Plant Science 176 (2009) 99–104

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Plant Science
journal homepage: www.elsevier.com/locate/plantsci

Compatibility of plant protein extraction methods with mass spectrometry

for proteome analysis
Inder S. Sheoran a, Andrew R.S. Ross b, Douglas J.H. Olson b, Vipen K. Sawhney a,*
a
Department of Biology, University of Saskatchewan, 112 Science Place, Saskatoon, SK, Canada S7N 5E2
b
National Research Council of Canada, Plant Biotechnology Institute, 110 Gymnasium Place, Saskatoon, SK, Canada S7N 0W9

A R T I C L E I N F O A B S T R A C T

Article history: Different protein extraction methods have been developed for plant proteome analysis but their
Received 26 June 2008 compatibility with mass spectrometry has rarely been tested. We evaluated four protein extraction
Received in revised form 5 September 2008 methods, i.e., trichloroacetic acid (TCA)–acetone, phenol, direct iso-electric focusing (IEF) buffer, and
Accepted 26 September 2008
Tris–HCl buffer, using tomato pollen for proteome analysis. The data presented show that the TCA–
Available online 14 October 2008
acetone and phenol protein extraction methods are superior to the other two tested methods for tomato
pollen proteome analysis, in terms of two-dimensional gel electrophoresis (2-DE) gel separation, mass
Keywords:
spectrometric analysis, and identification of proteins by peptide mass fingerprinting (PMF). These results
Protein extraction methods
Mass Spectrometry
highlight the importance of plant protein extraction method for subsequent MS analysis and protein
Pollen identification.
Proteomics ß 2008 Elsevier Ireland Ltd. All rights reserved.

1. Introduction downstream protein analysis by liquid chromatography and mass

The ongoing development in protein extraction, purification
and identification methods has significantly advanced our ability
to address an increasing number of biological questions using
proteomic approaches. Two-dimensional gel electrophoresis (2-
DE) is one of the most widely used techniques for resolving
complex protein extracts, although alternative, gel-free
approaches, including protein antibody arrays [1] and multi-
dimensional liquid chromatography with stable isotopic labeling
[2], have been developed. Sample quality is unquestionably a
critical factor in obtaining adequate 2-DE separation for proteome
analyses; hence, the protein extraction procedure is of prime
importance. Since cellular proteins have a range of biochemical
properties, including charge (pI), size (Mr), hydrophobicity,
susceptibility to proteolysis, post-translational modifications
and interactions with other molecules, and since these properties
vary with the species, developmental stage, cell and tissue type
and growth conditions, different extraction protocols have been
used for proteomic studies. However, the ideal extraction method
should not only capture the greatest possible number of proteins
from a biological sample, it should also be compatible with
* Corresponding author. Tel.: +1 306 966 4417; fax: +1 306 966 4461.
E-mail address: vipen.sawhney@usask.ca (V.K. Sawhney).
spectrometry and subsequent protein identification.
The extraction of comprehensive protein populations from
plants is particularly challenging due to the metabolic and
structural characteristics of plant tissues, including the plant cell
wall matrix [3–7]. With the exception of mature and dormant
structures, i.e., seeds and pollen, most plant cells have relatively
low protein content, and are also rich in proteases and oxidative
enzymes [6,8,9]. In addition, plant cells produce a broad spectrum
of metabolites, e.g., pigments, phenolic compounds, lipids and
polysaccharides that contaminate protein extracts contributing to
the difficulties in protein fractionation and downstream analysis.
A number of protein extraction protocols have been published
for various plant tissues [3–17] and a particular protocol is
generally optimized according to the aim of the study. Phenol and
trichloroacetic acid (TCA)-based extraction methods have been
found to give superior protein yield and good quality 2-DE gels for
certain plant tissues [3,5–7,14–17]. Whereas, the compatibility of
different staining methods of gels with mass spectrometry have
been investigated [18,19], little attempt has been made to evaluate
protein extraction methods for downstream analysis and identi-
fication of proteins using MS-based techniques. Here, we compare
four commonly used plant protein extraction methods for 2-DE
separation, followed by matrix-assisted laser desorption/ioniza-
tion time-of-flight mass spectrometry (MALDI-TOF-MS) analysis
and peptide mass fingerprinting (PMF) for proteome analysis,
using tomato pollen as an experimental system. Our results show

