- Email: [email protected]
Proteomic analysis of masticatory muscle tendon–aponeurosis hyperplasia: A preliminary study using a 2D-DIGE system
Journal of Oral and Maxillofacial Surgery, Medicine, and Pathology 24 (2012) 185–188
Contents lists available at SciVerse ScienceDirect
Journal of Oral and Maxillofacial Surgery, Medicine, and Pathology journal homepage: www.elsevier.com/locate/jomsmp
Oral and Maxillofacial Surgery/Original research
Proteomic analysis of masticatory muscle tendon–aponeurosis hyperplasia: A preliminary study using a 2D-DIGE system Tsuyoshi Sato a,∗ , Aya Nakamoto a , Naoko Hori a , Yuichiro Enoki a , Yousuke Fukushima a , Norimichi Nakamoto a , Yasuaki Sakata a , Hidenori Yamanaka b , Dai Chida c , Takahiro Abe d , Tetsuya Yoda a a
Department of Oral and Maxillofacial Surgery, Saitama Medical University, 38 Moro-hongou, Moroyama-machi, Iruma-gun, Saitama, 350-0495, Japan Chemicals Assessment Center, Chemicals Evaluation and Research Institute, 1600, Shimo-Takano, Sugito-machi, Kitakatsushika-gun, Saitama 345-0043, Japan c Department of Immunology and Pathology, Research Institute, National Center for Global Health and Medicine, 1-21-1, Toyama, Shinjuku-ku, Tokyo, 162-8655, Japan d Department of Oral and Maxillofacial Surgery, Faculty of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-0033, Japan b
a r t i c l e
i n f o
Article history: Received 8 September 2011 Received in revised form 6 February 2012 Accepted 20 February 2012 Available online 29 March 2012 Keywords: Masticatory muscle tendon-aponeurosis hyperplasia Two-dimensional fluorescence difference gel electrophoresis Liquid chromatography coupled with tandem mass spectrometry Collagen alpha-1(VI) chain
a b s t r a c t Masticatory muscle tendon–aponeurosis hyperplasia (MATAPHY) exhibits hyperplasia on tendon and aponeurosis of the masticatory muscles histopathologically. This disease is characterized by a palpable dense band of the anterior border of the masseter muscle on maximum mouth opening, strike root appearance on MR imaging and square mandible configuration. However, the common useful diagnostic markers of this disease are still unknown. Here we performed a preliminary study to identify potential therapeutic targets and diagnostic markers that may indicate disease progression of MATAPHY using a proteomic analysis method, two-dimensional fluorescence difference gel electrophoresis and liquid chromatography coupled with tandem mass spectrometry. We have found that myosin regulatory light chain 2 and myosin regulatory light chain 3 were down-regulated in MATAPHY. This result is consistent with the observation that muscle tissues showed atrophic changes. We have also demonstrated that aortic smooth muscle actin was down-regulated in MATAPHY, suggesting that this down-regulation is due to decreasing in muscle tissues that blood vessels are abundant and increasing in tendon tissues that blood vessels are exiguous. Furthermore, collagen alpha-1(VI) chain (COL6A1) was up-regulated in MATAPHY. COL6A1, which is essential for tendon fibrillogenesis, expressed in the masticatory muscle, indicating that tendon-aponeurosis hyperplasia is due to the overexpression of COL6A1. Facial deformity, which we choose as a control in this study, is sometimes congenital and may be complicated with deficiency of the skeletal proteins. Although this report has some limitations, this is the first report to describe the relationship between the variation of specific molecules and histopathological observation in MATAPHY. © 2012 Asian Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
1. Introduction Masticatory muscle tendon–aponeurosis hyperplasia (MATAPHY) is a new disease associated with limited mouth opening [1]. From the viewpoint of histopathology, both tendon and aponeurosis in MATAPHY exhibit hyperplasia [2]. This disease is classified into the masseter muscle type, which is accompanied by more severe hyperplasia of the masseter muscle aponeurosis with a square mandible due to hypertrophy of the mandibular angle as compared to the temporalis muscle tendon and the temporal muscle type, which does not show marked hyperplasia of the masseter
∗ Corresponding author. E-mail address: [email protected] (T. Sato).
muscle aponeurosis [3]. Previously, we demonstrated that good long-term results have been reliably obtained by resection of the hyperplastic masseter muscle aponeurosis with coronoidectomy for separation of the temporal muscle from the coronoid process in patients with MATAPHY [3]. The characteristics of this disease, which are a palpable dense band of the anterior border of the masseter muscle on maximum mouth opening, strike root appearance on magnetic resonance imaging, and square mandible configuration, are useful in its diagnosis [4,5]. However, common useful diagnostic markers of this disease are still unknown. Recent advances in proteomic analysis have allowed us to better understand the molecular basis of human diseases [6]. Two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) has been developed to quantify the differences between experimental pairs of samples resolved on the
2212-5558/$ – see front matter © 2012 Asian Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ajoms.2012.02.003
186
T. Sato et al. / Journal of Oral and Maxillofacial Surgery, Medicine, and Pathology 24 (2012) 185–188
Table 1 Basic characteristics of patients.
