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Protein denaturation improves enzymatic digestion efficiency for direct tissue analysis using mass spectrometry
Applied Surface Science 255 (2008) 1555–1559
Contents lists available at ScienceDirect
Applied Surface Science journal homepage: www.elsevier.com/locate/apsusc
Protein denaturation improves enzymatic digestion efficiency for direct tissue analysis using mass spectrometry M. Setou a,b,c,d,*, T. Hayasaka a,d, S. Shimma d, Y. Sugiura b,c, M. Matsumoto c a
Molecular Imaging Frontier Research Center, Department of Molecular Anatomy, Hamamatsu University School of Medicine, Hamamatsu, Shizuoka, Japan Department of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, Kanagawa, Japan c Mitsubishi Kagaku Institute of Life Sciences (MITILS), Machida, Tokyo, Japan d Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, Okazaki, Aichi, Japan b
A R T I C L E I N F O
A B S T R A C T
Article history:
Molecular identification using high-sensitivity tandem mass spectrometry is essential for protein analysis on the tissue surface. Here we report an improved digestion protocol for protein identification directly on the tissue surface using mass spectrometry. By denaturation process and the use of detergentsupplemented trypsin solution, we could successfully detect and identify many molecules such as tubulin, neurofilament, and synaptosomal-associated 25 kDa protein directly from a mouse cerebellum section. ß 2008 Elsevier B.V. All rights reserved.
Available online 13 May 2008 Keywords: MALDI-QIT–TOF MS Brain Peptide Denaturation Digestion MS/MS analysis
1. Introduction The advent of high-throughput measurement technologies has resulted in accumulation of the so-called ‘omics’ information including genome, transcriptome [1,2], proteome, and metabolome. However, each of their precise spatial information in the tissues is hard to obtain at present. The conventional labeling techniques, including the use of GFP and a microscope of in vivo[3] or in-incubator type application [4], are set only to detect molecules that are already known to be present in those tissues. There is also a ‘non-labeling’ technique even in electron microscopic level [5], yet, there still remains the limitation of the object preference. Imaging mass spectrometry and profiling for proteins based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI–TOF MS) [6–8] and high-spatialresolution imaging with secondary-ion mass spectrometry (SIMS) [9–11] are very important techniques for surface analysis of biological tissue sections. Because imaging mass spectrometry has little preference for objects to be seen, it is expected to be used to obtain the spatial
* Corresponding author at: Molecular Imaging Frontier Research Center, Department of Molecular Anatomy, Hamamatsu University School of Medicine, Hamamatsu, Shizuoka, Japan. Tel.: +81 53 435 2292; fax: +81 53 435 2292. E-mail address: [email protected] (M. Setou). 0169-4332/$ – see front matter ß 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.apsusc.2008.05.120
information of virtually all ‘omics’ listed above. Recently, several studies have been published reporting the use of imaging mass spectrometry based on MALDI–TOF MS for purposes such as pathological applications [12–15], biomarker discovery [16], and pharmaceuticals [17–20]. Despite this generous range of the objects to be seen with imaging mass spectrometry, the ability of MALDI–TOF MS to identify molecules on the tissue surface is limited. If identification of proteins is desired, we must resort to conventional proteomics approaches [15] or estimate the proteins from m/z values. To solve this problem, we developed an on-tissue digestion protocol based on denaturation and digestion [21–23]. In our previous studies, we performed ontissue digestion on polyvinylidene fluoride (PVDF). Due to the nonconductive characteristic of PVDF, the ionization efficiency was suppressed. In this paper, we optimized our on-tissue digestion protocol to be carried out directly on the tissue surface. The current paper describes the potential and advantages of our improved preparation procedure. We have performed molecular identification for mouse cerebellum sections using MALDI-quadrupole-ion-trap (QIT)–TOF MS. During the preparation of this manuscript, Groseclose et al. reported a similar digestion protocol for the identification of digested peptides on the tissue surface [24]. The main difference of our method from theirs is that our approach includes a denaturation process and the use of detergentsupplemented trypsin solution, both of which improve the efficiency of the enzymatic digestion.
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M. Setou et al. / Applied Surface Science 255 (2008) 1555–1559
Fig. 1. Workflow of the tissue denaturation process.
2. Experimental
chamber for 30 min. The dried tissues were rinsed with 70% ethanol for 30 s twice and then dehydrated for 15 min [28].
