Plant

Plant Science 176 (2009) 99–104

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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 b

Department of Biology, University of Saskatchewan, 112 Science Place, Saskatoon, SK, Canada S7N 5E2 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: Received 26 June 2008 Received in revised form 5 September 2008 Accepted 26 September 2008 Available online 14 October 2008

Different protein extraction methods have been developed for plant proteome analysis but their compatibility with mass spectrometry has rarely been tested. We evaluated four protein extraction methods, i.e., trichloroacetic acid (TCA)–acetone, phenol, direct iso-electric focusing (IEF) buffer, and Tris–HCl buffer, using tomato pollen for proteome analysis. The data presented show that the TCA– 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 spectrometric analysis, and identification of proteins by peptide mass fingerprinting (PMF). These results highlight the importance of plant protein extraction method for subsequent MS analysis and protein identification. ß 2008 Elsevier Ireland Ltd. All rights reserved.

Keywords: Protein extraction methods Mass Spectrometry Pollen Proteomics

1. Introduction 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 (2DE) is one of the most widely used techniques for resolving complex protein extracts, although alternative, gel-free approaches, including protein antibody arrays [1] and multidimensional 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: [email protected] (V.K. Sawhney). 0168-9452/$ – see front matter ß 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.plantsci.2008.09.015

downstream protein analysis by liquid chromatography and mass 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 identification 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/ionization 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

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that the choice of extraction procedure is a major factor in protein separation, analysis, and identification.

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. 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

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-polyacrylamide gels and separated using PROTEAN II XI multi-cell (BioRad, USA). The gels were stained with Colloidal Coomassie Brilliant Blue G250 (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 MALDITOF-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 recommended procedure. The resulting tryptic digests were concentrated 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 VoyagerDE 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.

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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

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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 dehydrogenase 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.

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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. Gene index number

Speciesa

Protein identity

MW/pIb

Methodsc

Rank

SCe (%)

Relative spot intensityf

2

45533923

NS

Glycine-rich RNA-binding protein

17.6/5.58

A B C D

1 1 3 2

80 71 42 62

7 7 4 6

45 45 27 39

++++ + ++ +++

6

30841938

SL

Thioredoxin peroxidase 1

17.4/5.17

A B C D

1 1 3 1

70 63 39 64

5 5 3 5

25 25 11 25

++ +++ + ++++

7

48209968

ST

Mitochondrial ATP synthase D-chain

19.8/5.34

A B C D

1 1 1 1

208 206 189 170

20 23 19 15

69 80 68 63

+++ ++ ++++ +

9

534916

ST

Soluble inorganic pyrophosphatase

24.4/5.59

A B C D

1 1 3 1

111 51 54 54

10 6 6 7

27 23 18 20

+++ ++++ + +++

13

1915974

SL

Fructokinase

35.0/5.76

A B C D

1 1 1 1

101 106 123 70

14 15 12 11

46 51 43 31

+++ +++ ++++ ++++

14

1419094

ST

Glutamine synthetase

39.4/5.38

A B C D

1 1 1 4

80 74 57 54

10 11 8 8

24 19 20 16

+++ ++++ +++ +

16

429108

SL

S-adenosyl-L-methionine synthase

43.1/5.76

A B C D

1 1 1 1

117 119 101 90

16 16 10 9

43 48 34 28

++++ +++ ++ +

17

19281

SL

Enolase

48.0/5.68

A B C D

1 1 1 1

168 111 128 137

20 15 15 15

46 41 41 41

+++ ++++ +++ +

18

19685

NP

ATP synthase beta subunit

59.9/5.95

A B C D

1 1 1 1

156 137 110 156

18 19 11 18

38 42 26 38

+ ++++ ++++ ++

19

12546

C

Chaperonin 60

61.4/6.28

A B C D

1 1 2 8

90 82 60 46

12 11 10 6

21 24 17 10

+ ++++ +++ ++

20

4582924

ST

Phospho-glycerate mutase

61.4/5.42

A B C D

1 1 1 8

104 107 85 40

15 19 12 7

29 33 23 11

+++ ++++ ++ +

Spot number

MOWSE score

PMd

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.

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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, MALDITOF-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 RNAbinding 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 compatibility 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

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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. References [1] R. Barry, M. Soloviev, Quantitative protein profiling using antibody arrays, Proteomics 4 (2004) 3717–3726. [2] M.R. Rose, T.J. Griffin, Gel-free mass spectrometry-based high throughput proteomics: tools for studying biological response of proteins and proteomes, Proteomics 6 (2006) 4678–4687. [3] S.C. Carpentier, E. Witters, K. Laukens, P. Deckers, R. Swennen, B. Panis, Preparation of protein extracts from recalcitrant plant tissues: an evaluation of different methods for two-dimensional gel electrophoresis analysis, Proteomics 5 (2005) 2497–2507. [4] P. Giavalisco, E. Nordhoff, H. Lehrach, J. Gobom, J. Klose, Extraction of proteins from plant tissues for two-dimensional electrophoresis analysis, Electrophoresis 24 (2003) 207–216. [5] J.K.C. Rose, S. Bashir, J.J. Giovannoni, M.M. Jahn, R.S. Saravanan, Tackling the plant proteome: practical approaches, hurdles and experimental tools, Plant J. 39 (2004) 715–733. [6] R.S. Saravanan, J.K.C. Rose, A critical evaluation of sample extraction techniques for enhanced proteomic analysis of recalcitrant plant tissues, Proteomics 4 (2004) 2522–2532. [7] W. Wang, M. Scali, R. Vignani, A. Spadafora, E. Sensi, S. Mazzuca, M. Cresti, Protein extraction for two-dimensional electrophoresis from olive leaf, a plant tissue containing high levels of interfering compounds, Electrophoresis 24 (2003) 2369–2375. [8] K. Gallardo, C. Job, S.P.C. Groot, M. Puype, H. Demol, J. Vandekerckhove, D. Job, Proteomic analysis of Arabidopsis seed germination and priming, Plant Physiol. 126 (2001) 835–848. [9] I.S. Sheoran, K.A. Sproule, D.J.H. Olson, A.R.S. Ross, V.K. Sawhney, Proteome profile and functional classification of proteins in Arabidopsis thaliana (Landsberg erecta) mature pollen, Sex, Plant Reprod. 19 (2006) 185–196.

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