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Bykova and Rampitsch
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REVIEW: Plant Redox Proteomics
Page 1 of 8
Supporting Information Table 1. Redox proteomics studies on reversible oxidative and nitrosative protein modifications in plants
Type of amino acid
modification, organism
and type of tissue
ROS/RNS/reduction
sources causing the
modification
Thiol-Cys Trx h targeted
proteins in peanut
(Arachis hypogaea L.)
seeds
In vitro reduction, either
 Trx h (reduced with
NADPH via NTR) or DTT
Thiol-Cys Trx targeted
proteins in the stroma of
spinach chloroplasts
In vitro reduction with
immobilised mutated
chloroplast Trx m
Thiol-Cys Trx targeted
proteins in the stroma of
spinach chloroplasts
In vitro reduction with
immobilised mutated
chloroplast Trx f or m
Thiol-Cys Trx h targeted
proteins in germinating
barley (Hordeum
vulgare L.) seeds
Intracellular induction
following imbibitions;
oxidation with H2O2
Thiol-Cys Trx-linked
proteins in endosperm of
wheat (Triticum aestivum
cv. Butte) seeds
In vitro reduction,  Trx
h (reduced with NADPH
via NTR)
Detection methods and
proteomic approaches
mBBr labelling of SHgroups; IEF 2DE or
nonreducing/reducing 2DE;
Edman degradation;
nanoESI-MS/MS
Mutated m-type Trx affinity
capture, 1DE, NH2-terminal
sequencing, recombinant
proteins
Physiological consequences/significance
1)The specificity of Trx in the reduction of
intramolecular vs. intermolecular disulfide bonds;
2) isolated 20 Trx targets and identified 5, three
allergens Ara h-type, desiccation-related and seed
maturation protein
1) Developed methodology for immobilised mutated mtype Trx affinity capture the target proteins;
2) identified four novel targets glutamine synthetase,
cyclophilin, Prx-Q, and the Rubisco small subunit.
1) Developed methodology for immobilised mutated
Mutated two types of Trxs
Trxs f and m affinity capture of the target proteins;
(f or m) affinity capture, IEF 2) identified 26 novel potential targets (35 total) that
2DE, LC-MS/MS
function in 10 chloroplast processes not known to be
Trx linked and in 5 established Trx-regulated processes
1) Disulfide proteins of both tissues undergo reduction;
Embryo and endosperm
2) Trx h abundance decreased in the endosperm and
total protein; SH-groups
increased in embryo
labelling with mBBr; 1D and
3) three Trx forms found in the scutellum and aleurone,
2DE; Western blotting;
and 2 forms in the root and the shoot
nano-ESI MS/MS
4) seven Trx target proteins were identified after H2O2
KCl-soluble proteins, mBBr 1) Found 23 Trx targets that function in metabolism (12
labelling of SH-groups, IEF
targets), protein storage (3), oxidative stress (3),
2DE, MS and MS/MS,
protein degradation (2), protein assembly/folding (1)
enzyme activity assays
and unknown reactions (2).
Reference
Yano et al.,
2001
[18]
Motohashi et
al., 2001
[24]
Balmer et al.,
2003
[25]
Marx et al.,
2003
[52]
Wong et al.,
2003
[53]
Bykova and Rampitsch
REVIEW: Plant Redox Proteomics
Page 2 of 8
Thiol-Cys Trx-linked
proteins in mitochondria
from photosynthetic (pea
and spinach leaves) and
heterotrophic (potato
tubers) sources
In vitro reduction with
immobilised mutated
chloroplast Trx m or h; or
E. coli Trx system; or
poplar Trx h2 and
Arabidopsis NTR
Soluble matrix proteins,
mutated Trxs (h or m)
affinity capture, differential
labelling with mBBr, IEF
2DE, ESI-MS/MS
Thiol-Cys Trx h1 and h2
targeted proteins in
mature and germinating
barley seeds
Intracellular induction,
treatment with HvTrx h1
and HvTrx h2
Cy5 maleimide and mBBr
labelling of free thiols, 2DE,
MALDI-TOF MS
Thiol-Cys Trx h targeted
proteins in endosperm of
wheat (Triticum aestivum
cv. Butte) seeds
In vitro reduction, either
 Trx h (reduced with
NADPH via NTR) or DTT
KCl-soluble proteins, mBBr
labelling of SH-groups, IEF
2DE, mutated poplar Trx h1
affinity, Q-TOF LC-MS/MS
Thiol-Cys Trx targeted
proteins in developing
wheat (Triticum aestivum
L., cv. Butte 86) seeds
Intracellular induction,
reduction with Trx or DTT
KCl-soluble, chloroformmethanol (CM) soluble and
insoluble albumin/globulin
fractions ; mBBr labelling,
2DE, nanoLC-MS/MS
1) In total, 16 different putative targets were identified
2) HvTrx h1 and HvTrx h2 were shown to have similar
target specificity
3) identified some of the previously known and novel
targets in both mature and germinating seeds
1) The two procedures were complementary: of the
total 68 targets, one-third were observed with both
procedures and one-third were unique to each;
2) young endosperm vs. mature endosperm revealed a
unique set of proteins in each developmental stage
1) Reduction by Trx alters protein solubility, thereby
promoting processes of the grain starchy endosperm;
2) the CM fraction contained LMW disulfide proteins,
stress enzymes, storage proteins and a component of
protein degradation
Thiol-Cys Trx targeted
proteins in dark-grown A.
