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Comparison of the protein profiles between the achilles and patella tendon in rats
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Materials Science and Engineering C 28 (2008) 1319 – 1321 www.elsevier.com/locate/msec
Comparison of the protein profiles between the achilles and patella tendon in rats Yoshimasa Ishii a,⁎, Masataka Deie b , Nobuo Adachi b , Osamu Hazeki c , Naoki Okamura c , Patrick Sharman b , Yuji Yasunaga a , Mitsuo Ochi b a
Department of Artificial Joints and Biomaterials, Hiroshima University, Japan b Department of Orthopaedic Surgery, Hiroshima University, Japan c Institute of Pharmaceutical Sciences, Hiroshima University, Japan Received 30 January 2008; accepted 3 February 2008 Available online 15 February 2008
Abstract We have examined and compared the protein profile of the achilles tendon with that of the patella tendon in 5 male Wistar rats using twodimensional polyacrylamide gel electrophoresis (2D-PAGE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). In the 2D-PAGE, we can plot the proteins of the achilles and the patella tendon, but not reveal the difference between the protein profiles of tendons in rat. However, in MALDI-TOF-MS, the difference in protein profiles between the patella and the achilles tendon can be detected. Although further analysis is required, our method to view protein profile has potential for the diagnosis of the nature and the pathological conditions of tendon. © 2008 Elsevier B.V. All rights reserved. Keywords: Protein profiles; Achilles tendon; Patella tendon
1. Introduction Many approaches have been used to characterize the biochemical and biological characteristics of tendon and ligament [1–3]. Studying proteomic approaches may identify tendon proteins, which would be important for elucidating the pathological conditions of disease and the healing mechanism after injury. Knowledge of different tendons' protein profiles would be advantageous for tissue selection in tendon graft operations. In the present study, we have examined and compared the protein profile of the achilles tendon with that of the patella tendon in rats using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and matrix-assisted laser desorp-
⁎ Corresponding author. Department of Artificial Joints and Biomaterials, Hiroshima University, 1-2-3, Kasumi, Minamiku, Hiroshima, Japan, 734-0022. Tel.: +81 82 2575232; fax: +81 82 2575234. E-mail address: [email protected] (Y. Ishii). 0928-4931/$ - see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2008.02.002
tion/ionization time-of-flight mass spectrometry (MALDI-TOFMS). 2. Materials and method Five 10-week- old wistar rats aged ten weeks were used in this study. Ligamentous parts of the right patella tendon and the achilles tendon were extracted. After homogenisation, tissues were mixed with a solution consisting of 5 M urea, 2 M thiourea, 5% CHAPS, Tris 40 mM, 50 mM dithiotheretol, 0.2% Bio-Lyte and a trace of bromophenol blue. Samples (125 microliter) containing 1.7 milligram of protein were loaded on 7 cm immobilized pH gradient strips (pH 3–10 Readystrip, Bio-rad). Proteins were separated by isoelectric focusing at 4000 V for 5 h, and were separated by SDS-PAGE at a constant voltage of 100 V. Separated proteins were visualized with Coomassie brilliant blue. Gels were scanned and the images were stored in a connected PC computor. The gels of the achilles tendon and the patella tendon were compared in each animal.
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Y. Ishii et al. / Materials Science and Engineering C 28 (2008) 1319–1321
Fig. 1. 2D-PAGE of the achilles tendon and the patella tendon are shown. (A) achilles tendon (B) patella tendon.
For further protein analysis, peptide mapping was undertaken using MALDI-TOF-MS. The peptide masses were determined using a Ultra Tof-Tof mass spectrometer (Bruker Daltonics, Billerica, MA, USA) and the spectrogram of the achilles tendon and the patella tendon were compared. 3. Results All tendons extracted were normal and showed no degenarative condition by gross appearance. The 2D-PAGE of
achilles tendon and the patella tendon are shown in Fig. 1. These two dimensional protein profiles showed the same pattern for both tendons. A double strong band at 120 (kDa) can be seen throughout pH 3-10. The other proteins were expressed by spots. The isoelectric point (pl) for approximately 90% of total proteins were between pH 5.0-7.0, and the relative molecular mass of approximately 90% of the total proteins were between 30-70 (kDa). Using MALDI-TOF-MS, we can show the protein profiles (3,000-17,000 m/z) (Fig. 2). From this, we cannot see the
Fig. 2. MALDI-TOF-MS of the achilles tendon (A) and the patella tendon (B) (3,000-17,000 m/z).
