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Plant Science
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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.
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 buffer comprising 8 M urea, 20 mM DTT, 4% CHAPS, 5 mM EDTA,
separation, analysis, and identification. 5 mM Tris base and 2% ampholyte (pH 3–10), as described in
Method A.
2. Methods
2.2.4. Method D (Tris–HCl buffer)
2.1. Plant material Proteins were extracted as described earlier [20], with some
modifications. Pollen were ground in liquid nitrogen and mixed
Tomato (Solanum lycopersicum cv. Rutgers) pollen grains were with Tris–HCl buffer consisting of 50 mM Tris–HCl pH 8.8, 5 mM
collected from the freshly open flowers from greenhouse-grown EDTA, 20 mM DTT, 100 mM KCl, and 2 mM PMSF. After thawing,
plants, placed on a glass slide, checked under a dissecting the mixture was ground for an additional 30 min at 4 8C and
microscope, and any debris removed with a needle. Pollen samples centrifuged at 25,000 g for 20 min. The supernatant was
were then pooled in an Eppendorf tube and either processed collected, the pellets re-extracted, and the supernatants pooled.
immediately or stored at 80 8C for later analysis. Proteins in the supernatant were then precipitated with 5
volume (v/v) of 100% acetone and incubated at 20 8C for 2 h.
2.2. Protein extraction After centrifugation, the pellet was washed twice with 80%
acetone and then re-suspended in IEF buffer as described in
Proteins from mature pollen (25 mg for each method) were Method A.
extracted using four different methods commonly used for protein Total protein in all the above extracts (A to D) was estimated
extraction from various plant tissues, including pollen, as detailed using the Bradford reagent (Bio-Rad, Hercules, CA, USA) before
below. immediate processing or storage at 80 8C for later analysis.
3. Results and discussion qualitative differences were observed between these gels. For
example, the average number of protein spots observed in gels
The total amount of protein extracted from equal amounts of using Method A was 555 61 (S.E.) and with Method B was
pollen tissues varied according to the protein extraction method 558 54. These were higher than with Methods C (318 40), and D
used. Considerably higher amount of protein was extracted with the (332 37). Also, with Methods A and B, there were more spots with
direct IEF buffer extraction method (Method C, 180.0 17.5 mg/g) high molecular mass compared to the other two methods. The lower
compared to other three methods (Method A, 138.4 16.4 mg/g; number of spots observed with Methods C and D could be related to
Method B, 114.8 13.6 mg/g; Method D, 104.6 11.2 mg/g { values the presence of impurities in these protein samples, as discussed
are S.E., n = 4}). This could be due to the simplicity of Method C; the above, and could also explain some horizontal and vertical streaking
single-step procedure avoiding losses that may occur with other in these gels (Fig 1C and D). Variations in spot number have also been
methods involving additional steps such as protein precipitation and reported in other plant proteomic studies which used different
re-solubilization, as suggested before [23]. Alternatively, it is possible methods of protein extraction [6].
that there is an over estimation of proteins in Method C due to the The 2-DE protein spot patterns obtained (using pH 4–7 IPG
presence of some impurities and small peptides in the extract which are strips) were similar for the four extraction methods (Fig. 1). Indeed,
removed in other methods involving additional steps. Variations in more than 90% of protein spots on the gel with minimum spots
protein recovery using different extraction methods have been (Method C) were matched across all other gels. Only a few
reported in other studies [5,16,24]. The amount of protein extracted differential spots were observed between gels from Methods A and
from different tomato plant tissues depends upon the tissue type and B as marked with small letters in Fig. 1A and B. Interestingly, one of
was somewhat greater for phenol than for TCA–acetone method [6]. On the spot marked ‘a’ in gel A was absent in gel B, but was present at
the other hand, TCA–acetone proved to be better than phenol for high levels in gels C and D. This protein was identified as
Brassica seeds [24]. However, in this study both these methods were calreticulin, a calcium binding protein involved in calcium
comparable in terms of total protein recovery, as is the case for banana signaling [25]. Phospho-glucomutase, phospho-glycerate dehy-
leaves [3]. drogenase and protein phosphatase C (spots g, h and j,
Equal amounts of the protein extracted from tomato pollen respectively) were identified only in the phenol method
using each of the four extraction Methods (A to D) were separated (Fig. 1B). Spot variations in 2-DE gels with different extraction
by 2-DE under identical conditions. Representative CCB-stained methods have been reported by others [3,5–7,13,15,16]. Only few
gels for each Method are shown in Fig. 1. All four methods resulted differences were observed in protein spots between gels from
in good quality, well-resolved gels; however, quantitative and 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 account for reduced variability in spot pattern when using
spot patterns for tomato using three different extraction protocols. different extraction methods.