FD MATAPHY
Sex
Age
Underlying disease
Female Female
22 56
Not particular Alcoholic liver injury
same 2D gel [7]. Liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) provides significant benefits to clinical diagnostic laboratories that conduct routine analyses [8]. We conducted a preliminary study to identify potential therapeutic targets, as well as diagnostic markers, that may indicate disease progression of MATAPHY by using a 2D-DIGE system. 2. Materials and methods 2.1. Patients and subjects Tissue specimens of temporal muscles and tendons, including 1 subject with MATAPHY and 1 with facial deformity (FD), were obtained from patients undergoing surgery (Table 1). As this MATAPHY patient has no sign of the masseter muscle type such as a palpable dense band of the anterior border of the masseter muscle on maximum mouth opening and strike root appearance on magnetic resonance imaging, this patient was diagnosed as the temporal muscle type. The patients were treated and followed up at Saitama Medical University. The study was performed in accordance with the Declaration of Helsinki and approved by the Institutional Review Board (approval number 595) at Saitama Medical University. The dentist in charge provided all patients or their guardians with an explanation of the study. Patients were free to withdraw from the study of their own free will at any time. Informed consent was obtained from all subjects. 2.2. Sample homogenization and protein labeling All samples were homogenized in 10 volumes of lysis buffer (LB) (4% (w/v) CHAPS, 2 M thiourea, 8 M urea, 10 mM Tris–HCl pH 8.8) and homogenized with a Polytron-type homogenizer. The sample solution was sonicated for a series of 20-s bursts on ice, and then centrifuged at 14,000 rpm for 20 min at 10 ◦ C. Protein concentration was determined using the Bradford method. Lysates were labeled with NHS ester-derivatives of the cyanine dyes Cy2, Cy3, and Cy5 (GE Healthcare, Little Chalfont, UK) following the manufacturer’s protocol. Typically, 100 g of lysate was “minimally” labeled with 200 pmol of Cy dye. Labeling reactions were performed on ice in the dark for 30 min, and then quenched with a 50-fold molar excess of free lysine to dye for 10 min on ice. Differentially labeled samples were reduced with 65 mM DTT and mixed with Pharmalytes pH 3–10 (GE Healthcare) for 10 min. Each control (specimens from FD) and test (specimens from MATAPHY) sample was labeled with Cy3 and Cy5. A pool of all samples was also prepared and labeled with Cy2 for use as a standard on all gels to aid image matching. 2.3. Gel electrophoresis and imaging Immobilized pH gradient (IPG) strips pH 3–10 L, 24 cm (GE Healthcare), were rehydrated, and mixed samples were applied with cup loading. Isoelectric focusing was performed using a Multiphor II (GE Healthcare) at 54 kVh at 20 ◦ C in the dark [9,10]. Strips were equilibrated for 10 min in 50 mM Tris–HCl pH 8.8, 6 M urea, 30% (v/v) glycerol, and 1% (w/v) SDS containing 65 mM DTT and then for a further 10 min in the same buffer containing 240 mM iodoacetamide. Equilibrated IPG strips were transferred onto 24 cm × 20 cm, 12% uniform polyacrylamide gels. Strips were
overlaid with 0.5% (w/v) low melting point agarose in running buffer containing bromophenol blue. Gels were run in Ettan DALT twelve (GE Healthcare) at 2 W per gel at 15 ◦ C, until the dye front had run off the bottom of the gels. The 2D gels were then scanned directly between glass plates using a Typhoon 9400 (GE Healthcare). The images generated were exported as tiff files for further protein profile analysis by DeCyderTM 7.0 (GE Healthcare). 2.4. Image analysis and statistical analysis Differential in-gel analysis (DIA) by DeCyderTM 7.0 was used to merge the Cy2, Cy3, and Cy5 images for each gel and to detect spot boundaries to calculate normalized spot volumes/protein abundance. At this stage, features resulting from non-protein sources (e.g., dust particles, and streaks) and faint spots (e.g., spot area <300, spot volume <10,000) were filtered out. The analysis was used to calculate abundant differences between samples run on the same gel. The biological variation analysis (BVA) of DeCyderTM 7.0 was then used to match all pair-wise image comparisons from DIA for comparative cross-gel statistical analysis. Comparison of normalized Cy3 and Cy5 spot volumes with the corresponding Cy2 standard spot volumes within each gel gave a standardized abundance. This value was compared across all gels for each matched spot, and average ratios were calculated from the standardized abundance of 3 gel images of each sample by the DeCyder software. Statistical analysis was performed using the triplicate values from each experimental condition to give Student’s P-value and a 2-tailed probability of less than 5% (P < 0.05) was considered statistically significant. 2.5. In-gel digestion and peptide extraction Gel electrophoresis for MS analysis was performed using 600 g of pooled lysate, following the same procedures as described above. After gel electrophoresis, the gel was stained with Sypro® Ruby, and each MS analysis gel was matched with the master gel for expression analysis by BVA software. Spots of interest were excised from 2D gels using an automated spot picker (GE Healthcare) following the manufacturer’s instructions. Spots were collected in 200 L of water in 96-well plates. The recovered gel pieces were washed with aqueous 50 mM ammonium bicarbonate and acetonitrile and then incubated with 12.5 ng/L trypsin (Promega, Southampton, UK) at 30 ◦ C for 15 h. The peptides generated were eluted with 50 mM ammonium bicarbonate followed by 10% (v/v) formic acid and acetonitrile. The combined fractions were dried in a SpeedVac (Thermo Fisher Scientific, Waltham, USA) and dissolved in 0.1% (v/v) formic acid. 2.6. Mass spectrometric analysis Mass spectrometric analysis was carried out using LC–MS/MS [11,12]. HPLC (CapLC, Waters, Milford, MA) was coupled with the Q-TOF micro mass spectrometer (Micromass, Manchester, UK). Instrument operation and data acquisition and analysis were performed using MassLynx3.2 software (Micromass). The tryptic peptides were concentrated and desalted on a 300-m i.d./5mm length C18 PepMap column (LC Packings, San Francisco, CA). The eluted peptide was analyzed by tandem mass spectrometric sequencing with an automated MS-to-MS/MS switching protocol. Online determination of precursor-ion masses was performed over the m/z range from 400 to 1600 atomic mass units in the positivecharge detection mode with a cone voltage of 50 V. The cone voltage, extraction voltage, microchannel plate (MCP) detector voltage, and collision energy were optimized before measurement of samples. A database search was performed with MASCOT Deamon (Matrix Science, London, UK) [13–15]. The generated pkl
T. Sato et al. / Journal of Oral and Maxillofacial Surgery, Medicine, and Pathology 24 (2012) 185–188
187
Fig. 1. Master number (no.) and its gel images of test and control, P-value, and Av. ratio (−X represents 1/X). (A) Master no. 207, (B) master no. 977, (C) master no. 1138, (D) master no. 1147, (E) master no. 1158, (F) master no. 1389, (G) master no. 1402, (H) master no. 1437, (I) master no. 1440, and (J) master no. 1442.