0168-9452/$ – see front matter ß 2008 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.plantsci.2008.09.015
100 I.S. Sheoran et al. / Plant Science 176 (2009) 99–104
that the choice of extraction procedure is a major factor in protein
separation, analysis, and identification.
2. Methods
2.1. Plant material
Tomato (Solanum lycopersicum cv. Rutgers) pollen grains were
collected from the freshly open flowers from greenhouse-grown
plants, placed on a glass slide, checked under a dissecting
microscope, and any debris removed with a needle. Pollen samples
were then pooled in an Eppendorf tube and either processed
immediately or stored at 80 8C for later analysis.
2.2. Protein extraction
Proteins from mature pollen (25 mg for each method) were
extracted using four different methods commonly used for protein
extraction from various plant tissues, including pollen, as detailed
below.
2.2.1. Method A (TCA–acetone)
Pollen grains were ground to a fine powder in liquid nitrogen
using a pestle and mortar, and extracted with acetone containing
10% TCA (w/v) and 1% DTT (w/v) as previously described [9,10]. The
samples were kept at 20 8C for 2 h (overnight, if necessary) before
centrifuging at 25,000  g for 20 min at 4 8C. The pellet was
washed twice by suspending in acetone containing 1% DTT, kept at
20 8C for 1 h, and centrifuged. The vacuum dried pellet was
dissolved in direct iso-electric focusing (IEF) buffer comprising 8 M
urea, 20 mM DTT, 4% CHAPS, and 2% ampholyte (pH 3–10) by
vortexing for 1 h at 20 8C. This solution was centrifuged at 20 8C for
20 min at 25,000  g. The supernatant was collected and the
residue re-extracted with IEF buffer. The combined supernatants
were centrifuged and used for protein estimation and 2-DE
analysis.
2.2.2. Method B (phenol extraction)
This procedure was based on that described by Hurkman and
Tanaka [11] with slight modifications. Pollen grains were ground to
a fine powder in liquid nitrogen using a pestle and mortar, and
extracted by further grinding for 5 min in 1 ml each of phenol (Tris
pH 8.8 buffered) and extraction buffer (0.1 M Tris–HCl pH 8.8,
5 mM EDTA, 20 mM DTT, 30% sucrose). The sample was transferred
into a centrifuge tube, vortexed for 30 min at 4 8C and centrifuged
at 25,000  g for 10 min. The upper, phenol layer was transferred
into another tube. The lower, aqueous phase was then re-extracted
with 1 ml each of phenol and extraction buffer, vortexed,
centrifuged, and the phenol layer combined with that collected
earlier. Proteins from the phenol extract were precipitated by
adding 5 volume (v/v) of 0.1 M ammonium acetate in 100%
methanol at 20 8C, vortexed, and kept at 20 8C (overnight if
necessary) before centrifuging at 25,000  g for 20 min at 4 8C. The
pellet was washed twice with 0.1 ammonium acetate in methanol,
twice with 80% acetone, and once with 80% acetone containing
10 mM DTT. In each case, the pellet was completely suspended by
vortexing, kept for 30 min at 20 8C, and then centrifuged at
25,000  g for 20 min at 4 8C. After the final wash, the pellet was
vacuum dried and proteins extracted using IEF buffer, as described
in Method A.
2.2.3. Method C (direct IEF buffer extraction)
Proteins were extracted directly in IEF buffer as described by
Gallardo et al. [8]. Pollen grains were ground to a fine powder in
liquid nitrogen using a pestle and mortar and extracted with an IEF
buffer comprising 8 M urea, 20 mM DTT, 4% CHAPS, 5 mM EDTA,
5 mM Tris base and 2% ampholyte (pH 3–10), as described in
Method A.
2.2.4. Method D (Tris–HCl buffer)
Proteins were extracted as described earlier [20], with some
modifications. Pollen were ground in liquid nitrogen and mixed
with Tris–HCl buffer consisting of 50 mM Tris–HCl pH 8.8, 5 mM
EDTA, 20 mM DTT, 100 mM KCl, and 2 mM PMSF. After thawing,
the mixture was ground for an additional 30 min at 4 8C and
centrifuged at 25,000  g for 20 min. The supernatant was
collected, the pellets re-extracted, and the supernatants pooled.
Proteins in the supernatant were then precipitated with 5
volume (v/v) of 100% acetone and incubated at 20 8C for 2 h.
After centrifugation, the pellet was washed twice with 80%
acetone and then re-suspended in IEF buffer as described in
Method A.
Total protein in all the above extracts (A to D) was estimated
using the Bradford reagent (Bio-Rad, Hercules, CA, USA) before
immediate processing or storage at 80 8C for later analysis.
2.3. Two-dimensional electrophoresis
Two-dimensional gel electrophoresis was carried out as
previously described [21]. Equal amount of protein (500 mg)
extracted with each of the four methods was loaded on 18 cm, pH
4–7, immobilized pH gradient strips through re-hydration and IEF
was performed using a Multiphor II horizontal electrophoresis
system (Amersham Biosciences, Uppsala, Sweden). The strips were
then equilibrated for 15 min in an equilibration buffer containing
0.05 M Tris–HCl (pH 8.8), 6 M urea, 30% (v/v) glycerol, 2% (w/v)
SDS, and 20 mM DTT, followed by another 15 min equilibration in
the same buffer containing 125 mM iodoacetamide without DTT.
The equilibrated strips were than loaded on 12.5% SDS-poly-
acrylamide gels and separated using PROTEAN II XI multi-cell (Bio-
Rad, USA).
The gels were stained with Colloidal Coomassie Brilliant Blue G-
250 (CCB) as described earlier [11], scanned, annotated and
analyzed using Phoretix 2D Image analysis software (UBI, Canada).
Three replicate gels were run for each of two different pollen
samples for each extraction method, of which one representative
set is presented here.
2.4. Mass spectrometry and protein identification
Twenty spots of varying intensity, Mr and pI that were
common to gels obtained using each of the four protein
extraction methods, were cut, digested, analyzed by MALDI-
TOF-MS and identified by PMF, as previously described [21,22].
In brief, excised protein spots were automatically de-stained,
dehydrated, reduced with DTT, alkylated with iodoacetamide,
and digested with trypsin using a MassPREP protein digest
station (Micromass, Manchester, UK) according to the recom-
mended procedure. The resulting tryptic digests were concen-
trated and desalted using C18 ZipTips (Millipore Corporation,
Bedford, MA, USA) according to the manufacturer’s instructions.
Samples were than analyzed by MALDI-TOF-MS on a Voyager-
DE STR (Applied biosystems, Framingham, MA, USA) operating
in the positive ion and reflectron. Spectra were acquired in
700–3000 m/z range, processed with Mascot Distiller 2.0.0
(www.matrixscience.com), and the resulting peak lists used to
identify the corresponding proteins in National Center for
Biotechnology Information non-redundant protein sequences
database by PMF using the Mascot (www.matrixscience.com)
search engine.
 I.S. Sheoran et al. / Plant Science 176 (2009) 99–104 101