These authors concluded that glycosylation may have contributed The comparison of extraction methods for MS compatibility
to the observed differences in 2-DE spot patterns by affecting was tested by analyzing 20 spots common to, and excised
protein solubility under different extraction conditions. It is also individually from, all four gels (Fig. 1, A to D). The results of
possible that variations in spot patterns observed by these authors protein identification by MALDI-TOF-MS and PMF are presented in
may be related to the developing tissues as opposed to mature Table 1. Of the 20 spots analyzed, 11 spots (55%) were identified
tissues. In this study, we observed similar 2-DE spot patterns for with a significant MOWSE score (i.e., a score greater than 67 at
mature tomato pollen using different extraction methods, as was p < 0.05) from gels using extraction Method A, 9 (45%) using
the case for mature soybean seeds [15]. Since both these tissues are Method B, 6 (33%) using Method C, and 5 (25%) using Method D
dormant and desiccated structures their non-active state may (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
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 In other studies, different protein extraction/solubilization
and B than for those identified in gels C and D (Table 1). The methods have also been evaluated. For example, different
procedures used to separate and identify proteins by 2-DE, MALDI- extraction methods were used for soybean seed proteins, but
TOF-MS and PMF were the same in all cases. Therefore, the MALDI-MS analysis was only performed on spots from the TCA–
observed differences in protein identification can only be acetone method [15]. In banana leaves, the effect of four extraction
attributed to different protein extraction procedures. The identity procedures on the relative abundance of protein spots was
of fewer protein spots from gels with Methods C and D can be reported, and 15 spots were selected for further analysis by
attributed to impurities in protein samples [26,27], and is not MALDI and MS/MS; however, it was not specified from which
directly related to spot intensity. For example, spot 6 is of relatively extraction method(s) the spots were selected [3]. The compat-
high intensity in Method D compared to other methods (Fig. 1), but ibility of TCA–acetone and phenol extraction methods with
had a higher MOWSE score in Method A (Table 1). downstream MS analysis was tested with respect to four spots
The MS spectra of trypsin digest peptides also varied with the from tomato leaves and two from tomato fruit [6]. All six spots
method of extraction. For example, the mass spectra of spot # 2 were identified using the TCA–acetone method and five using the
showed seven peptide peaks (Fig. 2A and B, marked with bold phenol extraction method [6].
numbers), but only four peaks were detected in Method C, and six
in Method D, and these were matched to glycine-rich RNA- 4. Conclusion
binding protein (Table 1 and Fig. 2). In Method A, the relative
intensity of the matching mass peaks was substantially high, In this study, the TCA–acetone and phenol protein extraction
and background noise low, compared to other methods (Fig. 2) methods were found to be superior to other two tested methods
and these factors might explain the low MOWSE score in for tomato pollen proteome analysis, in terms of 2-DE gel
Methods C and D. Thus, the different extraction methods also separation, mass spectrometric analysis, and identification of
show variations in mass spectra which would affect protein proteins by PMF. Both these methods are known to remove a large
identification by PMF. proportion of non-proteinaceous materials which can interfere
104 I.S. Sheoran et al. / Plant Science 176 (2009) 99–104
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