files were submitted to SWISS-PROT (release 57.6) and NCBInr (20090430). Search parameters were as follows: fixed modifications, carbamidomethyl; variable modifications, oxidation (M); missed cleavages, up to 1 (monoisotopic); peptide tolerance, 1.0 Da; and MS/MS tolerance, 0.5 Da. To automatically exclude all the very low scoring, random peptide matches, the ion score cutoff was set at 20. This means that homologous proteins are more likely to collapse into a single hit, avoiding the need to choose between them. Matches using mass values are always handled on a probabilistic basis. The total score is the absolute probability that the observed match is a random event. The MOWSE scores were the reported scores of −10 log10 (P), where P is the absolute probability. A probability of 10–20 thus becomes a score of 200 [16]. The automatically
identified proteins were checked individually to eliminate redundancy. 3. Results and discussion We performed 2D gel electrophoresis and 10 delineated protein spots showing remarkable changes including significant changes (P < 0.05) between MATAPHY and FD were observed (Fig. 1). These differentially expressed protein spots are shown: 5 spots were upregulated and 5 spots were downregulated in MATAPHY (Fig. 1A–J). A total of 10 differentially expressed spots were excised from gels and analyzed for protein identification by LC/MS/MS. As shown in Table 2, we identified 4 upregu-
188
T. Sato et al. / Journal of Oral and Maxillofacial Surgery, Medicine, and Pathology 24 (2012) 185–188
Table 2 Identification of differential proteins by searching in protein database. Master no. Up-regulated protein 1389 1158 1147 207 977 Down-regulated protein 1138 1402 1437 1442 1440
molecular pathogenesis. Further analysis will be needed to identify potential therapeutic targets and diagnostic markers of MATAPHY.
Protein name
Acknowledgments Apolipoprotein A-I Fibrinogen beta chain Fibrinogen beta chain Collagen alpha-1(VI) chain Vitamin D-binding protein Actin, aortic smooth muscle Myosin light chain 3 Myosin regulatory light chain 2, ventricular/cardiac muscle isoform Myosin regulatory light chain 2, ventricular/cardiac muscle isoform Myosin regulatory light chain 2, ventricular/cardiac muscle isoform
lated proteins—apolipoprotein AI (APOA1), fibrinogen beta chain (FGB), collagen alpha-1(VI) chain (COL6A1), and vitamin D-binding protein (GC)—and 3 downregulated proteins—aortic smooth muscle actin (ACTA2), myosin regulatory light chain 3 (MYL3), and myosin regulatory light chain 2 (ventricular/cardiac muscle isoform) (MYL2). Some spots appeared to correspond to the same protein, i.e., spots 1158 and 1147 were FGB and spots 1437, 1442, and 1440 were MYL2 (Table 2). This is the first report to describe the relationship between variation in specific molecules and histopathological observations in MATAPHY. Both MYL2 and MYL3 are expressed in skeletal muscles [17]. In patients with MATAPHY, histopathology shows atrophic changes in the muscles [2]. Consistent with this observation, we found that both MYL2 and MYL3 are downregulated in patients with MATAPHY. APOA1, FGB, and GC, which are expressed in the liver, were upregulated in patients with MATAPHY. We speculated that alcoholic liver injury in this MATAPHY patient may have caused the deposition of these proteins. ACTA2 is expressed in the smooth muscles of blood vessels [18]. It is possible that the downregulation of ACTA2 in MATAPHY may be due to a decrease in muscle tissue where blood vessels are abundant and an increase in tendon tissues in which blood vessels are exiguous. COL6, which is a heterotrimer of the alpha1, alpha2, and alpha3 chains and a nonfibrillar collagen expressed in developing and adult tendons [19], is upregulated in patients with MATAPHY. The tendons of COL6A1knockout mice showed a significant reduction in maximum load and stiffness as compared to wild-type tendons, suggesting that COL6A1 is essential for tendon fibrillogenesis [20]. Interestingly, Senga et al. reported that COL6 in mouse masseter tendon was detected in the external lamina of the muscle cells of myotendinous junction and fibrocartilage-like cells of the tendon-bone boundary [21]. This result leads us to conjecture that tendon–aponeurosis hyperplasia is caused by the overexpression of COL6A1. This study has two limitations as follows. First, it is difficult to obtain scientific sound results from each only one subject. As this is a preliminary study, our results do not have statistical meaning. Second, since some investigations suggested the possible correlation between jaw deformity and muscle characteristics, FD is sometimes congenital and may be complicated with deficiency of the skeletal proteins [22,23]. However, we think this study may be a meaningful report because no study has been conducted by 2D-DIGE methods for the comparison MATAPHY with FD. Application of proteomics analysis may help us focus on specific molecular abnormalities in diseases that have an unknown