3. Results and discussion
The total amount of protein extracted from equal amounts of
pollen tissues varied according to the protein extraction method
used. Considerably higher amount of protein was extracted with the
direct IEF buffer extraction method (Method C, 180.0  17.5 mg/g)
compared to other three methods (Method A, 138.4  16.4 mg/g;
Method B, 114.8  13.6 mg/g; Method D, 104.6  11.2 mg/g { values
are S.E., n = 4}). This could be due to the simplicity of Method C; the
single-step procedure avoiding losses that may occur with other
methods involving additional steps such as protein precipitation and
re-solubilization, as suggested before [23]. Alternatively, it is possible
that there is an over estimation of proteins in Method C due to the
presence of some impurities and small peptides in the extract which are
removed in other methods involving additional steps. Variations in
protein recovery using different extraction methods have been
reported in other studies [5,16,24]. The amount of protein extracted
from different tomato plant tissues depends upon the tissue type and
was somewhat greater for phenol than for TCA–acetone method [6]. On
the other hand, TCA–acetone proved to be better than phenol for
Brassica seeds [24]. However, in this study both these methods were
comparable in terms of total protein recovery, as is the case for banana
leaves [3].
Equal amounts of the protein extracted from tomato pollen
using each of the four extraction Methods (A to D) were separated
by 2-DE under identical conditions. Representative CCB-stained
gels for each Method are shown in Fig. 1. All four methods resulted
in good quality, well-resolved gels; however, quantitative and
qualitative differences were observed between these gels. For
example, the average number of protein spots observed in gels
using Method A was 555  61 (S.E.) and with Method B was
558  54. These were higher than with Methods C (318  40), and D
(332  37). Also, with Methods A and B, there were more spots with
high molecular mass compared to the other two methods. The lower
number of spots observed with Methods C and D could be related to
the presence of impurities in these protein samples, as discussed
above, and could also explain some horizontal and vertical streaking
in these gels (Fig 1C and D). Variations in spot number have also been
reported in other plant proteomic studies which used different
methods of protein extraction [6].
The 2-DE protein spot patterns obtained (using pH 4–7 IPG
strips) were similar for the four extraction methods (Fig. 1). Indeed,
more than 90% of protein spots on the gel with minimum spots
(Method C) were matched across all other gels. Only a few
differential spots were observed between gels from Methods A and
B as marked with small letters in Fig. 1A and B. Interestingly, one of
the spot marked ‘a’ in gel A was absent in gel B, but was present at
high levels in gels C and D. This protein was identified as
calreticulin, a calcium binding protein involved in calcium
signaling [25]. Phospho-glucomutase, phospho-glycerate dehy-
drogenase and protein phosphatase C (spots g, h and j,
respectively) were identified only in the phenol method
(Fig. 1B). Spot variations in 2-DE gels with different extraction
methods have been reported by others [3,5–7,13,15,16]. Only few
differences were observed in protein spots between gels from
soybean seed extracts using different extraction methods [15]. In