2.1. Materials 2.4. On-tissue digestion and matrix application Ammonium hydrogencarbonate, 2-propanol, acetonitrile, and trifluoroacetic acid (TFA) were purchased from Kanto Chemical Co., Inc. (Tokyo, Japan). Sodium dodecyl sulfate (SDS) and 1,4-dithio-DLthreitol (DTT) were purchased from Nakarai Tesque (Kyoto, Japan). Sequencing-grade modified trypsin was purchased from Promega (Madison, WL). Octyl b-D-glucopyranoside (N-octylglucoside) was obtained from Sigma (St. Louis, MO). The calibration standard peptide mixture (1000–4000 Da) and 2,5-dihydroxybenzoic acid (2,5-DHB) were purchased from Bruker Daltonics (Leipzig, Germany). Milli-Q water (Millipore, Bedford, MA) was used for the preparation of all buffers and solvents. 2.2. Conductive film Indium-tin-oxide (ITO) film was purchased from Tobi Co., Ltd. (Osaka, Japan). We used a film of 125-mm thickness (OTEC-210). The conductivity was 100 V and transparency was 80% (l = 550 nm). These properties were equivalent to those of the ITO glasses reported by Galicia et al. [25] and Chaurand et al. [26]. The ITO film is flexible. Since this film was supplied as an A4-size flat sheet, the problem of curl could be almost disregarded. Uncurled film improved the adhesion between the flexible material and the tissue section. In order to avoid cracking in the conductive layer, it is desirable that the ITO film be carefully handled in a clean place when it is cut out after attaching the protection sheet to the surface.
On-tissue digestion consisted of two steps, denaturation and digestion. The denaturing solution consisted of 2% SDS, 0.5 M Tris/ HCl (pH 6.8), 40 mM DTT, and 40% ethanol. A workflow of the denaturation process is shown in Fig. 1. Explanation of each step follows: (i) An incubation bag with Parafilm was prepared. A small piece of laboratory paper (e.g., Kimwipes1) was put inside the bag. The paper piece had the role of absorbing the denaturing solution to prevent drying. (ii) Denaturing solution was sprayed on the sample surface. Spraying was performed by a 0.2-mm nozzle caliber air brush, Procon Boy FWA Platinum (Mr. Hobby, Tokyo, Japan). The sprayed sample was placed inside the bag, and the bag was tightly sealed. (iii) A spacer was prepared. Here, we usually used a pipette tip holder. The white paper was laboratory paper. (iv) The closed bag was placed onto the spacer. (v) The spacer was wrapped to avoid contact with steam. (vi) The wrapped spacer was placed into a heat-proof box. A wet piece of thick laboratory paper was put in the box. The box was placed on the heat block, and the temperature was set to 80 8C. (vii) Incubation was performed for 10 h. After incubation, the ITO sheet with the tissue section was washed with 70% ethanol, and then dehydrated in the vacuum chamber.
2.3. Section preparation Eight-week-old male C57BL/6Cr mice were used in this study. The mice were purchased from Japan SLC Inc. (Shizuoka, Japan). The mice were sacrificed and dissected under diethyl ether anesthesia. The extirpated brain blocks were immediately frozen in powdered dry ice and stored at 80 8C until needed. Frozen sections were sliced at 16 8C with a cryostat (Leica CM 3050) at a thickness of 5 mm [27]. The frozen sections were then thawmounted on ITO sheets and allowed to dehydrate in a vacuum
After denaturation, enzymatic digestion was performed. In this experiment, a trypsin solution was prepared at 200 mg/ml in 25 mM NH4HCO3 and 20 mM N-octylglucoside [29]. N-Octylglucoside is known as a nonionic detergent which does not interfere with mass spectra. The trypsin solution and 2,5-DHB were dispensed by a chemical inkjet printer (CHIP-1000, Shimadzu, Kyoto, Japan) [30]. The ITO films with tissue sections were adhered to the MS target plates with double-faced conductive tape and installed in the CHIP-1000. The trypsin solution was dispensed as
M. Setou et al. / Applied Surface Science 255 (2008) 1555–1559
10 iterations onto the denatured tissue at 1 nl per iteration. Tryptic digestion was performed for 10 h at 37 8C in a humid environment. After digestion, 5 nl of 2,5-DHB prepared by 12.5 mg/ml in 0.1% TFA containing 25% (v/v) acetonitrile was printed onto each position in the same manner as that described above. 2.5. Mass spectrometry In this study, MALDI-QIT–TOF MS, AXIMA-QIT (Shimadzu, Kyoto, Japan), was used. This instrument was equipped with a 337nm N2 laser capable of operation at a 5 Hz repetition rate. All data acquisitions were performed in the mid-mass range mode (m/z 750–3000) under application of a stage voltage of +18 V (positiveion detection). An external calibration method was used. Singlestage MS spectrum shown in Section 3 were obtained from 200 laser irradiations. In MS/MS analysis, the conditions of data acquisition (i.e., laser power, collision energy and the number of laser irradiations) were changed in order to obtain product ion mass spectra with high intensity and a high signal-to-noise ratio of fragment peaks. Identification based on the MS/MS spectrum was carried out using MS/MS ion search in Mascot1 (Matrix Science Inc.). The spectra shown in Section 3 were processed with real-time smoothing and baseline subtraction.