The authors declare no conflict of interest. This work was supported by grants-in-aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan (20592344 for T.Y.) References [1] Kakudo K, Yoda T. New concept of limited mouth opening associated with square mandible: diagnosis and treatment for masticatory muscle tendon–aponeurosis hyperplasia. J Jpn Soc TMJ 2009;21:28–30 (in Japanese with English abstract). [2] Chiba R. Histopathologic evaluation of aponeurectomy materials in masticatory muscle tendon–aponeurosis hyperplasia. J Jpn Soc TMJ 2009;21:51–4 (in Japanese with English abstract). [3] Yoda T, Sato T, Abe T, Sakamoto I, Tomaru Y, Omura K, et al. Long-term results of surgical therapy for masticatory muscle tendon–aponeurosis hyperplasia accompanied by limited mouth opening. Int J Oral Maxillofac Surg 2009;38:1143–7. [4] Arika T, Kakudo K. Hyperplasia of tendon and aponeurosis of masticatory muscles (HyTAM)—clinical appearance. J Jpn Soc TMJ 2009;21:31–4 (in Japanese with English abstract). [5] Kobayashi K, Shimoda S, Yoda T, Kakudo K. Current status of MR imaging for the masticatory muscle tendon–aponeurosis hyperplasia. J Jpn Soc TMJ 2009;21:35–9 (in Japanese with English abstract). [6] Dowling P, Meleady P, Henry M, Clynes M. Recent advances in clinical proteomics using mass spectrometry. Bioanalysis 2010;2:1609–15. [7] Timms JF, Cramer R. Difference gel electrophoresis. Proteomics 2008;8:4886–97. [8] Shushan B. A review of clinical diagnostic applications of liquid chromatography-tandem mass spectrometry. Mass Spectrom Rev 2010;29:930–44. [9] Sanchez JC, Rouge V, Pisteur M, Ravier F, Tonella L, Moosmayer M, et al. Improved and simplified in-gel sample application using reswelling of dry immobilized pH gradients. Electrophoresis 1997;18:324–7. [10] Rabilloud T, Adessi C, Giraudel A, Lunardi J. Improvement of the solubilization of proteins in two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis 1997;18:307–16. [11] Mann M. A shortcut to interesting human genes: peptide sequence tags, expressed-sequence tags and computers. Trends Biochem Sci 1996;21:494–5. [12] Yates 3rd JR, Eng JK, McCormack AL. Mining genomes: correlating tandem mass spectra of modified and unmodified peptides to sequences in nucleotide databases. Anal Chem 1995;67:3202–10. [13] Perkins DN, Pappin DJ, Creasy DM, Cottrell JS. Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 1999;20:3551–67. [14] Mann M, Wilm M. Error-tolerant identification of peptides in sequence databases by peptide sequence tags. Anal Chem 1994;66:4390–9. [15] Eng JK, McCormack AL, Yates 3rd JR. An approach to correlate MS/MS data to amino acid sequences in a protein database. J Am Soc Mass Spectrom 1994;5:976–89. [16] Pappin DJ, Hojrup P, Bleasby AJ. Rapid identification of proteins by peptidemass fingerprinting. Curr Biol 1993;3:327–32. [17] Sweeney HL, Bowman BF, Stull JT. Myosin light chain phosphorylation in vertebrate striated muscle: regulation and function. Am J Physiol 1993;264:C1085–95. [18] Milewicz DM, Guo DC, Tran-Fadulu V, Lafont AL, Papke CL, Inamoto S, Kwartler CS, et al. Genetic basis of thoracic aortic aneurysms and dissections: focus on smooth muscle cell contractile dysfunction. Annu Rev Genomics Hum Genet 2008;9:283–302. [19] Contri MB, Guerra D, Vignali N, Taparelli F, Marcuzzi A, Caroli A, et al. Ultrastructural and immunocytochemical study on normal human palmar aponeuroses. Anat Rec 1994;240:314–21. [20] Izu Y, Ansorge HL, Zhang G, Soslowsky LJ, Bonaldo P, Chu ML, et al. Dysfunctional tendon collagen fibrillogenesis in collagen VI null mice. Matrix Biol 2011;30:53–61. [21] Senga K, Kobayashi M, Hattori H, Yasue K, Mizutani H, Ueda M, et al. Type VI collagen in mouse masseter tendon, from osseous attachment to myotendinous junction. Anat Rec 1995;243:294–302. [22] Lim D, Beitzel F, Lynch G, Woods MG. Myosin heavy chain isoform composition of human masseter muscle from subjects with different mandibular plane angles. Aust Orthod J 2006;22:105–14. [23] Raoul G, Rowlerson A, Sciote J, Codaccioni E, Stevens L, Maurage CA, et al. Masseter myosin heavy chain composition varies with mandibular asymmetry. J Craniofac Surg 2011;22:1093–8.