Y. Ishii et al. / Materials Science and Engineering C 28 (2008) 1319–1321
1321
Fig. 3. MALDI-TOF-MS of the achilles tendon (A) and the patella tendon (B) (13,000-17,000 m/z).
variation between the patella tendon and the achilles tendon. However, in the range of 13,000-17,000 m/z, we can see the three different peaks between the achilles tendon and the patella tendon (Fig. 3). Peak 1 is expressed by the Achilles tendon only; peaks 2 and 3 appear to be expressed by the patella tendon also, but whereas these peaks were measured at 550 m/Z in the patella tendon, they were only measured at 75 m/z in the achilles tendon. However, we cannot identify the protein by linear mode. 4. Discussion Tendons and ligaments consist of mostly collagen and elastin embedded in a proteoglycan-water matrix, with collagen accounting for 65% to 75% and elastin approximately 2% [1,2]. However, there are few reports to detect the constitution and the difference in protein between the different tendons or ligaments. Yahia LH et al. reported the difference between the collagen structure of the canine anterior cruciate ligament (ACL) and patellar tendon (PT) by using light and scanning electron microscopy [3]. The collagen waviness known as a crimping was found to occur in both ACL and PT fascicles. However, while the helical wave pattern occurs in both PT and ACL, the planar waveform is found only in the centrally located ACL fascicles. In addition, there is less variability in fascicular size and density of the PT cross-section than of the ACL. Sung KL et. al studied the differences between the adult anterior cruciate ligament (ACL) and the medial collateral ligament (MCL) by characterizing the roles of several cytoskeletal proteins in liga-
ment fibroblast adhesion [4]. They found that both ACL and MCL fibroblast adhesion depends on cytoskeletal assembly; however, this dependence differs between ACL and MCL fibroblasts especially concerning the role of tropomodulin. Evans CE et al. examined the in vitro phenotype of cells cultured from both flexor and extensor tendons [5]. The cells obtained from the two different types of tendon behaved similarly when exposed to identical environmental conditions in vitro. From these findings, different tendons may consist of different protein compositions. However, the protein profile of different tendons has not been studied in details before. This is the first report of a two dimensional protein map and a MALDI-TOF-MS of tendon. In the 2D-PAGE, we cannot reveal the difference between the protein profiles of the achilles and the patella tendon in rat. However, in MALDI-TOF-MS, we can suggest the differences of protein profiles between the patella and the achilles tendon. Nevertheless further studies are required. Our method to detect protein profile has potential to diagnose the nature and the pathological conditions of tendon. References [1] C.J. Hooley, N.G. McCrum, R.E. Cohen, J. Biomech. 13 (6) (1980) 521. [2] G.P. Hess, W.L. Cappiello, R.M. Poole, S.C. Hunter, Sports Med. 8 (6) (Dec 1989) 371. [3] L.H. Yahia, G. Drouin, J. Orthop. Res. 7 (2) (1989) 243. [4] K.L. Sung, L. Yang, D.E. Whittemore, Y. Shi, G. Jin, A.H. Hsieh, W.H. Akeson, L.A. Sung, Proc. Natl. Acad. Sci. U. S. A. 20 (93) (1996) 9182. [5] C.E. Evans, I.A. Trail, J. Hand Surg. [Br] 26 (4) (2001) 307.
Materials Science and Engineering C 28 (2008) 1319 – 1321 www.elsevier.com/locate/msec
Comparison of the protein profiles between the achilles and patella tendon in rats Yoshimasa Ishii a,⁎, Masataka Deie b , Nobuo Adachi b , Osamu Hazeki c , Naoki Okamura c , Patrick Sharman b , Yuji Yasunaga a , Mitsuo Ochi b a
Department of Artificial Joints and Biomaterials, Hiroshima University, Japan b Department of Orthopaedic Surgery, Hiroshima University, Japan c Institute of Pharmaceutical Sciences, Hiroshima University, Japan Received 30 January 2008; accepted 3 February 2008 Available online 15 February 2008
Abstract We have examined and compared the protein profile of the achilles tendon with that of the patella tendon in 5 male Wistar rats using twodimensional polyacrylamide gel electrophoresis (2D-PAGE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). In the 2D-PAGE, we can plot the proteins of the achilles and the patella tendon, but not reveal the difference between the protein profiles of tendons in rat. However, in MALDI-TOF-MS, the difference in protein profiles between the patella and the achilles tendon can be detected. Although further analysis is required, our method to view protein profile has potential for the diagnosis of the nature and the pathological conditions of tendon. © 2008 Elsevier B.V. All rights reserved. Keywords: Protein profiles; Achilles tendon; Patella tendon
1. Introduction Many approaches have been used to characterize the biochemical and biological characteristics of tendon and ligament [1–3]. Studying proteomic approaches may identify tendon proteins, which would be important for elucidating the pathological conditions of disease and the healing mechanism after injury. Knowledge of different tendons' protein profiles would be advantageous for tissue selection in tendon graft operations. In the present study, we have examined and compared the protein profile of the achilles tendon with that of the patella tendon in rats using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and matrix-assisted laser desorp-
⁎ Corresponding author. Department of Artificial Joints and Biomaterials, Hiroshima University, 1-2-3, Kasumi, Minamiku, Hiroshima, Japan, 734-0022. Tel.: +81 82 2575232; fax: +81 82 2575234. E-mail address: [email protected] (Y. Ishii). 0928-4931/$ - see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2008.02.002
tion/ionization time-of-flight mass spectrometry (MALDI-TOFMS). 2. Materials and method Five 10-week- old wistar rats aged ten weeks were used in this study. Ligamentous parts of the right patella tendon and the achilles tendon were extracted. After homogenisation, tissues were mixed with a solution consisting of 5 M urea, 2 M thiourea, 5% CHAPS, Tris 40 mM, 50 mM dithiotheretol, 0.2% Bio-Lyte and a trace of bromophenol blue. Samples (125 microliter) containing 1.7 milligram of protein were loaded on 7 cm immobilized pH gradient strips (pH 3–10 Readystrip, Bio-rad). Proteins were separated by isoelectric focusing at 4000 V for 5 h, and were separated by SDS-PAGE at a constant voltage of 100 V. Separated proteins were visualized with Coomassie brilliant blue. Gels were scanned and the images were stored in a connected PC computor. The gels of the achilles tendon and the patella tendon were compared in each animal.
