Biology Reference
In-Depth Information
tant for identifying the 1,000-1,500 proteins thought to be
present in plant mitochondria. Mitochondria have also provided
an excellent model system through which a number of novel pro-
teomic analyses have been developed. They are relatively discrete
membrane-bound organelles that are found in signifi cant numbers
in most plant tissues and typically they represent 2-5 % of total cel-
lular protein. Procedures for mitochondrial isolations from differ-
ent plants are well established and are capable of producing
milligrams of mitochondrial proteins from 10 to 100 g of fresh
plant material. The majority of the protein complement is within
the dynamic range of standard proteomic techniques, although
low-abundance and hydrophobic proteins still represent challeng-
ing targets.
A variety of studies have now begun to uncover the proteome
of mitochondria from Arabidopsis [
1
-
24
], rice [
5
,
25
-
29
], wheat
[
30
,
31
], maize [
32
], barley [
33
], pea [
34
,
35
], soybean [
36
,
37
],
medicago [
38
], and Chlamydomonas [
39
]. Gel-based protein sep-
aration of proteins followed by mass spectrometry identifi cation
has been the most widely used strategy to date to determine pro-
teome composition. Isoelectric focusing (IEF)/SDS-PAGE is val-
ued for its reproducible and well-resolved separation of soluble
proteins, while blue native (BN)/SDS-PAGE has been deployed
to defi ne the protein components of the large protein complexes
I-V of the respiratory chain, and has proven particularly successful
for visualizing hydrophobic proteins not amenable to IEF [
8
,
18
,
19
]. Gel-bound proteins are routinely identifi ed by a wide range of
tandem mass spectrometry techniques, while traditional approaches
such as Edman degradation and peptide mass fi ngerprinting (PMF)
[
1
,
2
,
40
] have been largely superseded by ESI-MS/MS [
3
,
12
,
19
,
20
,
24
] and MALDI-TOF/TOF [
41
]. Larger scale analyses
have digested complex mitochondrial samples in the liquid phase,
and used high-performance liquid chromatography (HPLC) of
peptides coupled to tandem mass spectrometry to provide many
hundreds of identifi cation of mitochondrial proteins [
6
,
7
,
24
,
28
].
Critical to studies of mitochondria is the deduction of which pro-
teins are of mitochondrial origin and which are non-mitochondrial.
Contaminants can co-localize with mitochondrial fractions during
isolation due to similarities in density (as with Percoll
®
centrifuga-
tion) or charge (as with free fl ow electrophoresis (FFE)) or may be
connected or associated biochemically such as components of gly-
colysis [
42
,
43
] or ascorbic acid (vitamin C) biosynthesis [
44
]. The
use of quantitative proteomics platforms to compare protein abun-
dance in enriched versus depleted mitochondrial fractions provides
a high-throughput basis for determining the subcellular localiza-
tion of proteins [
9
,
17
,
24
]. The following techniques will outline
a basic procedure for isolating plant mitochondria, investigating
the proteome using 2D-SDS-PAGE-DIGE or LC-MS/MS of pep-
tides, and defi ning enrichment or contamination by differential