Fig. 1. 2-DE gels of tomato pollen protein extracted with the TCA–acetone [A], Phenol [B], direct IEF Buffer [C], and Tris–HCl [D] methods. Equal amount of protein (500 mg)
was separated on 18 cm, pH 4–7 IPG strips in the first dimension and visualized using CCB staining. Identical protein spots with numbered arrow from each gel were used for
MALDI-TOF-MS analysis and identified proteins are listed in Table 1. Some of the differential spots between gels are encircled and marked with small letters. The right hand
bottom corner numbers indicate the total number of spots in the gel.
102 I.S. Sheoran et al. / Plant Science 176 (2009) 99–104

contrast, Saravanan and Rose [6] reported large differences in 2-DE
spot patterns for tomato using three different extraction protocols.
These authors concluded that glycosylation may have contributed
to the observed differences in 2-DE spot patterns by affecting
protein solubility under different extraction conditions. It is also
possible that variations in spot patterns observed by these authors
may be related to the developing tissues as opposed to mature
tissues. In this study, we observed similar 2-DE spot patterns for
mature tomato pollen using different extraction methods, as was
the case for mature soybean seeds [15]. Since both these tissues are
dormant and desiccated structures their non-active state may
account for reduced variability in spot pattern when using
different extraction methods.
The comparison of extraction methods for MS compatibility
was tested by analyzing 20 spots common to, and excised
individually from, all four gels (Fig. 1, A to D). The results of
protein identification by MALDI-TOF-MS and PMF are presented in
Table 1. Of the 20 spots analyzed, 11 spots (55%) were identified
with a significant MOWSE score (i.e., a score greater than 67 at
p < 0.05) from gels using extraction Method A, 9 (45%) using
Method B, 6 (33%) using Method C, and 5 (25%) using Method D
(Table 1). The number of peptides matched and protein sequence

Table 1
Identification of tomato pollen protein spots selected from 2-DE gels for four protein extraction methods (A, B, C and D) by MALDI-TOF-MS and PMF. Bold MOWSE scores
indicate significant protein identification.

Spot number Gene index Speciesa Protein identity MW/pIb Methodsc Rank MOWSE PMd SCe (%) Relative spot
number score intensityf