thaliana plants
In vitro reduction with
immobilised mutated five
cytoplasmic Trx h1-h5
isoforms
Whole tissue cell lysate
proteins, mutated isoforms
Trxs h1-h5 affinity capture,
IEF 2DE, MALDI-MS PMF
1) Identified cytosolic proteins of the anti-oxidative
stress system (3), involved in protein biosynthesis (2),
metabolic enzymes (5), and chloroplast proteins (4);
nine of them novel targets
Yamazaki et
al., 2004
[58]
Thiol-Cys Trx targeted
proteins in A. thaliana
leaves
In vitro reduction,  Trx
h3 (reduced with NADPH
via NTR)
Rubisco depletion,
differential alkylation with
[14C]iodoacetamide, IEF
2DE, autoradiography,
MALDI TOF MS
1) Identified 19 novel potential targets (about 40 total)
involved in the Calvin cycle, metabolism, protein
folding, photosynthesis, defense against oxidative
stress and amino acid synthesis, 4 proteins of GDC
complex.
Marchand et
al., 2004
[59]
1) Identified 50 potential targets functional in 12
processes including photorespiration, TCA cycle and
associated reactions, lipid metabolism, electron
transport, ATP synthesis, membrane transport, protein
synthesis, nitrogen and sulfur metabolism, hormone
synthesis, and stress reactions
Balmer et al.,
2004
[54]
Maeda et al.,
2004
[55]
Wong et al.,
2004a
[56]
Wong et al.,
2004b
[57]
Bykova and Rampitsch
Thiol-Cys Trx h reducible
disulfides in proteins
from barley seeds
REVIEW: Plant Redox Proteomics
In vitro reduction, 
HvTrx h (reduced with
NADPH via NTR)
Differential labelling of SHgroups by iodoacetamide
and 4-vinylpyridine, soluble
extracts and recombinant
protein, 2DE, MALDI-TOF
MS
Page 3 of 8
1) Identified Trx h-reducible disulfide bonds in
individual target proteins;
2) showed specific reduction of nine disulfides in amylase/subtilisin inhibitor, four -amylase/trypsin
inhibitors and a protein of unknown function.
Thiol-Cys Grx-interacting
proteins from leaves and
stems of poplar P.
trichocarpa or leaves of
P. sativum, and A.
thaliana, or tubers of S.
