Topic:

Comprehensive proteome analysis of lettuce latex using

multidimensional protein-identification technology

Presented to:
Dr. Murtaza Hasan
Presented by :
Haleema Sikandar
Department :
Biochemistry and biotechnology
Semester :
4th

Roll no :
76
 Outline of presentation

 Title page
 Abstract
 Introduction
 Material & Method
 Result & Discussion
 Acknowledgments
 References
 Title page
Title :
Comprehensive proteome analysis of lettuce latex using
multidimensional protein –identification technology
Author :
Won Kyong Choa.1 ,Xiong -Yan Chena,1 , Nazim Mohamad Uddina,1 ,
Yeonggil Rima , Juyeon Moona, Jin- Hee Junga ,Chunlin Shi a,Hyosub
Chua ,Suwha Kimb ,Seon-Won kima , Zee- Young Parkb ,Jae-Yean Kima* .
1 These authors contributed equally to this work.
Corresponding author:
Jae-Yean Kim a*
Affiliation :

Division of Applied Life Science ,Environmental

Biotechnology ,National Core Research Centre ,PMBBRC, Gyeongsang
National University ,Jinju 660-701, Republic of Korea
Department of Life Science ,Gwangju Institute of Science and
Technology ,Gwangju 500-711,Republic of Korea .

Keywords :

Lactuca sativa
Lettuce
Asteraceae
Latex
Proteome
 Abstract:
Component of Abstract :
 Problem:
Lettuce produce a milky latex of variable chemical compositions.
 Objective :
Main physiological roles of the latex in higher plants , we
embarked on its proteome analysis
 Method :
Multidimensional protein identification technology
Bioinformatics analysis
 Result :
Analysis shows most frequently encountered proteins in the latex
were organellar proteins from plastid &mitochondria
 Application :
Resistance & viral proteins were present in the latext perform a
possible function defense and pathogenesis .
 Introduction:
M1:(Importance)

• Lettuce is one of the most important leafy vegetable .Lettuce

belong to family Asteracea and contain nine pairs of chromosome
.The lettuce secret a colored juice that is often milky called latex .
• The latex accumulate in specialized continuous tube called
laticifers . The primary role of laticifers is to provide a place to
deposit numerous metabolites .
• Laticifer is important for defense mechanism against insects .
Move 2 :(current knowledge )

•In recent review paper it was describe very well that laticifers might
act as a putative storage of excess atmospheric carbon and that they
can regulate levels of atmospheric isoprene gas.

•Many of other latex –containing plants are important to the global

economy because poppy is a source of opium and the rubber tree is
well known as being a source of natural rubber .

Move 3 :(Application )

•To achieve a detailed understanding of the latex proteome MudPIT

has been used to identify proteins in lettuce latex .
 Material and Method:
Chemicals and Reagents :
• PBS solution
• Liquid N2
• H2O
• Urea
• Coomassie brilliant blue
• Ammonium bicarbonate
• CH3CN
• Iodoacetamide
• Trypsin solution
• General experimental procedures
• Lettuce latex sampling
• Sodium dodecyl sulfate polyacrylamide gel electrophoresis
• In- gel digestion and peptide sample preparation
• Micro- LC/LC-MS/MS analysis
• Database search
 Results & Discussion
 Protein identification by MS/MS analysis:
MS/MS spectra obtained from the three gel sections were used to
search for sequence similarity within a protein database constructed
from the lettuce EST database and the lettuce protein database
obtained from the NCBI .

 Subcellular localization of the identified latex proteins :

Protein that were predicted to localized in
1. Plastids (17%)
2. Mitochondria(13%)
3. Cytoplasm(6%)
4. Nucleus (4%)
Fig .1.SDS-PAGE of latex proteins from lettuce .(A) SDS-PAGE analysis of the
latex proteins from lettuce .lanes : molecular marker ,1(first experiment)
,2(second experiment);the molecular masses are indicated on the left .The
one-dimensional gel of the latex proteins was excised into three sections ; H
(high),M(middle),and L (low).(B) Venn diagram of proteins identified in each
gel section .A total of 587 proteins were identified by MudPIT analysis ,and
138 proteins were found in at least two gel sections .Among them, 28
proteins were detected in all gel sections .
 Organellar proteins related to various metabolites :

•14 proteins composing main protein complexes of the oxidative

phosphorylation in mitochondria , such as.
•cytochrome oxidase
•ATP synthase
•NADH dehydrogenase
were detected in the lettuce latex .

 Functional classification of lettuce latex proteins :

The identified lettuce latex proteins were classified into functional
classes using FunCat .
1. Metabolism related proteins(20%)
2. Cell rescue, defense and virulence proteins (12%)
3. Proteins with binding function (10%)
4. Cellular transport proteins (8%)
Fig.2. Functional classification and subcellular localization of the 587 latex
proteins identified .Each of the unique proteins was classified according to
the FunCat groups (A)Functional classification of the 587 latex proteins
identified .A , transcription ;B, protein synthesis ;C,intrection with the
environment ;D , development ; E , cellular communication ,F,cell cycle
(B)Subcellular localization of 587 latex proteins identified.A,plasma
membrane :B , cell wall ;C , cytoskeleton ;D ,Endoplasmic reticulum,E,Golgi;
 Metabolism –related proteins :
• The most interesting latex protein related to metabolism is
hydroxymethylglutaryl coenzyme A synthase which was identified
in H.brasiliensis and is involved in the early steps of rubber
biosynthesis and functions as a regulatory enzyme is isoprenoid
biosynthesis.

 Protein related to cell rescue ,defense , and virulence

• Ring necrosis is a serious disease of lettuce which results in necrotic
rings and ring patterns in leaves . This disease is caused by LRNV
,which is transmitted by the chytrid fungus Olpidium brassicae as a
vector from the soil.
• In lettuce ,a large no of RGC2 genes have been identified from
seven genotypes of three lactuca species .RGC2 are resistance
protein .
Fig.3. The identified lettuce latex proteins involve in oxidative phosphorylation
derived from KEGG pathway implemented in Blas2go .lettuce latex proteins
were converted to the corresponding Arabidopsis locus and were mapped on
the corresponding KEGG pathway .
 Concluding remarks :
The main purpose of this study was to give the first comprehensive
overview of the latex proteins of lettuce based on the available
lettuce EST database. Successful application of the lettuce EST and
NCBI database led to the identification of a large no of lettuce latex
proteins .
 Acknowledgments:
We thank Mrs . Sunseon kim for technical assistance , Prof . Ju
Hyeon Kim for the lettuce seeds ,and the composite genome
project team for EST data. This work was supported by KOSFE
grants to the National Research Lab Program (M10600000205-
06j000020510),the WCU program(R33-2008-000-10002-0) and the
Environmental Biotechnology National core research
center(R152003-012-01003-0).X.Y.C. and Y.R were supported by
fellowships from the Brain Korea 21 Program ,a program of the
Korean Ministry of Education ,Science ,and Technology.
 References :
• Aki ,T .,Shigyo,M.,Nakano ,R.,Yoneyama ,S.,2008 .Nano scale
proteomics revealed the presence of regulatory proteins including
three FT-like proteins in phloem and xylem saps from rice . Plant cell
physiol .49, 767-709
• Alvarez , S , Goodger ,J.Q ,March ,E.I, Chen , S , Asirvatham ,V.S ,
Schachtman ,D.P , 2006.Characterization of the maize xylem sap
proteome .J . Proteome Res .5,963-972