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following protein fragments: actin, a- and b-tubulin, respectively. Tubulin is a subunit of microtubule [31]. Using MALDI-QIT–TOF MS, we already evaluated the reductive state of polyglutamylation onto tubulins in the mutant mice lacking a molecule involved in btubulin polyglutamylation [32,33]. Furthermore, for the peaks of product ion mass spectra with lower intensity than those shown in Fig. 3B, we also successfully obtained good MS/MS results (Fig. 3C). These peptides were identified as fragments of creatine kinase (a), stromal-cell-derived factor receptor 1 (SDR 1) (b), synaptosomal-associated 25K protein (SNAP25) (c), and neurofilament triplet L protein (d). The corresponding sequences are also shown in each product ion mass spectrum. SNAP25 is a member of soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs) that are involved in synaptic vesicle exocytosis which, we have shown, also involves a novel presynaptic kinase as its regulator [34]. The product ion mass spectra of precursor ions at m/z 1460.7, 1800.8, 2141.2, and 2798.3 are not shown, but we have identified that
3. Results and discussion A spectrum obtained from a trypsin-digested mouse cerebellum section is shown in Fig. 2. Since the purpose of this experiment was to evaluate the capability of molecular identification on the tissue surface, tryptic digestion was performed in a limited area (Fig. 2 inset). The spectrum obtained from the tissue section treated by our protocol, which includes a denaturation process and the use of detergent-supplemented trypsin solution (Fig. 2a), shows a large number of signal peaks derived from trypsindigested products. On the other hand, the spectrum obtained without detergent-supplemented solution (Fig. 2b) has a smaller number of detectable peaks and a decreased signal-to-noise ratio. Moreover, the spectra obtained without the denaturation process (Fig. 2c), and with neither the denaturation process nor the detergent-supplemented solution (Fig. 2d) have no significant peaks above the noise level. Fig. 2e shows the signal-to-noise ratio of the peak at m/z 1198.7, corresponding to the trypsin-digested fragments of actin, obtained from each spectrum; it clearly demonstrates the high-efficiency enzymatic digestion of our procedure. These results indicate that the combination of a denaturation process and the use of detergent-supplemented trypsin solution, and particularly the former, was very effective for enzymatic digestion on the tissue surface. Our interpretation is that temperature-induced unfolding of analyte proteins increases the accessibility of enzymes to the proteins, and such an effect was further promoted by the detergent added to the trypsin solution. Employing our procedure, we successfully obtained product ion mass spectra and identified peptides corresponding to the peaks shown in Fig. 3A. In MS/MS analysis of a complex mixture of trypsin-digested peptides, QIT instrument played an important role in obtaining high-quality MS/MS data. Since the ionization efficiency of the tissue section became low, the storage of interested precursor ions and dissociation with adequate collision-induced dissociation power were helpful for MS/MS analysis. To improve the mass resolution for extracted ions, TOF instrument was also needed. We consider a combination of QIT and TOF instruments to be essential for direct tissue analysis. Fig. 3B shows the spectra with ion signals at m/z 1198.7 (a), 1701.9 (b), and 1871.0 (c). A Mascot-MS/MS ion search assigned the peaks to the
Fig. 2. Mass spectra obtained from 10-mm mouse cerebellum sections of trypsindigested position (see inset). Tissue sections were prepared with the proposed protocol (denaturation process and detergent) (a), with denaturation and without detergent (b), without denaturation and with detergent (c), and with neither denaturation nor detergent (d). Asterisks represent the mass peaks at m/z 1198.7. Signal-to-noise ratio of peak at m/z 1198.7 was obtained from each tissue section (e). Bar, 1 mm.