Contents lists available at SciVerse ScienceDirect
Journal of Oral and Maxillofacial Surgery, Medicine, and Pathology journal homepage: www.elsevier.com/locate/jomsmp
Oral and Maxillofacial Surgery/Original research
Proteomic analysis of masticatory muscle tendon–aponeurosis hyperplasia: A preliminary study using a 2D-DIGE system Tsuyoshi Sato a,∗ , Aya Nakamoto a , Naoko Hori a , Yuichiro Enoki a , Yousuke Fukushima a , Norimichi Nakamoto a , Yasuaki Sakata a , Hidenori Yamanaka b , Dai Chida c , Takahiro Abe d , Tetsuya Yoda a a
Department of Oral and Maxillofacial Surgery, Saitama Medical University, 38 Moro-hongou, Moroyama-machi, Iruma-gun, Saitama, 350-0495, Japan Chemicals Assessment Center, Chemicals Evaluation and Research Institute, 1600, Shimo-Takano, Sugito-machi, Kitakatsushika-gun, Saitama 345-0043, Japan c Department of Immunology and Pathology, Research Institute, National Center for Global Health and Medicine, 1-21-1, Toyama, Shinjuku-ku, Tokyo, 162-8655, Japan d Department of Oral and Maxillofacial Surgery, Faculty of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-0033, Japan b
a r t i c l e
i n f o
Article history: Received 8 September 2011 Received in revised form 6 February 2012 Accepted 20 February 2012 Available online 29 March 2012 Keywords: Masticatory muscle tendon-aponeurosis hyperplasia Two-dimensional fluorescence difference gel electrophoresis Liquid chromatography coupled with tandem mass spectrometry Collagen alpha-1(VI) chain
a b s t r a c t Masticatory muscle tendon–aponeurosis hyperplasia (MATAPHY) exhibits hyperplasia on tendon and aponeurosis of the masticatory muscles histopathologically. This disease is characterized by a palpable dense band of the anterior border of the masseter muscle on maximum mouth opening, strike root appearance on MR imaging and square mandible configuration. However, the common useful diagnostic markers of this disease are still unknown. Here we performed a preliminary study to identify potential therapeutic targets and diagnostic markers that may indicate disease progression of MATAPHY using a proteomic analysis method, two-dimensional fluorescence difference gel electrophoresis and liquid chromatography coupled with tandem mass spectrometry. We have found that myosin regulatory light chain 2 and myosin regulatory light chain 3 were down-regulated in MATAPHY. This result is consistent with the observation that muscle tissues showed atrophic changes. We have also demonstrated that aortic smooth muscle actin was down-regulated in MATAPHY, suggesting that this down-regulation is due to decreasing in muscle tissues that blood vessels are abundant and increasing in tendon tissues that blood vessels are exiguous. Furthermore, collagen alpha-1(VI) chain (COL6A1) was up-regulated in MATAPHY. COL6A1, which is essential for tendon fibrillogenesis, expressed in the masticatory muscle, indicating that tendon-aponeurosis hyperplasia is due to the overexpression of COL6A1. Facial deformity, which we choose as a control in this study, is sometimes congenital and may be complicated with deficiency of the skeletal proteins. Although this report has some limitations, this is the first report to describe the relationship between the variation of specific molecules and histopathological observation in MATAPHY. © 2012 Asian Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
1. Introduction Masticatory muscle tendon–aponeurosis hyperplasia (MATAPHY) is a new disease associated with limited mouth opening [1]. From the viewpoint of histopathology, both tendon and aponeurosis in MATAPHY exhibit hyperplasia [2]. This disease is classified into the masseter muscle type, which is accompanied by more severe hyperplasia of the masseter muscle aponeurosis with a square mandible due to hypertrophy of the mandibular angle as compared to the temporalis muscle tendon and the temporal muscle type, which does not show marked hyperplasia of the masseter
∗ Corresponding author. E-mail address: [email protected] (T. Sato).
muscle aponeurosis [3]. Previously, we demonstrated that good long-term results have been reliably obtained by resection of the hyperplastic masseter muscle aponeurosis with coronoidectomy for separation of the temporal muscle from the coronoid process in patients with MATAPHY [3]. The characteristics of this disease, which are a palpable dense band of the anterior border of the masseter muscle on maximum mouth opening, strike root appearance on magnetic resonance imaging, and square mandible configuration, are useful in its diagnosis [4,5]. However, common useful diagnostic markers of this disease are still unknown. Recent advances in proteomic analysis have allowed us to better understand the molecular basis of human diseases [6]. Two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) has been developed to quantify the differences between experimental pairs of samples resolved on the
2212-5558/$ – see front matter © 2012 Asian Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ajoms.2012.02.003
186
T. Sato et al. / Journal of Oral and Maxillofacial Surgery, Medicine, and Pathology 24 (2012) 185–188
Table 1 Basic characteristics of patients.