tuberosum
In vitro reduction with
immobilised mutated
poplar Grx
Soluble proteins from
whole extracts, purified
mitochondria or
chloroplasts; mutated
poplar Grx affinity capture,
nano-ESI QTOF MS/MS
Thiol-Cys Trx m-linked
proteins in amyloplasts
isolated from wheat
starchy endosperm
In vitro reduction of
oxidised Cys with
immobilised mutated
spinach Trx m; or E. coli
Trx system
Soluble amyloplast
proteins, differential mBBr
labelling of disulfide bonds,
mutant spinach Trx m
affinity capture, IEF 2DE,
nanoLC-MS/MS
Thiol-Cys Trx-interacting
proteins in rice bran
In vitro reduction with
endogenous Trx
activated by NADPH and
E. coli NTR, or
recombinant E. coli Trx
system
Dry/imbibed rice bran with
GA and  leupeptin, mBBr
labelling of reduced SHgroups, IEF 2DE, N-terminal
amino acid sequencing
1) Identified fragments of embryo-specific protein and
dienelactone hydrolase as putative targets;
2) Trx activates cysteine protease with a concurrent
unfolding of its substrate, the embryo-specific protein
Yano and
Kuroda, 2006
[63]
Intracellular induction
and  Trx h3 treatment
Labelling with radioactive
iodoacetamide or PEOiodoacetylbiotin and avidin
affinity, or mutated Trx h3
affinity, 2DE, and MALDITOF MS
1) Three differential labelling methods identified
complementary sets of proteins, in total 73 Trx targets;
2) each method had its own shortcomings and biases;
3) the identified proteins were localized in various
compartments and control major processes in
photosynthetic cells
Marchand et
al., 2006
[64]
Thiol-Cys Trx-interacting
proteins in A. thaliana
leaves
1) Identified 94 putative targets, involved in various
cellular processes such as oxidative stress response,
nitrogen, sulfur, and carbon metabolisms, translation,
protein folding, and other;
2) some of the proteins were previously found to
interact with Trx or to be glutathiolated, but others
could be more specific partners of Grx
1) Found components of the ferredoxin/Trx system
(ferredoxin, ferredoxin–Trx reductase, and Trx),
originally described for chloroplasts, in amyloplasts;
2) 42 potential Trx target proteins, 13 novel, function in
starch metabolism and a range of processes including a
major membrane ADP-glucose transporter
Maeda et al.,
2005
[60]
Rouhier et
al., 2005
[61]
Balmer et al.,
2006
[62]
Bykova and Rampitsch
REVIEW: Plant Redox Proteomics
Thiol-Cys Trx-linked
proteins in germinating
Medicago truncatula
seeds
Intracellular induction
and  Trx h treatment, in
vitro aerobic oxidation of
protein fractions
Thiol-Cys intra- and
intermolecular disulfide
bonding in Arabidopsis
cell culture mitochondrial
proteome
In vitro treatment of
mitochondria with
cytotoxic aldehyde 4hydroxy-2-nonenal
Thiol-Cys Trx h reducible
proteins in embryos from
germinating barley seeds
Intracellular induction
and  Trx h treatment
Fractionation with NaCl,
labelling of free thiols with
mBBr, or mutated pea Trx
h3 affinity, 2DE and LCMS/MS analysis
Soluble and membrane
proteins, 2-D
oxidant/reductant
diagonal-SDS-PAGE with
diamide and DTT; LCMS/MS analysis
Quantitative approach
based on differential ICAT
labelling, avidin affinity
capture and LC-MS analysis
Thiol-Cys redox-regulated Intracellular induction
proteins in Arabidopsis
after treatment with
plants treated with
MeJA
methyl jasmonate (MeJA)
Shoot and root proteins,
differential mBBr labelling
of reversibly oxidized thiol
groups, IEF 2DE, nanoLCMS/MS
In vitro reduction with
immobilised mutated
plastidial Trx y2
Crude extract; mutated
plastidial Trx y2 affinity
capture, IEF 2DE, MALDITOF MS PMF, activity
measurements for phenylammonia-lyase (PAL) and
monodehydroascorbate
reductase (MDHAR)
Thiol-Cys Trx h targeted
proteins in Arabidopsis
roots
Page 4 of 8
1) Total 111 proteins with 59 of them novel targets
were identified with few differences between axes and
cotyledons; function in major processes of the seed;
2) more than half of the potential targets were reduced
during germination providing evidence for Trx function.