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Fig. 3. (A) Mass spectrum obtained from a trypsin-digested mouse cerebellum section sections. (B) Product ion mass spectra of m/z: (a) 1198.7, (b) 1701.9, and (c) 1871.0, which had relatively high intensities in the mass spectrum. (C) Product ion mass spectra of m/z: (a) 1303.7, (b) 1527.8, (c) 1669.9, and (d) 1747.9, which had relatively low intensities in the mass spectrum.
M. Setou et al. / Applied Surface Science 255 (2008) 1555–1559
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Table 1 Proteins identified directly from digested mouse cerebellum section using MALDI-QIT–TOF MS Protein identified
Matched sequence
Sequence position
Mass observed
Dm (calculated-observed)
Actin Creatine kinase MBP SDR 1 SNAP 25 a-Tubulin Neurofilament triplet L protein MBP b-Tubulin MBP b-Tubulin
AVFPSIVGRPR VLTPELYAELR TQDENPVVHFFK SVGYPHPEWIWR AWGNNQDGVVASQPAR AVFVDLEPTVIDEVR SAYSSYSAPVSSSLSVR FSWGAEGQKPGFGYGGR INVYYNEAAGNKYVPR TQDENPVVHFFKNIVIPR SGPFGQIFRPDNFGQSGAGNNWAK
26–36 33–43 237–248 34–45 104–119 65–79 37–53 271–287 47–62 237–254 78–103
1198.71 1303.72 1460.73 1527.83 1669.88 1701.91 1747.89 1800.84 1870.95 2141.17 2798.32
0.00 0.06 0.02 0.03 0.03 0.01 0.04 0.01 0.00 0.05 0.03
these peaks were derived from abundant proteins. The results of the identification are summarized in Table 1. In conclusion, we showed the feasibility of an improved protocol for protein identification directly on a mouse section. The use of the improved protocol enabled to detect and identify more molecules from the trypsin-digested tissue surface. Thus, this protocol will complement imaging mass spectrometry in the future. Acknowledgements We thank all members of the SENTAN project for useful comments and discussions. This work was supported by a SENTAN program of Japan Science and Technology Agency grant-in-aid to M.S. References [1] H. Miki, M. Setou, N. Hirokawa, Genome Res. 13 (2003) 1455. [2] M. Matsumoto, M. Setou, K. Inokuchi, Neurosci. Res. 57 (2007) 411. [3] Y. Fukuda, Y. Kawano, Y. Tanikawa, M. Oba, M. Koyama, H. Takagi, M. Matsumoto, K. Nagayama, M. Setou, Neurosci. Lett. 400 (2006) 53. [4] T. Hatanaka, Y. Hatanaka, J. Tsuchida, V. Ganapathy, M. Setou, J. Biol. Chem. 281 (2006) 39273. [5] M. Setou, D. Radostin, K. Atsuzawa, I. Yao, Y. Fukuda, N. Usuda, K. Nagayama, Med. Mol. Morphol. 39 (2006) 176. [6] P. Chaurand, S.A. Schwartz, R.M. Caprioli, Anal. Chem. 76 (2004) 87A. [7] R.M. Caprioli, T.B. Farmer, J. Gile, Anal. Chem. 69 (1997) 4751. [8] Y. Sugiura, S. Shimma, M. Setou, Anal. Chem. 78 (2006) 8227. [9] D. Touboul, F. Halgand, A. Brunelle, R. Kersting, E. Tallarek, B. Hagenhoff, O. Laprevote, Anal. Chem. 76 (2004) 1550. [10] H. Nygren, P. Malmberg, C. Kriegeskotte, H.F. Arlinghaus, FEBS Lett. 566 (2004) 291.
