Download Proteome analysis of Arabidopsis thaliana mitochondrial proteins

Innovations Forum: Proteome analysis of A. thaliana
Proteome analysis of Arabidopsis thaliana
mitochondrial proteins
L. Sweetlove*, J. Flensburg†, O. Rönn†, H.-O. Gustavsson†, and E. Forsberg†
*Department of Plant Sciences, University of Oxford, Oxford, UK; †Amersham Biosciences AB, Uppsala, Sweden
Mitochondria were isolated from Arabidopsis thaliana cells and the cell extracts analyzed
by 2-D electrophoresis-mass spectrometry (2D-MS). Low-abundance proteins were
analyzed by peptide-mass fingerprinting (PMF) using Ettan™ MALDI-ToF Pro. Peptide-mass
fingerprinting of 50 selected spots from the 2-D electrophoresis gel resulted in a 74%
protein identification rate. Proteins not successfully identified by PMF were analyzed by
chemically assisted fragmentation MALDI using Ettan CAF™ MALDI Sequencing Kit in
combination with Ettan MALDI-ToF Pro. Using this approach increased the identification
rate of the selected mitochondrial proteins to 86%.
Introduction
During the last decade, Arabidopsis thaliana, a small flowering
weed, has developed into a model system for plant development,
physiology, and genetics. Scientists all over the world are using
new tools to investigate the plant’s proteome to discover the
processes common to all plants. A. thaliana is a member of the
mustard (Brassicaceae) family, which includes cultivated species
such as cabbage and radish. The sequencing of the whole genome
of A. thaliana was completed during 2000 by the international
Arabidopsis Genome Initiative (1).
In this study, an analysis of the mitochondrial proteome was
undertaken. Plant mitochondria have a multiplicity of functions
other than that of ATP production. Many of these, such as synthesis
of ascorbic acid and oxidation of the amino acids proline and glycine,
are unique to plants. However, the identity of the complete set of
mitochondrial proteins that constitute the mitochondrial proteome
has not been resolved. Hence, the complete set of metabolic
functions of plant mitochondria remains undefined.
Some 95% of the genes encoding mitochondrial proteins are
located in the nucleus and the proteins are targeted back to the
mitochondrion using encrypted targeting information in the
protein sequence. Identification of all genes carrying these
targeting sequences is one way of defining the complete set of
mitochondrial proteins. However, the presence of multiple types
of targeting sequences, and the lack of homology between
consensus targeting sequences in plants and animals, diminish
confidence in this approach.
The genome of A. thaliana has been analyzed in this fashion and the
number of gene products with a predicted mitochondrial location
range from as little as 349 to as many as 2897 (1).
Since the initial launch of the complete genome sequence of
A. thaliana, however, attempts have been made to improve the
accuracy of gene models. Introduction of improved gene prediction
algorithms as well as annotation based on increased availability of
full-length cDNAs has dramatically improved the accuracy of the
A.thaliana genome information (2). To ascertain whether this
improves the ability to identify proteins by PMF, we repeated the
analysis of some A. thaliana mitochondrial proteins, described earlier
in reference 3. A total of 50 proteins were analyzed using twodimensional electrophoresis-mass spectrometry (2D-MS).
Peptide-mass fingerprinting using Ettan MALDI-ToF Pro was used
for the first tier of protein identification. To further improve the
protein identification rate using MALDI-ToF, chemically assisted
fragmentation MALDI was used to analyze proteins that were not
successfully identified by PMF. This technique enables peptide
sequence data to be acquired simply, quickly, and with good
sensitivity using MALDI ToF MS.
Materials and Methods
First-dimension electrophoresis was performed using Immobiline™
DryStrip pH 3–10 NL, 18 cm on Multiphor™ II IEF System. Seconddimension separation was performed using Ettan DALTtwelve Large
Vertical Electrophoresis System using large format, lab-cast SDS gels.
The gel was stained with colloidal Coomassie™ Brilliant Blue
(American Bioanalytical) and fifty spots of low staining intensities
were selected for further manipulation.
Life Science News 15, 2003 Amersham Biosciences 13
Innovations Forum: Proteome analysis of A. thaliana
Using Ettan Spot Handling Workstation, selected spots were
automatically picked, tryptically digested, and the resultant tryptic
peptides co-spotted with α-cyano-4-hydroxycinnamic acid matrix
solution onto the MALDI sample slide.
In the current analysis, identification of these six proteins was
confirmed and the eight previously unidentified proteins were this
time successfully identified (Table 1). This clearly demonstrates that
Protein identification was performed by PMF using the Ettan
MALDI-ToF Pro mass spectrometer. The entire process for
protein identification by PMF was fully automated from data
acquisition, through calibration, spectral processing, and
subsequent databsase searching.
Proteins not successfully identified by PMF were further analyzed by
chemically assisted fragmentation MALDI using Ettan CAF MALDI
Sequencing Kit in combination with Ettan MALDI-ToF Pro. The
relevant peptide digests were derivatized using the kit according to
the manufacturer’s instructions.
Results and discussion
We were able to consistently resolve approximately 100 highabundance proteins and 250 low-abundance proteins in an
experimental setup using a wide pH 3–10 nonlinear gradient
on the gels in the first dimension (Fig 1). Fifty of these proteins,
most of them representing low-abundance proteins on the 2-D
electrophoresis gel, were analyzed. After automated spot handling,
PMF identified 74% of these proteins (37 of 50 [Fig 2]). Fourteen
of the 50 proteins in this study had been analyzed earlier (3), where
only six proteins (43%) were successfully identified.
Fig 2. Peptide-mass fingerprinting of selected mitochondrial proteins yielded an
unambiguous match from an enquiry of the A. thaliana database.
improvements in genomic database annotations, as well as the
superior mass accuracy of Ettan MALDI-ToF Pro contribute to a very
high success rate of protein identification through PMF.