1) Intermolecular disulfide bonds in alternative oxidase,
O-acetylserine (thiol) lyase, citrate synthase and
between subunits of the ATP synthase were found;
2) intramolecular disulfide bonds were observed in a
range of mitochondrial dehydrogenases, elongation
factor Tu, adenylate kinase and phosphate translocator
1) Significancly reduced disulfide targets were
identified in 104 peptides out 199 total labelled;
2) targeted protein translation, the ascorbateglutathione cycle and redox control, metabolism,
protein folding and seed storage proteins
1) Identified 15 proteins in shoots and 6 proteins in
roots significantly affected in redox state or abundance
2) stress and defense proteins constitute a large group
that responded to MeJA
3) Cys residues involved in the disulfide dynamics were
mapped in 21 proteins
1) Isolated 24 putative new targets out of 72 total,
functioning in metabolism, detoxification and response
to stress, protein processing and signal transduction;
2) the mevalonic acid-dependent biosynthesis of
isoprenoids and phenylpropanoid biosynthesis were
shown as new redox-mediated processes;
3) the enzymatic activities of PALK and plastidial
MDHAR are regulated by in vitro reduction of Trx y
Alkhalfioui et
al., 2007
[65]
Winger et al.,
2007
[66]
Hägglund et
al., 2008
[67]
Alvarez et al.,
2009
[68]
Marchand et
al., 2010
[69]
Bykova and Rampitsch
REVIEW: Plant Redox Proteomics
Page 5 of 8
1) The expression of trx h decreased, and transgenic
wheat had high resistance to pre-harvest sprouting;
2) found 36 differential proteins involved in regulation
of carbohydrates, esters, nucleic acid, proteins and
energy metabolism, and biological stress.
1) 193 reactive Cys were found in 79 unique proteins
from various functional groups responding differentially
in dormant, non-dormant, ABA or GA treated seeds;
2) a dramatic increase in protein thiol redox state in
seeds during imbibition and hormonal treatment;
3) higher antioxidant capacity in dry dormant versus
non-dormant seeds
1) Quantitative redox differences between the
genotypes were found for 31 proteins (64 total)
containing 78 unique redox active cysteines;
2) a biochemical shift in dormant seeds in the
accumulation of proteins to those with roles in storage
and protection against biotic and abiotic stresses.
Response to anti-Trx s
gene changes in wheat
(Triticum aestivum L.)
seeds
Intracellular induction
Differential proteome of
transgenic and WT seeds,
IEF 2DE, MALDI-TOF-MS
PMF, quantitative RT-PCR
Thiol-Cys oxidation in
seeds of high dormancy
wheat (Triticum aestivum
L.) genotype RL4137
Intracellular induction
during seed afterripening, or/and ABA/GA
treatments of seeds
Solubility-based protein
fractions, differential
labelling of free thiols with
mBBr, IEF 2DE, quantitative
analysis, nanoLC-MS/MS,
wheat EST sequences
Thiol-Cys oxidation in
harvest-ripe grains of
dormant and nondormant hybrid
genotypes of wheat
(Triticum aestivum L.)
Intracellular induction
Solubility-based protein
fractions, differential
labelling of free thiols with
mBBr, IEF 2DE, quantitative
analysis, nanoLC-MS/MS,
wheat EST sequences
Prxs and NADPHdependent Trx systems in
the legume Lotus
japonicus trated with
phytohormones and NO
Plants treated with ABA,
GA3, JA, indole-3-acetic
acid, 1-aminocyclopropane-1-carboxylic
acid, or CK (kinetin and 6benzyl-aminopurine
mixture); seeds treated
with CK; and roots with
SNAP and GSNO
Quantitative RT-PCR,
immunological and
gel-free proteomic nanoLCMS/MS approaches
1) Identified 7Prxs, 14 Trxs, and 3 NTR functional genes;
2) reveal a complex regulation of Prxs that is dependent
on the isoform, tissue, and signalling molecule
TovarMéndez et
al., 2011
[46]
Thiol-Cys oxidation in A.
thaliana cells and plants
Treatments with a
sublethal dose of H2O2,
salicylic acid or the
Differential direct and
blocking IAF or BIAM/NEM
labelling of thiols, 2-DE,
1) Five out of 84 detected putative redox-sensitive
proteins were confirmed to undergo oxidative
modification in the oxidant-treated leaves;
Wang et al.,
2012
[71]
Guo et al.,
2011
[70]
Bykova et al.,
2011a
[19]
Bykova et al.,
2011b
[20]
Bykova and Rampitsch
REVIEW: Plant Redox Proteomics
Page 6 of 8
peptide elicitor flg22
electrophoretic mobility
shift assay, nanoLC-MS/MS,
FLAG-tag fused proteins,
immunoprecipitation
and Western blotting
Cys S-nitrosylation in A.