[11] P. Sjovall, J. Lausmaa, H. Nygren, L. Carlsson, P. Malmberg, Anal. Chem. 75 (2003) 3429. [12] S.A. Schwartz, R.J. Weil, M.D. Johnson, S.A. Toms, R.M. Caprioli, Clin. Cancer Res. 10 (2004) 981. [13] K. Yanagisawa, Y. Shyr, B.J. Xu, P.P. Massion, P.H. Larsen, B.C. White, J.R. Roberts, M. Edgerton, A. Gonzalez, S. Nadaf, et al. Lancet 362 (2003) 433. [14] P. Chaurand, S. Fouchecourt, B.B. DaGue, B.J. Xu, M.L. Reyzer, M.C. Orgebin-Crist, R.M. Caprioli, Proteomics 3 (2003) 2221. [15] M. Stoeckli, P. Chaurand, D.E. Hallahan, R.M. Caprioli, Nat. Med. 7 (2001) 493. [16] M.L. Reyzer, R.M. Caprioli, J. Proteome Res. 4 (2005) 1138. [17] Y. Hsieh, R. Casale, E. Fukuda, J. Chen, I. Knemeyer, J. Wingate, R. Morrison, W. Korfmacher, Rapid Commun. Mass Spectrom. 20 (2006) 965. [18] L. Crossman, N.A. McHugh, Y. Hsieh, W.A. Korfmacher, J. Chen, Rapid Commun. Mass Spectrom. 20 (2006) 284. [19] M. Rudin, M. Rausch, M. Stoeckli, Mol. Imaging Biol. 7 (2005) 5. [20] T.C. Rohner, D. Staab, M. Stoeckli, Mech. Ageing Dev. 126 (2005) 177. [21] S. Shimma, M. Furuta, K. Ichimura, Y. Yoshida, M. Setou, Surf. Int. Anal. 38 (2006) 1712. [22] S. Shimma, M. Furuta, K. Ichimura, J. Mass Spectrom. Soc. Jpn. 54 (2006) 133. [23] S. Shimma, Y. Sugiura, M. Setou, J. Mass Spectrom. Soc. Jpn. 54 (2006) 210. [24] M.R. Groseclose, M. Andersson, W.M. Hardesty, R.M. Caprioli, J. Mass Spectrom. 42 (2007) 254. [25] M.C. Galicia, A. Vertes, J.H. Callahan, Anal. Chem. 74 (2002) 1891. [26] P. Chaurand, S.A. Schwartz, D. Billheimer, B.J. Xu, A. Crecelius, R.M. Caprioli, Anal. Chem. 76 (2004) 1145. [27] Y. Sugiura, S. Shimma, M. Setou, J. Mass Spectrom. Soc. Jpn. 54 (2006) 45. [28] S.A. Schwartz, M.L. Reyzer, R.M. Caprioli, J. Mass Spectrom. 38 (2003) 699. [29] H. Katayama, T. Nagasu, Y. Oda, Rapid Commun. Mass Spectrom. 15 (2001) 1416. [30] A.J. Sloane, J.L. Duff, N.L. Wilson, P.S. Gandhi, C.J. Hill, F.G. Hopwood, P.E. Smith, M.L. Thomas, R.A. Cole, N.H. Packer, et al. Mol. Cell Proteomics 1 (2002) 490. [31] S. Westermann, K. Weber, Nat. Rev. Mol. Cell Biol. 4 (2003) 938. [32] K. Ikegami, R.L. Heier, M. Taruishi, H. Takagi, M. Mukai, S. Shimma, S. Taira, K. Hatanaka, N. Morone, I. Yao, et al. Proc. Natl. Acad. Sci. U.S.A. 104 (2007) 3213. [33] K. Ikegami, M. Mukai, J. Tsuchida, R.L. Heier, G.R. Macgregor, M. Setou, J. Biol. Chem. 281 (2006) 30707. [34] E. Inoue, S. Mochida, H. Takagi, S. Higa, M. Deguchi-Tawarada, E. Takao-Rikitsu, M. Inoue, I. Yao, K. Takeuchi, I. Kitajima, et al. Neuron 50 (2006) 261.
Contents lists available at ScienceDirect
Applied Surface Science journal homepage: www.elsevier.com/locate/apsusc
Protein denaturation improves enzymatic digestion efficiency for direct tissue analysis using mass spectrometry M. Setou a,b,c,d,*, T. Hayasaka a,d, S. Shimma d, Y. Sugiura b,c, M. Matsumoto c a
Molecular Imaging Frontier Research Center, Department of Molecular Anatomy, Hamamatsu University School of Medicine, Hamamatsu, Shizuoka, Japan Department of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, Kanagawa, Japan c Mitsubishi Kagaku Institute of Life Sciences (MITILS), Machida, Tokyo, Japan d Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, Okazaki, Aichi, Japan b
A R T I C L E I N F O
A B S T R A C T
Article history:
Molecular identification using high-sensitivity tandem mass spectrometry is essential for protein analysis on the tissue surface. Here we report an improved digestion protocol for protein identification directly on the tissue surface using mass spectrometry. By denaturation process and the use of detergentsupplemented trypsin solution, we could successfully detect and identify many molecules such as tubulin, neurofilament, and synaptosomal-associated 25 kDa protein directly from a mouse cerebellum section. ß 2008 Elsevier B.V. All rights reserved.
