complementation
GAL4 regulatory proteins has two domains
3. Differential Display Proteomics

Tools used are:

a) Two-dimensional polyacrylamide gel electrophoresis

(2-D-PAGE)
b) Antibody-based screening (ABS)
c) Protein chip technology/Protein Microarray
a) Two-Dimensional Polyacrylamide Gel Electrophoresis
(2-D-PAGE)

• 2D-PAGE is a form of gel electrophoresis in which proteins are

separated and identified in two dimensions oriented at right angles
to each other
• In this technique, proteins are separated by two different physical
properties
o The first dimension, isoelectric focusing (IEF), separates
proteins on the basis of their net charge
o The second dimension, sodium dodecyl sulfate (SDS)-PAGE,
further separates the proteins by their mass

Small changes in charge and mass can easily be detected by this

method, because it is rare that two different proteins will resolve to
the same place in both dimensions
 b) Antibody-Based Screening (ABS)

ABS is used to explore functional relationship between

proteins by using individual proteins directly as assay for
their building partner in biological samples coated within
microwell plates using  Enzyme-linked Immunosorbent
Assay (ELISA) test techniques
 c) Protein Chip Technology/Protein Microarray

• A variety of bait proteins such as antibodies, are immobilized

in an array format onto specially treated surface
• The surface is then probed with the sample of interest and
only the proteins that bind to the relevant antibodies remain
bound to the chip
• These chips are then placed in a chip reader which includes a
mass spectrophotometer for analysis of the proteins
• The protein chip is probed with fluorescently labelled proteins
from two different cell states
• Cell lysates are labelled by different fluorophores and mixed
such that the colour acts as a reader for the change in
abundance of the protein bound to the antibody
Protein Chip Technology (Microarray)
• Protein-Protein Interactions
• Protein Localization and Compartmentalization
• Protein Post-Translational Modifications (PTMs)
• Protein Function
• Protein Expression Studies
• Protein identification
What proteomics can do…
 Protein Identification

• 57 protein spots identified

o 24 upregulated
o 28 downregulated
o 5 induced by salt stress
 Novel identified - U8, U9, U13, D1, U11
 Recently Identified Proteins In Different Crop Species
Species Technique Number of proteins Reference
identified
Glycine max L. Merr High-throughput LC– 4975 proteins from nuclear preparations Copper et al (2011)
MS/MS of Phakopsora pachyrhizi infected
soybean leaves
Manihot esculenta Nano LC–MALDI- 1387 protein groups from starch-rich Qwiti et al (2011)
Crantz TOF/TOF cassava roots
Musa paradisiaca L. Nano LC–MS/MS 3477 proteins from plantain Yang et al (2012)
seedlings

Musa ssp. CPLL+nanoLCMS/MS 1131 proteins from banana fruit Esteve et al (2013)
Zea mays nanoLC–MS/MS 1105 proteins from chloroplasts Friso et al (2010)
Phaseolus vulgaris L. LC–MS/MS Over 3000 proteins from bean leaves Lee et al (2009)

Phoenix dactylifera L 2-D PAGE + MALDI- 58 proteins from date palm leaves Sghaier-Hammami
TOF/TOF et al (2012)
Solanum tuberosum RP-LC+ nano LCMS/MS 4463 proteins from potato tubers Yang et al (2011)

Triticum aestivum 2-D PAGE + LC–MS/MS 230 proteins from grains Gao et al (2009)

Extracted from Vanderschuren et al (2013) J Proteomics 93: 5-19

 Protein Expression Studies

J. Exp. Bot. (2014) 65 (2): 655-71

• 27 proteins differentially expressed on 2-DE

o 25 identified by MALDI –TOF/TOF MS
• expression of 10 representative proteins was analyzed
 Differential-expression proteomics studies in the field of
plant response to abiotic stress factors

Stress factor Main results at proteome level

Cold Up:cp29, SAM , ROS, COR/LEA, HSP70
Down: HSP90
Heat Up: OEE1 ,Trx h, sHSPs, chloroplast chaperonins, eIF5A-3 (PCD)

Drought Up: Photosynthesis- and carbon-related proteins, GAPDH, PGK, PGM, HSP70
Down: Homoeobox-leucine zipper protein, AP2/EREBP transcription factor
Waterlogging Up: GAPDH, glucose fermentation, signal transduction, PCD, RNA processing, ROS
scavenging, ascorbate and GSH metabolism
Down: Rubisco LSU

Salinity Up: APX, DHAR, Trx h, peroxiredoxin, SOD, NDPK, ACPreductase

Down: GAPDH, fructokinase 2, Rubisco LSU and SSU, PGK

Excessive mineral nutrients:

Aluminium Up: Cu/Zn-SOD, GST, CS
Boron Up: IDS2, IDS3, MTK
Copper Up: germin-like proteins, CAX,tubulin α-6, actin 8
Down: F15H18.8, actin-related protein 4

Extracted from Kosová et al (2011) J Proteomics 74: 1301-22

 Protein Function

Assignment of 190 identified nuclear proteins to functional categories

Protein Post-translational Modifications (PTMs)

P = in vitro phosphorylated RPG1 protein positive control; U = uninoculated control;

PS = phosphoserine antibody; PT = phosphothreonine antibody.
 Protein PTM studies in plants

Modification Species Reference

Phosphorylation Arabidopsis Nuhse et al (2004)
Arabidopsis Schulze (2012)
Spinach Carlberg et al (2003)
Rice Scafaro et al (2010)
Barley Horie et al (2011)
Glycosylphosphatidyl Arabidopsis Elortza et al (2003)
inositol modification
Ubiquitination Arabidopsis Vierstra (2004)
Tyrosine nitration Pea Morales (2013)
Protein Localization and Compartmentalization