FD MATAPHY
Sex
Age
Underlying disease
Female Female
22 56
Not particular Alcoholic liver injury
same 2D gel [7]. Liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) provides significant benefits to clinical diagnostic laboratories that conduct routine analyses [8]. We conducted a preliminary study to identify potential therapeutic targets, as well as diagnostic markers, that may indicate disease progression of MATAPHY by using a 2D-DIGE system. 2. Materials and methods 2.1. Patients and subjects Tissue specimens of temporal muscles and tendons, including 1 subject with MATAPHY and 1 with facial deformity (FD), were obtained from patients undergoing surgery (Table 1). As this MATAPHY patient has no sign of the masseter muscle type such as a palpable dense band of the anterior border of the masseter muscle on maximum mouth opening and strike root appearance on magnetic resonance imaging, this patient was diagnosed as the temporal muscle type. The patients were treated and followed up at Saitama Medical University. The study was performed in accordance with the Declaration of Helsinki and approved by the Institutional Review Board (approval number 595) at Saitama Medical University. The dentist in charge provided all patients or their guardians with an explanation of the study. Patients were free to withdraw from the study of their own free will at any time. Informed consent was obtained from all subjects. 2.2. Sample homogenization and protein labeling All samples were homogenized in 10 volumes of lysis buffer (LB) (4% (w/v) CHAPS, 2 M thiourea, 8 M urea, 10 mM Tris–HCl pH 8.8) and homogenized with a Polytron-type homogenizer. The sample solution was sonicated for a series of 20-s bursts on ice, and then centrifuged at 14,000 rpm for 20 min at 10 ◦ C. Protein concentration was determined using the Bradford method. Lysates were labeled with NHS ester-derivatives of the cyanine dyes Cy2, Cy3, and Cy5 (GE Healthcare, Little Chalfont, UK) following the manufacturer’s protocol. Typically, 100 g of lysate was “minimally” labeled with 200 pmol of Cy dye. Labeling reactions were performed on ice in the dark for 30 min, and then quenched with a 50-fold molar excess of free lysine to dye for 10 min on ice. Differentially labeled samples were reduced with 65 mM DTT and mixed with Pharmalytes pH 3–10 (GE Healthcare) for 10 min. Each control (specimens from FD) and test (specimens from MATAPHY) sample was labeled with Cy3 and Cy5. A pool of all samples was also prepared and labeled with Cy2 for use as a standard on all gels to aid image matching. 2.3. Gel electrophoresis and imaging Immobilized pH gradient (IPG) strips pH 3–10 L, 24 cm (GE Healthcare), were rehydrated, and mixed samples were applied with cup loading. Isoelectric focusing was performed using a Multiphor II (GE Healthcare) at 54 kVh at 20 ◦ C in the dark [9,10]. Strips were equilibrated for 10 min in 50 mM Tris–HCl pH 8.8, 6 M urea, 30% (v/v) glycerol, and 1% (w/v) SDS containing 65 mM DTT and then for a further 10 min in the same buffer containing 240 mM iodoacetamide. Equilibrated IPG strips were transferred onto 24 cm × 20 cm, 12% uniform polyacrylamide gels. Strips were
overlaid with 0.5% (w/v) low melting point agarose in running buffer containing bromophenol blue. Gels were run in Ettan DALT twelve (GE Healthcare) at 2 W per gel at 15 ◦ C, until the dye front had run off the bottom of the gels. The 2D gels were then scanned directly between glass plates using a Typhoon 9400 (GE Healthcare). The images generated were exported as tiff files for further protein profile analysis by DeCyderTM 7.0 (GE Healthcare). 2.4. Image analysis and statistical analysis Differential in-gel analysis (DIA) by DeCyderTM 7.0 was used to merge the Cy2, Cy3, and Cy5 images for each gel and to detect spot boundaries to calculate normalized spot volumes/protein abundance. At this stage, features resulting from non-protein sources (e.g., dust particles, and streaks) and faint spots (e.g., spot area <300, spot volume <10,000) were filtered out. The analysis was used to calculate abundant differences between samples run on the same gel. The biological variation analysis (BVA) of DeCyderTM 7.0 was then used to match all pair-wise image comparisons from DIA for comparative cross-gel statistical analysis. Comparison of normalized Cy3 and Cy5 spot volumes with the corresponding Cy2 standard spot volumes within each gel gave a standardized abundance. This value was compared across all gels for each matched spot, and average ratios were calculated from the standardized abundance of 3 gel images of each sample by the DeCyder software. Statistical analysis was performed using the triplicate values from each experimental condition to give Student’s P-value and a 2-tailed probability of less than 5% (P < 0.05) was considered statistically significant. 2.5. In-gel digestion and peptide extraction Gel electrophoresis for MS analysis was performed using 600 g of pooled lysate, following the same procedures as described above. After gel electrophoresis, the gel was stained with Sypro® Ruby, and each MS analysis gel was matched with the master gel for expression analysis by BVA software. Spots of interest were excised from 2D gels using an automated spot picker (GE Healthcare) following the manufacturer’s instructions. Spots were collected in 200 L of water in 96-well plates. The recovered gel pieces were washed with aqueous 50 mM ammonium bicarbonate and acetonitrile and then incubated with 12.5 ng/L trypsin (Promega, Southampton, UK) at 30 ◦ C for 15 h. The peptides generated were eluted with 50 mM ammonium bicarbonate followed by 10% (v/v) formic acid and acetonitrile. The combined fractions were dried in a SpeedVac (Thermo Fisher Scientific, Waltham, USA) and dissolved in 0.1% (v/v) formic acid. 2.6. Mass spectrometric analysis Mass spectrometric analysis was carried out using LC–MS/MS [11,12]. HPLC (CapLC, Waters, Milford, MA) was coupled with the Q-TOF micro mass spectrometer (Micromass, Manchester, UK). Instrument operation and data acquisition and analysis were performed using MassLynx3.2 software (Micromass). The tryptic peptides were concentrated and desalted on a 300-m i.d./5mm length C18 PepMap column (LC Packings, San Francisco, CA). The eluted peptide was analyzed by tandem mass spectrometric sequencing with an automated MS-to-MS/MS switching protocol. Online determination of precursor-ion masses was performed over the m/z range from 400 to 1600 atomic mass units in the positivecharge detection mode with a cone voltage of 50 V. The cone voltage, extraction voltage, microchannel plate (MCP) detector voltage, and collision energy were optimized before measurement of samples. A database search was performed with MASCOT Deamon (Matrix Science, London, UK) [13–15]. The generated pkl
T. Sato et al. / Journal of Oral and Maxillofacial Surgery, Medicine, and Pathology 24 (2012) 185–188
187
Fig. 1. Master number (no.) and its gel images of test and control, P-value, and Av. ratio (−X represents 1/X). (A) Master no. 207, (B) master no. 977, (C) master no. 1138, (D) master no. 1147, (E) master no. 1158, (F) master no. 1389, (G) master no. 1402, (H) master no. 1437, (I) master no. 1440, and (J) master no. 1442.