The remaining unidentified samples were analyzed by chemically
assisted fragmentation MALDI. The CAF reagent included in Ettan
CAF MALDI Sequencing Kit, and initially reported by Keough and
colleagues in 1999 (4), allows the rapid and efficient acquisition of
peptide sequence information using MALDI-ToF.
Chemically assisted fragmentation MALDI offers two key benefits.
One is that fragmentation efficiency of singly charged peptides
produced during MALDI post-source decay is enhanced, thereby
increasing sensitivity. The other is that only a single fragment ion
series (y-ion series) is observed, thereby increasing sensitivity and
simplifying the spectrum (applicable to any MALDI MS/MS spectrum).
In this study, six of the 13 proteins not identified by PMF were
unambiguously identified by chemically assisted fragmentation
MALDI using Ettan CAF MALDI Sequencing Kit. In the example
shown in Figure 4, nine consecutive residues of sequence were
obtained and used to identify the protein. Using this technique, the
protein identification rates from a single MALDI were increased to
86%. Ettan CAF MALDI Sequencing Kit is equally applicable to any
MS instrument equipped with a MALDI ionization source.
Fig 1. Two-dimensional electrophoresis of the A. thaliana mitochondrial proteome. Firstdimension separation: 18 cm Immobiline DryStrip pH 3–10 NL IPG strips, Ettan IPGphor™
IEF System. Second-dimension separation: Large-format lab-cast gels, Ettan DALTtwelve
Large Vertical Electrophoresis System. Staining: Coomassie™ Brilliant Blue.
14 Life Science News 15, 2003 Amersham Biosciences
Innovations Forum: Proteome analysis of A. thaliana
OCH3
A
C
NH
N2N
CH
NH
CO
CH
NH
O-Methylisourea-hydrogen sulfate
CH2
CO
CH2
CH2
CH2
CH2
CH2
CH2
CH2
NH2
NH2
lysine
C
H2N
NH
homoarginine (+42 amu)
O
O
N
O
H
O
R1
S
OH + N2H
R2
O
O
O
Pep
OH
S
R1
O
O
B
Conclusions
H
NH
Fig 4. Chemically assisted fragmentation MALDI spectrum. Nine consecutive residues of
sequence were acquired, which allowed successful identification of mitochondrial proteins
in the A. thaliana database.
Pep
R2
sulfonated N-terminal (+136 amu)
Fig 3. Reaction scheme for the chemically assisted fragmentation derivatization step of
Ettan CAF MALDI Sequencing Kit. (A) The first step is protection of the ε-amino group of
the lysine side chain. (B) The second step is sulfonation of the α-amino N-terminus of
the peptide.
The 2-D MS workflow is a valuable tool to characterize a plant
mitochondrial proteome, enabling low-abundance proteins to
be detected and identified. Automated PMF analysis gave an
identification rate of 74% (37 proteins of 50 total). The automated
software of Ettan MALDI-ToF Pro enables protein identification with
minimal manual intervention. Chemically assisted fragmentation
MALDI, which is a relatively simple technique, further improved
protein identification rates to 86%.
Reference
Table 1. Improved protein identification of A. thaliana mitochondrial proteins using Ettan
MALDI-ToF Pro
Spot
number
Protein identity
(see reference 2)
Protein identity
(Ettan MALDI-ToF Pro)
1. Arabidopsis Genome Initiative-Analysis of the genome sequence of the
flowering plant Arabidopsis thaliana. Nature 408, 796–815 (2000).
2. Wortman, J. R., et al. Annotation of the Arabidopsis genome. Plant Physiol
132, 461–468 (2003).
3. Millar, A. H., et al. Analysis of the Arabidopsis mitochondrial proteome. Plant
Physiol 127, 1711–1727 (2001).
1
glycine-rich RNA-binding protein
glycine-rich RNA-binding protein
2
manganese superoxide
dismutase (MSD1)
manganese superoxide
dismutase (MSD1)
3
ATP-dependent Clp protease
proteolytic subunit (ClpP2), putative
ATP-dependent Clp protease
proteolytic subunit (ClpP2), putative
4
glutathione transferase, putative
glutathione transferase, putative
5
glutathione transferase, putative
glutathione transferase, putative
6
glutathione transferase, putative
glutathione transferase, putative
Ordering Information
7
not identified
10 kDa chaperonin (CPN10)
Multiphor II IEF System
18-1018-06
8
not identified
hypothetical protein
Immobiline DryStrip pH 3–10 NL, 18 cm
17-1235-01
9
not identified
F1F0-ATPase inhibitor protein
80-6466-46
10
not identified
armadillo repeat containing protein
Ettan DALTtwelve Large
Vertical System, 115 V
11
not identified
expressed protein
Ettan DALTtwelve Large
Vertical System, 220 V
80-6466-27
12
not identified
unknown protein
Ettan CAF MALDI Sequencing Kit
17-6002-97
13
not identified
plastid protein –related
18-1156-54
14
not identified
expressed protein
Ettan MALDI-ToF Pro
Mass Spectrometer, 120 VAC
Ettan MALDI-ToF Pro
Mass Spectrometer, 240 VAC
18-1156-53
Ettan Spot Handling Workstation
18-1164-05
4. Keough, T., et. al. A method for high-sensitivity peptide sequencing using
postsource decay matrix-assisted laser desorption ionization mass
spectrometry. Proc. Natl. Acad. Sci. USA 96, 7131–7136 (1999).
To obtain a brochure on Ettan MALDI-ToF Pro, please circle 3 on the reader reply card,
or download it at www.lsn-online.com/info.
Life Science News 15, 2003 Amersham Biosciences 15