Two ER proteins – calnexin and cytochrome b5; and

One Golgi protein, gtl-6;
found in both ER and Golgi due to the cross-contamination
 Proteins localized using proteomics

Proteins Predicted Reference

localization
Glycosyltransferase family 29 Golgi Henrissat et al (2001)
Glycosyltransferase family 34 (AtGT5) Golgi Faik et al (2002)
Glycosyltransferase family 8 (quasimodo1) Golgi Bouton et al (2002)
Obtusifoliol 14-demethylase (CYP51) ER Kushiro et al (2001)
β-ketoacyl reductase (glossy8) ER Xu et al (2002)
Protein disulfide isomerase ER Sevier et al (2002)
NADH dehydrogenase family (ndb4) Mitochondria Michalecka et al (2003)
Mitochondrial phosphate transporter (AT5) Mitochondria Hamel et al (2004)
Long chain acyl-CoA synthetase 9 (LACS9) Plastid Schnurr et al (2002)
TOC75 homolog Plastid Hinnah et al (1997)

Extracted from Dunkley et al (2004) Mol. Cell Proteomics 3 (11): 1128-34

 Protein-Protein Interaction

Study conducted using the publicly available program STRING 9.05

Fourteen key proteins involved in cowpea resistance

FSD1, APX1, ATTRX2, RBCL, AT2G45290, TPI, GAPA, CA1, PSBP-1,RCA,

TRX-M4 , PSAG, PSAD-2 , AT4G37230

interact with each other

Protein–protein interaction network analyzed by STRING software
 Main Protein-Protein Interaction (PPI) resources for plants

Database Plant Reference

BioGRID Mainly Arabidopsis Stark et al (2011)
DIP Arabidopsis Xenarios et al (2000)
MINT Arabidopsis Licata et al (2012)
APID Arabidopsis Prieto et al (2006)
PAIR Arabidopsis Lin et al (2011)
BAR Arabidopsis, Oryza Geisler-Lee et al (2007)
PRIN Oryza Gu et al (2011)
STRING All Szklarczyk et al (2011)
IntAct All Kerrien et al (2012)
iPfam All Finn et al (2005)

Extracted from Braun et al (2013) Annu. Rev. Plant Biol. 64: 161-187
 Applications of Proteomics
• Salt-responsive proteins in rice (Malakshah et al. 2007)
• Proteomic profiles of transgenic wheat cultivars (Scossa et al. 2008)
• Identification of novel allergens in maize (Fasoli et al. 2009)
• Improving plant protection (Komatsu et al. 2013)
• Plant cell wall proteome (Printz et al. 2015)
 Real time PCR analysis of three genes that were salt responsive at
protein level.

 No concordance detected between change in level of genes and

proteins expression
 With strong increase in the amount of the transgenic protein, the endogenous
glutenin subunit, all subclasses of gliadins, and metabolic as well as chloroform/
methanol soluble proteins were diminished in the transgenic genotype
Use of proteomic techniques to evaluate genetically modified crops

Crop Transgenic protein/gene Reference

Maize CryIA(b)/cryIA(b) Coll et al (2011)

Pea α-amylaseinhibitor-1/αai1 Slam et al (2009)

Potato Glucanbranchingenzyme/W2 Lehesranta et al (2005)
Rice PAT/bar Gong et al (2012 )
Soybean CP4EPSPS/cp4epsps Barbosa et al (2012)

Tobacco S c FvB9/ sc FvB9 Carli et al (2009)

Tomato ScF vG4/sc FvG4 Carli et al (2010)
Wheat LMW -GS /LMW -GS gene Scossa et al (2008)

Extracted from Gong and Wang (2013) Front. in Plant Sci. 4:1-8
 Novel Allergens Identified

 Vicilin
 Globulin-2
 50 kDa gamma-zein
 Endochitinase
 Thioredoxin
 Trypsin inhibitor
 Proteomic studies on identification of novel plant allergens

Plant name Allergen Name Reference

Hazelnuts Cor a 9 (11S globulin) Beyer et al (2002)

Latex Hev b 1-8 ,Hev b 13 Wagner et al (2007)
Peanuts Ara h 1,Ara h 3, Ara h ¾, Ara h 4,Gly 1,iso-Ara h 3 Boldt et al (2005)

Tomato Vicilin , Legumin Bässler et al (2009)

Wheat α-amylase inhibitor, β-amylase, profilin , Serpin Sotkovský et al (2008)

Maize Vicilin 2D, globulin-2, 50 kDa γ-zein, Fasoli et al (2009)

Endochitinase, thioredoxin, trypsin inhibitor

Sweet orange Cyt s 1 Pignataro et al (2010)

Rice 52 kDa globulin, 63 kDa globulin Satoh et al (2011)

Extracted from Nakamura and Teshima (2013) J Proteomics 93: 40-49

• Gradual utilization of nutritional reserves (in rice seed germination)
• Comparative proteomic analyses of C4 chloroplasts influencing
sunlight conversion efficiency
• Identifying the proteins regulating male sterility
• Analyzing the interaction between crops and bacteria
• Identifying the protein accumulated during abiotic stress
• Alfalfa stems cell wall proteins extracted using three-steps
extraction procedure
• LC-MS analysis identified highest number of cell wall proteins
 What has proteomics done
for crop improvement?
 Greatly advanced our understanding of proteome composition,
modulation and modification
 Unveiled a number of proteins potentially relevant for crop breeding
 Contributed better understanding of physiological mechanisms
underlying plant stress response
 Cross-species (ortho proteomics) and cross-genotype (comparative
proteomics) proteome information holds significant potential for
advanced crop breeding programs