files were submitted to SWISS-PROT (release 57.6) and NCBInr (20090430). Search parameters were as follows: fixed modifications, carbamidomethyl; variable modifications, oxidation (M); missed cleavages, up to 1 (monoisotopic); peptide tolerance, 1.0 Da; and MS/MS tolerance, 0.5 Da. To automatically exclude all the very low scoring, random peptide matches, the ion score cutoff was set at 20. This means that homologous proteins are more likely to collapse into a single hit, avoiding the need to choose between them. Matches using mass values are always handled on a probabilistic basis. The total score is the absolute probability that the observed match is a random event. The MOWSE scores were the reported scores of −10 log10 (P), where P is the absolute probability. A probability of 10–20 thus becomes a score of 200 [16]. The automatically
identified proteins were checked individually to eliminate redundancy. 3. Results and discussion We performed 2D gel electrophoresis and 10 delineated protein spots showing remarkable changes including significant changes (P < 0.05) between MATAPHY and FD were observed (Fig. 1). These differentially expressed protein spots are shown: 5 spots were upregulated and 5 spots were downregulated in MATAPHY (Fig. 1A–J). A total of 10 differentially expressed spots were excised from gels and analyzed for protein identification by LC/MS/MS. As shown in Table 2, we identified 4 upregu-
188
T. Sato et al. / Journal of Oral and Maxillofacial Surgery, Medicine, and Pathology 24 (2012) 185–188
Table 2 Identification of differential proteins by searching in protein database. Master no. Up-regulated protein 1389 1158 1147 207 977 Down-regulated protein 1138 1402 1437 1442 1440
molecular pathogenesis. Further analysis will be needed to identify potential therapeutic targets and diagnostic markers of MATAPHY.
Protein name
Acknowledgments Apolipoprotein A-I Fibrinogen beta chain Fibrinogen beta chain Collagen alpha-1(VI) chain Vitamin D-binding protein Actin, aortic smooth muscle Myosin light chain 3 Myosin regulatory light chain 2, ventricular/cardiac muscle isoform Myosin regulatory light chain 2, ventricular/cardiac muscle isoform Myosin regulatory light chain 2, ventricular/cardiac muscle isoform
lated proteins—apolipoprotein AI (APOA1), fibrinogen beta chain (FGB), collagen alpha-1(VI) chain (COL6A1), and vitamin D-binding protein (GC)—and 3 downregulated proteins—aortic smooth muscle actin (ACTA2), myosin regulatory light chain 3 (MYL3), and myosin regulatory light chain 2 (ventricular/cardiac muscle isoform) (MYL2). Some spots appeared to correspond to the same protein, i.e., spots 1158 and 1147 were FGB and spots 1437, 1442, and 1440 were MYL2 (Table 2). This is the first report to describe the relationship between variation in specific molecules and histopathological observations in MATAPHY. Both MYL2 and MYL3 are expressed in skeletal muscles [17]. In patients with MATAPHY, histopathology shows atrophic changes in the muscles [2]. Consistent with this observation, we found that both MYL2 and MYL3 are downregulated in patients with MATAPHY. APOA1, FGB, and GC, which are expressed in the liver, were upregulated in patients with MATAPHY. We speculated that alcoholic liver injury in this MATAPHY patient may have caused the deposition of these proteins. ACTA2 is expressed in the smooth muscles of blood vessels [18]. It is possible that the downregulation of ACTA2 in MATAPHY may be due to a decrease in muscle tissue where blood vessels are abundant and an increase in tendon tissues in which blood vessels are exiguous. COL6, which is a heterotrimer of the alpha1, alpha2, and alpha3 chains and a nonfibrillar collagen expressed in developing and adult tendons [19], is upregulated in patients with MATAPHY. The tendons of COL6A1knockout mice showed a significant reduction in maximum load and stiffness as compared to wild-type tendons, suggesting that COL6A1 is essential for tendon fibrillogenesis [20]. Interestingly, Senga et al. reported that COL6 in mouse masseter tendon was detected in the external lamina of the muscle cells of myotendinous junction and fibrocartilage-like cells of the tendon-bone boundary [21]. This result leads us to conjecture that tendon–aponeurosis hyperplasia is caused by the overexpression of COL6A1. This study has two limitations as follows. First, it is difficult to obtain scientific sound results from each only one subject. As this is a preliminary study, our results do not have statistical meaning. Second, since some investigations suggested the possible correlation between jaw deformity and muscle characteristics, FD is sometimes congenital and may be complicated with deficiency of the skeletal proteins [22,23]. However, we think this study may be a meaningful report because no study has been conducted by 2D-DIGE methods for the comparison MATAPHY with FD. Application of proteomics analysis may help us focus on specific molecular abnormalities in diseases that have an unknown