Available online 13 May 2008 Keywords: MALDI-QIT–TOF MS Brain Peptide Denaturation Digestion MS/MS analysis
1. Introduction The advent of high-throughput measurement technologies has resulted in accumulation of the so-called ‘omics’ information including genome, transcriptome [1,2], proteome, and metabolome. However, each of their precise spatial information in the tissues is hard to obtain at present. The conventional labeling techniques, including the use of GFP and a microscope of in vivo[3] or in-incubator type application [4], are set only to detect molecules that are already known to be present in those tissues. There is also a ‘non-labeling’ technique even in electron microscopic level [5], yet, there still remains the limitation of the object preference. Imaging mass spectrometry and profiling for proteins based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI–TOF MS) [6–8] and high-spatialresolution imaging with secondary-ion mass spectrometry (SIMS) [9–11] are very important techniques for surface analysis of biological tissue sections. Because imaging mass spectrometry has little preference for objects to be seen, it is expected to be used to obtain the spatial
* Corresponding author at: Molecular Imaging Frontier Research Center, Department of Molecular Anatomy, Hamamatsu University School of Medicine, Hamamatsu, Shizuoka, Japan. Tel.: +81 53 435 2292; fax: +81 53 435 2292. E-mail address: [email protected] (M. Setou). 0169-4332/$ – see front matter ß 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.apsusc.2008.05.120
information of virtually all ‘omics’ listed above. Recently, several studies have been published reporting the use of imaging mass spectrometry based on MALDI–TOF MS for purposes such as pathological applications [12–15], biomarker discovery [16], and pharmaceuticals [17–20]. Despite this generous range of the objects to be seen with imaging mass spectrometry, the ability of MALDI–TOF MS to identify molecules on the tissue surface is limited. If identification of proteins is desired, we must resort to conventional proteomics approaches [15] or estimate the proteins from m/z values. To solve this problem, we developed an on-tissue digestion protocol based on denaturation and digestion [21–23]. In our previous studies, we performed ontissue digestion on polyvinylidene fluoride (PVDF). Due to the nonconductive characteristic of PVDF, the ionization efficiency was suppressed. In this paper, we optimized our on-tissue digestion protocol to be carried out directly on the tissue surface. The current paper describes the potential and advantages of our improved preparation procedure. We have performed molecular identification for mouse cerebellum sections using MALDI-quadrupole-ion-trap (QIT)–TOF MS. During the preparation of this manuscript, Groseclose et al. reported a similar digestion protocol for the identification of digested peptides on the tissue surface [24]. The main difference of our method from theirs is that our approach includes a denaturation process and the use of detergentsupplemented trypsin solution, both of which improve the efficiency of the enzymatic digestion.
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M. Setou et al. / Applied Surface Science 255 (2008) 1555–1559
Fig. 1. Workflow of the tissue denaturation process.
2. Experimental
chamber for 30 min. The dried tissues were rinsed with 70% ethanol for 30 s twice and then dehydrated for 15 min [28].
2.1. Materials 2.4. On-tissue digestion and matrix application Ammonium hydrogencarbonate, 2-propanol, acetonitrile, and trifluoroacetic acid (TFA) were purchased from Kanto Chemical Co., Inc. (Tokyo, Japan). Sodium dodecyl sulfate (SDS) and 1,4-dithio-DLthreitol (DTT) were purchased from Nakarai Tesque (Kyoto, Japan). Sequencing-grade modified trypsin was purchased from Promega (Madison, WL). Octyl b-D-glucopyranoside (N-octylglucoside) was obtained from Sigma (St. Louis, MO). The calibration standard peptide mixture (1000–4000 Da) and 2,5-dihydroxybenzoic acid (2,5-DHB) were purchased from Bruker Daltonics (Leipzig, Germany). Milli-Q water (Millipore, Bedford, MA) was used for the preparation of all buffers and solvents. 2.2. Conductive film Indium-tin-oxide (ITO) film was purchased from Tobi Co., Ltd. (Osaka, Japan). We used a film of 125-mm thickness (OTEC-210). The conductivity was 100 V and transparency was 80% (l = 550 nm). These properties were equivalent to those of the ITO glasses reported by Galicia et al. [25] and Chaurand et al. [26]. The ITO film is flexible. Since this film was supplied as an A4-size flat sheet, the problem of curl could be almost disregarded. Uncurled film improved the adhesion between the flexible material and the tissue section. In order to avoid cracking in the conductive layer, it is desirable that the ITO film be carefully handled in a clean place when it is cut out after attaching the protection sheet to the surface.