thaliana cell suspension
cultures and plants
Treatments with GSNO
(NO-donor and Snitrosylating agent) or
gaseous NO
Biotin-switch technique;
neutravidin affinity; 1D
PAGE; Western blot;
nanoLC-MS/MS
1) The starting point for investigation of cellular
processes regulated by protein S-nitrosylation in plants;
2) identified 115 candidates, including stress-related,
redox-related, signalling/regulating, cytoskeleton, and
metabolic proteins, many of which were identified as
targets in animals
Lindermayr
et al., 2005
[27]
Cys S-nitrosylation in A.
thaliana plants
undergoing
hypersensitive response
Leaves infiltrated with
Pseudomonas syringae
pv. tomato (Pst) or with
Pst carrying the avrB
avirulence gene
Biotin-switch technique;
neutravidin affinity; 1DE
and Western blot; IEF 2DE;
MALDI-TOF/TOF MS/MS
analysis
1) 16 S-nitrosylated proteins identified were mostly
enzymes serving in general metabolism (33%),
photosynthetic process (28%), signalling and regulation
(17%), antioxidant defense (17%), and unknown
function
RomeroPuertas et
al., 2008
[142]
1) Nineteen target proteins associated with carbon,
nitrogen and sulfur metabolism, the cytoskeleton,
stress and photosynthesis
2) Rubisco activity was inhibited by S-nitrosylation
Abat et al.,
2008
[85]
1) Cold stress modulates S-nitrosoproteome;
2) this study showed that at least 40% Rubisco
inactivation by low temperature can be explained by Snitrosylation
Abat and
Deswal, 2009
[102]
Cys S-nitrosylation in a
medicinal CAM plant
Kalanchoe pinnata
Treatment with GSNO
Cys S-nitrosylation in
Brassica juncea induced
by low temperature
Intracellular induction by
cold stress
Cys S-nitrosylation and
protein carbonylation in
citrus plants (Citrus
aurantium L.) in response
to salinity stress
Intracellular induction by
salinity stress and/or pretreatments with H2O2 or
an NO donor SNP
Biotin-switch technique;
neutravidin affinity; 1D
PAGE; Western blot;
MALDI-TOF MS or LCMS⁄MS
Biotin-switch technique;
neutravidin affinity; IEF 2D
PAGE; Western blot; PMF
by MALDI-TOF MS or LCMS/MS
Biotin-switch assay;
immunoprecipitation; IEF
2D PAGE; Western blot;
nanoLC-MS/MS
2) different labelling approaches led to partially
overlapping sets of proteins;
3) cytokine-induced apoptosis inhibitor 1 was also
oxidised after treatment of leaves with SA and flg22
1) Indicate an overlap between H2O2- and ·NO-signalling
pathways in acclimation to salinity;
Tanou et al.,
2) pre-treatments of citrus plants with H2O2 or SNP can 2009
prime an enhanced plant defense to salinity
[99]
Bykova and Rampitsch
REVIEW: Plant Redox Proteomics
Cys S-nitrosylation
and/or glutathionylation
in mitochondria from
Arabidopsis thaliana
leaves
Treatment of
mitochondria with GSNO
or GSH
Biotin-switch technique;
MALDI-TOF MS; nanoLCMS/MS; neutral loss-driven
nano-HPLC-MS2/3
Cys S-nitrosylation and
protein carbonylation in
recalcitrant Antiaris
toxicaria seeds
Intracellular induction by
treatments with
desiccation or NO gas
Biotin-switch assay;
immunoprecipitation; IEF
2D PAGE; Western blot for
both PTMs, nanoLC-MS/MS
Cys S-nitrosylation in a
WT and nitric oxide
excess1 (noe1) mutant, in
rice (Oryza sativa) leaves
Cys S-nitrosylation in pea
(Pisum sativum L.) leaf
homogenates and
peroxisomes
3-nitroTyr in sunflower
(Helianthus annuus L.)
hypocotyls
1) In vivo oxygendependent chemical
reactions;
2) in vivo transnitrosylation via the
transfer of NO from
intracellular SNO to Cys
1) Total or peroxisomal
proteins treated with an
NO donor, SNAP or GSNO
2) plants subject to
cadmium and herbicide
2,4-D abiotic stress
1) Intracellular induction:
a) reaction of NO and
O2•– to yield ONOO-; b)
decay of NO and O2•–
into NO2 and H2O2 and
interaction with heme
peroxidases.