The authors declare no conflict of interest. This work was supported by grants-in-aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan (20592344 for T.Y.) References [1] Kakudo K, Yoda T. New concept of limited mouth opening associated with square mandible: diagnosis and treatment for masticatory muscle tendon–aponeurosis hyperplasia. J Jpn Soc TMJ 2009;21:28–30 (in Japanese with English abstract). [2] Chiba R. Histopathologic evaluation of aponeurectomy materials in masticatory muscle tendon–aponeurosis hyperplasia. J Jpn Soc TMJ 2009;21:51–4 (in Japanese with English abstract). [3] Yoda T, Sato T, Abe T, Sakamoto I, Tomaru Y, Omura K, et al. Long-term results of surgical therapy for masticatory muscle tendon–aponeurosis hyperplasia accompanied by limited mouth opening. Int J Oral Maxillofac Surg 2009;38:1143–7. [4] Arika T, Kakudo K. Hyperplasia of tendon and aponeurosis of masticatory muscles (HyTAM)—clinical appearance. J Jpn Soc TMJ 2009;21:31–4 (in Japanese with English abstract). [5] Kobayashi K, Shimoda S, Yoda T, Kakudo K. Current status of MR imaging for the masticatory muscle tendon–aponeurosis hyperplasia. J Jpn Soc TMJ 2009;21:35–9 (in Japanese with English abstract). [6] Dowling P, Meleady P, Henry M, Clynes M. Recent advances in clinical proteomics using mass spectrometry. Bioanalysis 2010;2:1609–15. [7] Timms JF, Cramer R. Difference gel electrophoresis. Proteomics 2008;8:4886–97. [8] Shushan B. A review of clinical diagnostic applications of liquid chromatography-tandem mass spectrometry. Mass Spectrom Rev 2010;29:930–44. [9] Sanchez JC, Rouge V, Pisteur M, Ravier F, Tonella L, Moosmayer M, et al. Improved and simplified in-gel sample application using reswelling of dry immobilized pH gradients. Electrophoresis 1997;18:324–7. [10] Rabilloud T, Adessi C, Giraudel A, Lunardi J. Improvement of the solubilization of proteins in two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis 1997;18:307–16. [11] Mann M. A shortcut to interesting human genes: peptide sequence tags, expressed-sequence tags and computers. Trends Biochem Sci 1996;21:494–5. [12] Yates 3rd JR, Eng JK, McCormack AL. Mining genomes: correlating tandem mass spectra of modified and unmodified peptides to sequences in nucleotide databases. Anal Chem 1995;67:3202–10. [13] Perkins DN, Pappin DJ, Creasy DM, Cottrell JS. Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 1999;20:3551–67. [14] Mann M, Wilm M. Error-tolerant identification of peptides in sequence databases by peptide sequence tags. Anal Chem 1994;66:4390–9. [15] Eng JK, McCormack AL, Yates 3rd JR. An approach to correlate MS/MS data to amino acid sequences in a protein database. J Am Soc Mass Spectrom 1994;5:976–89. [16] Pappin DJ, Hojrup P, Bleasby AJ. Rapid identification of proteins by peptidemass fingerprinting. Curr Biol 1993;3:327–32. [17] Sweeney HL, Bowman BF, Stull JT. Myosin light chain phosphorylation in vertebrate striated muscle: regulation and function. Am J Physiol 1993;264:C1085–95. [18] Milewicz DM, Guo DC, Tran-Fadulu V, Lafont AL, Papke CL, Inamoto S, Kwartler CS, et al. Genetic basis of thoracic aortic aneurysms and dissections: focus on smooth muscle cell contractile dysfunction. Annu Rev Genomics Hum Genet 2008;9:283–302. [19] Contri MB, Guerra D, Vignali N, Taparelli F, Marcuzzi A, Caroli A, et al. Ultrastructural and immunocytochemical study on normal human palmar aponeuroses. Anat Rec 1994;240:314–21. [20] Izu Y, Ansorge HL, Zhang G, Soslowsky LJ, Bonaldo P, Chu ML, et al. Dysfunctional tendon collagen fibrillogenesis in collagen VI null mice. Matrix Biol 2011;30:53–61. [21] Senga K, Kobayashi M, Hattori H, Yasue K, Mizutani H, Ueda M, et al. Type VI collagen in mouse masseter tendon, from osseous attachment to myotendinous junction. Anat Rec 1995;243:294–302. [22] Lim D, Beitzel F, Lynch G, Woods MG. Myosin heavy chain isoform composition of human masseter muscle from subjects with different mandibular plane angles. Aust Orthod J 2006;22:105–14. [23] Raoul G, Rowlerson A, Sciote J, Codaccioni E, Stevens L, Maurage CA, et al. Masseter myosin heavy chain composition varies with mandibular asymmetry. J Craniofac Surg 2011;22:1093–8.