On-tissue digestion consisted of two steps, denaturation and digestion. The denaturing solution consisted of 2% SDS, 0.5 M Tris/ HCl (pH 6.8), 40 mM DTT, and 40% ethanol. A workflow of the denaturation process is shown in Fig. 1. Explanation of each step follows: (i) An incubation bag with Parafilm was prepared. A small piece of laboratory paper (e.g., Kimwipes1) was put inside the bag. The paper piece had the role of absorbing the denaturing solution to prevent drying. (ii) Denaturing solution was sprayed on the sample surface. Spraying was performed by a 0.2-mm nozzle caliber air brush, Procon Boy FWA Platinum (Mr. Hobby, Tokyo, Japan). The sprayed sample was placed inside the bag, and the bag was tightly sealed. (iii) A spacer was prepared. Here, we usually used a pipette tip holder. The white paper was laboratory paper. (iv) The closed bag was placed onto the spacer. (v) The spacer was wrapped to avoid contact with steam. (vi) The wrapped spacer was placed into a heat-proof box. A wet piece of thick laboratory paper was put in the box. The box was placed on the heat block, and the temperature was set to 80 8C. (vii) Incubation was performed for 10 h. After incubation, the ITO sheet with the tissue section was washed with 70% ethanol, and then dehydrated in the vacuum chamber.
2.3. Section preparation Eight-week-old male C57BL/6Cr mice were used in this study. The mice were purchased from Japan SLC Inc. (Shizuoka, Japan). The mice were sacrificed and dissected under diethyl ether anesthesia. The extirpated brain blocks were immediately frozen in powdered dry ice and stored at 80 8C until needed. Frozen sections were sliced at 16 8C with a cryostat (Leica CM 3050) at a thickness of 5 mm [27]. The frozen sections were then thawmounted on ITO sheets and allowed to dehydrate in a vacuum
After denaturation, enzymatic digestion was performed. In this experiment, a trypsin solution was prepared at 200 mg/ml in 25 mM NH4HCO3 and 20 mM N-octylglucoside [29]. N-Octylglucoside is known as a nonionic detergent which does not interfere with mass spectra. The trypsin solution and 2,5-DHB were dispensed by a chemical inkjet printer (CHIP-1000, Shimadzu, Kyoto, Japan) [30]. The ITO films with tissue sections were adhered to the MS target plates with double-faced conductive tape and installed in the CHIP-1000. The trypsin solution was dispensed as
M. Setou et al. / Applied Surface Science 255 (2008) 1555–1559
10 iterations onto the denatured tissue at 1 nl per iteration. Tryptic digestion was performed for 10 h at 37 8C in a humid environment. After digestion, 5 nl of 2,5-DHB prepared by 12.5 mg/ml in 0.1% TFA containing 25% (v/v) acetonitrile was printed onto each position in the same manner as that described above. 2.5. Mass spectrometry In this study, MALDI-QIT–TOF MS, AXIMA-QIT (Shimadzu, Kyoto, Japan), was used. This instrument was equipped with a 337nm N2 laser capable of operation at a 5 Hz repetition rate. All data acquisitions were performed in the mid-mass range mode (m/z 750–3000) under application of a stage voltage of +18 V (positiveion detection). An external calibration method was used. Singlestage MS spectrum shown in Section 3 were obtained from 200 laser irradiations. In MS/MS analysis, the conditions of data acquisition (i.e., laser power, collision energy and the number of laser irradiations) were changed in order to obtain product ion mass spectra with high intensity and a high signal-to-noise ratio of fragment peaks. Identification based on the MS/MS spectrum was carried out using MS/MS ion search in Mascot1 (Matrix Science Inc.). The spectra shown in Section 3 were processed with real-time smoothing and baseline subtraction.
