Biology Reference
In-Depth Information
Chapter 33
Shotgun Proteomics of Plant Plasma Membrane
and Microdomain Proteins Using Nano-LC-MS/MS
Daisuke Takahashi , Bin Li , Takato Nakayama , Yukio Kawamura ,
and Matsuo Uemura
Abstract
Shotgun proteomics allows the comprehensive analysis of proteins extracted from plant cells, subcellular
organelles, and membranes. Previously, two-dimensional gel electrophoresis-based proteomics was used
for mass spectrometric analysis of plasma membrane proteins. In order to get comprehensive proteome
profi les of the plasma membrane including highly hydrophobic proteins with a number of transmembrane
domains, a mass spectrometry-based shotgun proteomics method using nano-LC-MS/MS for proteins
from the plasma membrane proteins and plasma membrane microdomain fraction is described. The results
obtained are easily applicable to label-free protein semiquantifi cation.
Key words
Plasma membrane, Detergent-resistant membrane, Microdomain, Nano-LC-MS/MS,
Shotgun proteomics, Label-free semiquantifi cation, In-gel digestion, In-solution digestion
1
Introduction
Comprehensive protein identifi cation consists of solubilization and
pre-separation of proteins, peptide digestion and fragmentation
using trypsin, and separation and detection of each peptide with
liquid chromatography-tandem mass spectrometer (LC-MS/MS)
[
1
-
4
]. Compared with soluble protein proteomics, the solubiliza-
tion and pre-separation steps for proteomics of cellular membranes
including the plasma membrane (PM) is diffi cult because of a large
number of the highly hydrophobic properties of the proteins and
its highly hydrophobic lipid environments [
5
-
7
]. Although mem-
brane proteomics has been performed by two-dimensional gel
electrophoresis (2-DE)-based proteomics [
5
,
7
,
8
], PM proteins
are particularly diffi cult to solubilize and 2-DE-based proteomics
requires a large amount of valuable PM proteins [
9
]. Microdomains,
which are considered to exist as extremely hydrophobic compart-
ments in the PM because of the enrichment of specifi c lipids and
