Biology Reference
In-Depth Information
Chapter 21
Analysis of Rice Proteins Using SDS-PAGE
Shotgun Proteomics
Karlie A. Neilson , Iniga S. George , Samantha J. Emery ,
Sridevi Muralidharan , Mehdi Mirzaei , and Paul A. Haynes
Abstract
In this chapter we describe the workfl ow used in our laboratory to analyze rice leaf samples using label-free
shotgun proteomics based on SDS-PAGE fractionation of proteins. Rice proteomics has benefi tted sub-
stantially from successful execution of shotgun proteomics techniques. We describe steps on how to pro-
ceed starting from rice protein extraction, SDS-PAGE, in-gel protein digestion with trypsin, nanoLC-MS/
MS, and database searching using the GPM. Data from these experiments can be used for spectral count-
ing, where simultaneous quantitation of several thousand proteins can be obtained.
Key words
Shotgun proteomics, Rice, Label-free, Plant proteomics, SDS-PAGE
Abbreviations
2-DE Two-dimensional electrophoresis
ACN Acetonitrile
BCA Bicinchoninic acid
BSA Bovine serum albumin
DTT Dithiothreitol
FDR False discovery rate
GO Gene ontology
GPM Global proteome machine
IAA Iodoacetamide
MS Mass spectrometry
MS/MS Tandem mass spectrometry
MudPIT Multidimensional protein identifi cation technology
NSAF Normalized spectral abundance factor
RP Reversed phase
SDS-PAGE Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
TCA
Trichloroacetic acid
WEGO
Web gene ontology annotation plot