Page 7 of 8
1) Identified 11 mitochondrial target proteins including
the major photorespiratory cycle enzymatic system
GDC and SHMT;
2) GDC complex activity was inhibited by GSNO due to
S-nitrosylation/S-glutathionylation of several Cys
residues
1) Promoted antioxidant ascorbate-glutathione cycle
enzyme activities via the regulation of antioxidant
protein carbonylation and S-nitrosylation;
2) enhanced desiccation tolerance by activating the
antioxidant pathway and reducing H2O2 accumulation
Palmieri et
al., 2010
[84]
Bai et al.,
2011
[100]
Biotin-switch assay and
isolation of biotinylated
proteins, nanoLC-MS/MS
1)S-nitrosylation is involved in the control of lightmediated H2O2-induced leaf cell death in rice;
2) uncovered a series of S-nitrosylated
proteins in both noe1 and wild-type plants
Lin et al.,
2012
[94]
Biotin-switch; Western
blot; immunoprecipitation,
MALDI TOF/TOF and 2DnanoLC-MS/MS
1) Assessed the presence of S-nitrosylation in pea-leaf
peroxisomes and identified six putative targets;
2) S-nitrosylation levels of CAT and glycolate oxidase
changed under cadmium and 2,4-D treatments
OrtegaGalisteo et
al., 2012
[97]
MRM detection of Tyr and
NO2-Tyr using LC-MS/MS
Ion Trap; 1D, 2D PAGE and
Western blot of NO2-Tyr;
MALDI TOF/TOF MS/MS;
immunohistochemistry of
NO2-Tyr and fluorescein
1) Identified 21 protein targets involved in
photosynthesis, and in antioxidant, ATP, carbohydrate,
and nitrogen metabolism;
2) an in vitro analyses showed that the Tyr nitration of
S-adenosyl-homocysteine hydrolase inhibited its
activity;
3) an in silico analysis revealed that Tyr448 from barley
Chaki et al.,
2009
[115]
Bykova and Rampitsch
REVIEW: Plant Redox Proteomics
2) Treatment with SIN-1
(ONOO- donor)
3-nitroTyr in A. thaliana
seedlings
Intracellular induction (as
described above)
Cys S-glutathionylation in
A. thaliana suspension
culture cells
In vitro glutathionylation
with biotinylated GSH
ethyl ester (BioGEE) and
H2O2 treatment
Cys S-glutathionylation in
A. thaliana suspension
culture cells induced by
stress
Intracellular induction or
treatment with the
oxidant tert-butylhydroperoxide
3
4
Page 8 of 8
ONOO- detection by CLMS
SAHH could be the potential nitration target
Immunoprecipitation with
an anti-3-nitroTyr antibody;
shotgun LC-MS/MS QTOF
analysis; 2D PAGE;
Western blotting; MALDI
TOF MS and MS/MS
Biotinylated GSH labelling,
ion exchange and
hydrophobic interaction LC,
1-DE, Western blotting,
peptide sequencing
In vivo labelling of the thiol
pool with L-[35S]Cys; in vitro
labelling with biotinylated,
oxidized glutathione (GSSGbiotin), affinity streptavidin
purification, IEF 2DE,
MALDI-TOF MS analysis
1) Identified 127 putative in vivo protein targets;
2) about 35% corresponded to homologues of proteins
reported to be nitrated in non-plant organisms;
3) identified 7 nitrated peptides from 6 proteins
4) in vivo nitration sites among putative targets could
not be identified by MS/MS
1) Biotinylated GSH was incorporated into about 20
proteins, two of which were identified as the key
enzymes for sugar metabolism, TPI and putative
plastidic aldolase;
2) TPI was inactivated by GSSG, and reactivated by GSH.
1) 79 polypeptides were identified, representing a
mixture of proteins that underwent direct thiolation as
well as proteins complexed with thiolated polypeptides
2) the mechanism of thiolation in five proteins showed
modification of active-site cysteines in 3 proteins, and 2
others required proteins/protein complexes for activity.
Lozano-Juste
et al., 2011
[125]
Ito et al.,
2003
[131]
Dixon et al.,
2005
[128]