2 45533923 NS Glycine-rich RNA-binding protein 17.6/5.58 A 1 80 7 45 ++++

B 1 71 7 45 +
C 3 42 4 27 ++
D 2 62 6 39 +++

6 30841938 SL Thioredoxin peroxidase 1 17.4/5.17 A 1 70 5 25 ++

B 1 63 5 25 +++
C 3 39 3 11 +
D 1 64 5 25 ++++

7 48209968 ST Mitochondrial ATP synthase D-chain 19.8/5.34 A 1 208 20 69 +++

B 1 206 23 80 ++
C 1 189 19 68 ++++
D 1 170 15 63 +

9 534916 ST Soluble inorganic pyrophosphatase 24.4/5.59 A 1 111 10 27 +++

B 1 51 6 23 ++++
C 3 54 6 18 +
D 1 54 7 20 +++

13 1915974 SL Fructokinase 35.0/5.76 A 1 101 14 46 +++

B 1 106 15 51 +++
C 1 123 12 43 ++++
D 1 70 11 31 ++++

14 1419094 ST Glutamine synthetase 39.4/5.38 A 1 80 10 24 +++

B 1 74 11 19 ++++
C 1 57 8 20 +++
D 4 54 8 16 +

16 429108 SL S-adenosyl-L-methionine synthase 43.1/5.76 A 1 117 16 43 ++++

B 1 119 16 48 +++
C 1 101 10 34 ++
D 1 90 9 28 +

17 19281 SL Enolase 48.0/5.68 A 1 168 20 46 +++

B 1 111 15 41 ++++
C 1 128 15 41 +++
D 1 137 15 41 +

18 19685 NP ATP synthase beta subunit 59.9/5.95 A 1 156 18 38 +

B 1 137 19 42 ++++
C 1 110 11 26 ++++
D 1 156 18 38 ++

19 12546 C Chaperonin 60 61.4/6.28 A 1 90 12 21 +

B 1 82 11 24 ++++
C 2 60 10 17 +++
D 8 46 6 10 ++

20 4582924 ST Phospho-glycerate mutase 61.4/5.42 A 1 104 15 29 +++

B 1 107 19 33 ++++
C 1 85 12 23 ++
D 8 40 7 11 +
a
Abbreviations for various species: C, Cucurbita sp.; NP, Nicotiana plumbaginifolia; NS, Nicotiana sylvestries; NT, Nicotiana tobacum; SL, Solanum lycopersicum; and ST,
Solanum tuberosum.
b
Theoretical molecular mass and pI of the identified proteins.
c
Pollen protein extraction methods; A, TCA–acetone; B, phenol; C, direct IEF buffer; and D, Tris–HCl.
d
PM = number of peptide matched.
e
SC = protein sequence coverage.
f
Relative spot intensity; (+) least, (++++) highest.
 I.S. Sheoran et al. / Plant Science 176 (2009) 99–104 103

Fig. 2. MALDI-TOF-MS spectra of the tryptic digest of spot # 2 from gels with protein extracted by the TCA–acetone [A], Phenol [B], direct IEF Buffer [C], and Tris–HCl [D]
methods. The peptide mass peaks marked with bold numbers are those matched to glycine-rich RNA-binding protein.

coverage obtained were also higher for proteins identified in gels A
and B than for those identified in gels C and D (Table 1). The
procedures used to separate and identify proteins by 2-DE, MALDI-
TOF-MS and PMF were the same in all cases. Therefore, the
observed differences in protein identification can only be
attributed to different protein extraction procedures. The identity
of fewer protein spots from gels with Methods C and D can be
attributed to impurities in protein samples [26,27], and is not
directly related to spot intensity. For example, spot 6 is of relatively
high intensity in Method D compared to other methods (Fig. 1), but
had a higher MOWSE score in Method A (Table 1).
The MS spectra of trypsin digest peptides also varied with the
method of extraction. For example, the mass spectra of spot # 2
showed seven peptide peaks (Fig. 2A and B, marked with bold
numbers), but only four peaks were detected in Method C, and six
in Method D, and these were matched to glycine-rich RNA-
binding protein (Table 1 and Fig. 2). In Method A, the relative
intensity of the matching mass peaks was substantially high,
and background noise low, compared to other methods (Fig. 2)
and these factors might explain the low MOWSE score in
Methods C and D. Thus, the different extraction methods also
show variations in mass spectra which would affect protein
identification by PMF.
In other studies, different protein extraction/solubilization
methods have also been evaluated. For example, different
extraction methods were used for soybean seed proteins, but
MALDI-MS analysis was only performed on spots from the TCA–
acetone method [15]. In banana leaves, the effect of four extraction
procedures on the relative abundance of protein spots was
reported, and 15 spots were selected for further analysis by
MALDI and MS/MS; however, it was not specified from which
extraction method(s) the spots were selected [3]. The compat-
ibility of TCA–acetone and phenol extraction methods with
downstream MS analysis was tested with respect to four spots
from tomato leaves and two from tomato fruit [6]. All six spots
were identified using the TCA–acetone method and five using the
phenol extraction method [6].
4. Conclusion
In this study, the TCA–acetone and phenol protein extraction
methods were found to be superior to other two tested methods
for tomato pollen proteome analysis, in terms of 2-DE gel
separation, mass spectrometric analysis, and identification of
proteins by PMF. Both these methods are known to remove a large
proportion of non-proteinaceous materials which can interfere
104 I.S. Sheoran et al. / Plant Science 176 (2009) 99–104
with plant proteome analysis and are superior to other methods for
the extraction and separation of proteins. Our study demonstrates
that these methods are also efficient for downstream proteome
analysis, and emphasizes the importance of protein extraction
method in achieving optimal separation and identification of
proteins using 2-DE and mass spectrometry.
Acknowledgements
This research was supported by a Discovery grant from the
Natural Sciences and Engineering Research Council of Canada to
V.K.S., and by funding for mass spectrometry and proteomics
equipment from the National Research Council of Canada.
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