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following protein fragments: actin, a- and b-tubulin, respectively. Tubulin is a subunit of microtubule [31]. Using MALDI-QIT–TOF MS, we already evaluated the reductive state of polyglutamylation onto tubulins in the mutant mice lacking a molecule involved in btubulin polyglutamylation [32,33]. Furthermore, for the peaks of product ion mass spectra with lower intensity than those shown in Fig. 3B, we also successfully obtained good MS/MS results (Fig. 3C). These peptides were identified as fragments of creatine kinase (a), stromal-cell-derived factor receptor 1 (SDR 1) (b), synaptosomal-associated 25K protein (SNAP25) (c), and neurofilament triplet L protein (d). The corresponding sequences are also shown in each product ion mass spectrum. SNAP25 is a member of soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs) that are involved in synaptic vesicle exocytosis which, we have shown, also involves a novel presynaptic kinase as its regulator [34]. The product ion mass spectra of precursor ions at m/z 1460.7, 1800.8, 2141.2, and 2798.3 are not shown, but we have identified that
3. Results and discussion A spectrum obtained from a trypsin-digested mouse cerebellum section is shown in Fig. 2. Since the purpose of this experiment was to evaluate the capability of molecular identification on the tissue surface, tryptic digestion was performed in a limited area (Fig. 2 inset). The spectrum obtained from the tissue section treated by our protocol, which includes a denaturation process and the use of detergent-supplemented trypsin solution (Fig. 2a), shows a large number of signal peaks derived from trypsindigested products. On the other hand, the spectrum obtained without detergent-supplemented solution (Fig. 2b) has a smaller number of detectable peaks and a decreased signal-to-noise ratio. Moreover, the spectra obtained without the denaturation process (Fig. 2c), and with neither the denaturation process nor the detergent-supplemented solution (Fig. 2d) have no significant peaks above the noise level. Fig. 2e shows the signal-to-noise ratio of the peak at m/z 1198.7, corresponding to the trypsin-digested fragments of actin, obtained from each spectrum; it clearly demonstrates the high-efficiency enzymatic digestion of our procedure. These results indicate that the combination of a denaturation process and the use of detergent-supplemented trypsin solution, and particularly the former, was very effective for enzymatic digestion on the tissue surface. Our interpretation is that temperature-induced unfolding of analyte proteins increases the accessibility of enzymes to the proteins, and such an effect was further promoted by the detergent added to the trypsin solution. Employing our procedure, we successfully obtained product ion mass spectra and identified peptides corresponding to the peaks shown in Fig. 3A. In MS/MS analysis of a complex mixture of trypsin-digested peptides, QIT instrument played an important role in obtaining high-quality MS/MS data. Since the ionization efficiency of the tissue section became low, the storage of interested precursor ions and dissociation with adequate collision-induced dissociation power were helpful for MS/MS analysis. To improve the mass resolution for extracted ions, TOF instrument was also needed. We consider a combination of QIT and TOF instruments to be essential for direct tissue analysis. Fig. 3B shows the spectra with ion signals at m/z 1198.7 (a), 1701.9 (b), and 1871.0 (c). A Mascot-MS/MS ion search assigned the peaks to the
Fig. 2. Mass spectra obtained from 10-mm mouse cerebellum sections of trypsindigested position (see inset). Tissue sections were prepared with the proposed protocol (denaturation process and detergent) (a), with denaturation and without detergent (b), without denaturation and with detergent (c), and with neither denaturation nor detergent (d). Asterisks represent the mass peaks at m/z 1198.7. Signal-to-noise ratio of peak at m/z 1198.7 was obtained from each tissue section (e). Bar, 1 mm.
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Fig. 3. (A) Mass spectrum obtained from a trypsin-digested mouse cerebellum section sections. (B) Product ion mass spectra of m/z: (a) 1198.7, (b) 1701.9, and (c) 1871.0, which had relatively high intensities in the mass spectrum. (C) Product ion mass spectra of m/z: (a) 1303.7, (b) 1527.8, (c) 1669.9, and (d) 1747.9, which had relatively low intensities in the mass spectrum.
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Table 1 Proteins identified directly from digested mouse cerebellum section using MALDI-QIT–TOF MS Protein identified
Matched sequence
Sequence position
Mass observed
Dm (calculated-observed)
Actin Creatine kinase MBP SDR 1 SNAP 25 a-Tubulin Neurofilament triplet L protein MBP b-Tubulin MBP b-Tubulin
AVFPSIVGRPR VLTPELYAELR TQDENPVVHFFK SVGYPHPEWIWR AWGNNQDGVVASQPAR AVFVDLEPTVIDEVR SAYSSYSAPVSSSLSVR FSWGAEGQKPGFGYGGR INVYYNEAAGNKYVPR TQDENPVVHFFKNIVIPR SGPFGQIFRPDNFGQSGAGNNWAK
26–36 33–43 237–248 34–45 104–119 65–79 37–53 271–287 47–62 237–254 78–103
1198.71 1303.72 1460.73 1527.83 1669.88 1701.91 1747.89 1800.84 1870.95 2141.17 2798.32
0.00 0.06 0.02 0.03 0.03 0.01 0.04 0.01 0.00 0.05 0.03
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