1320
Y. Ishii et al. / Materials Science and Engineering C 28 (2008) 1319–1321
Fig. 1. 2D-PAGE of the achilles tendon and the patella tendon are shown. (A) achilles tendon (B) patella tendon.
For further protein analysis, peptide mapping was undertaken using MALDI-TOF-MS. The peptide masses were determined using a Ultra Tof-Tof mass spectrometer (Bruker Daltonics, Billerica, MA, USA) and the spectrogram of the achilles tendon and the patella tendon were compared. 3. Results All tendons extracted were normal and showed no degenarative condition by gross appearance. The 2D-PAGE of
achilles tendon and the patella tendon are shown in Fig. 1. These two dimensional protein profiles showed the same pattern for both tendons. A double strong band at 120 (kDa) can be seen throughout pH 3-10. The other proteins were expressed by spots. The isoelectric point (pl) for approximately 90% of total proteins were between pH 5.0-7.0, and the relative molecular mass of approximately 90% of the total proteins were between 30-70 (kDa). Using MALDI-TOF-MS, we can show the protein profiles (3,000-17,000 m/z) (Fig. 2). From this, we cannot see the
Fig. 2. MALDI-TOF-MS of the achilles tendon (A) and the patella tendon (B) (3,000-17,000 m/z).
Y. Ishii et al. / Materials Science and Engineering C 28 (2008) 1319–1321
1321
Fig. 3. MALDI-TOF-MS of the achilles tendon (A) and the patella tendon (B) (13,000-17,000 m/z).
variation between the patella tendon and the achilles tendon. However, in the range of 13,000-17,000 m/z, we can see the three different peaks between the achilles tendon and the patella tendon (Fig. 3). Peak 1 is expressed by the Achilles tendon only; peaks 2 and 3 appear to be expressed by the patella tendon also, but whereas these peaks were measured at 550 m/Z in the patella tendon, they were only measured at 75 m/z in the achilles tendon. However, we cannot identify the protein by linear mode. 4. Discussion Tendons and ligaments consist of mostly collagen and elastin embedded in a proteoglycan-water matrix, with collagen accounting for 65% to 75% and elastin approximately 2% [1,2]. However, there are few reports to detect the constitution and the difference in protein between the different tendons or ligaments. Yahia LH et al. reported the difference between the collagen structure of the canine anterior cruciate ligament (ACL) and patellar tendon (PT) by using light and scanning electron microscopy [3]. The collagen waviness known as a crimping was found to occur in both ACL and PT fascicles. However, while the helical wave pattern occurs in both PT and ACL, the planar waveform is found only in the centrally located ACL fascicles. In addition, there is less variability in fascicular size and density of the PT cross-section than of the ACL. Sung KL et. al studied the differences between the adult anterior cruciate ligament (ACL) and the medial collateral ligament (MCL) by characterizing the roles of several cytoskeletal proteins in liga-
ment fibroblast adhesion [4]. They found that both ACL and MCL fibroblast adhesion depends on cytoskeletal assembly; however, this dependence differs between ACL and MCL fibroblasts especially concerning the role of tropomodulin. Evans CE et al. examined the in vitro phenotype of cells cultured from both flexor and extensor tendons [5]. The cells obtained from the two different types of tendon behaved similarly when exposed to identical environmental conditions in vitro. From these findings, different tendons may consist of different protein compositions. However, the protein profile of different tendons has not been studied in details before. This is the first report of a two dimensional protein map and a MALDI-TOF-MS of tendon. In the 2D-PAGE, we cannot reveal the difference between the protein profiles of the achilles and the patella tendon in rat. However, in MALDI-TOF-MS, we can suggest the differences of protein profiles between the patella and the achilles tendon. Nevertheless further studies are required. Our method to detect protein profile has potential to diagnose the nature and the pathological conditions of tendon. References [1] C.J. Hooley, N.G. McCrum, R.E. Cohen, J. Biomech. 13 (6) (1980) 521. [2] G.P. Hess, W.L. Cappiello, R.M. Poole, S.C. Hunter, Sports Med. 8 (6) (Dec 1989) 371. [3] L.H. Yahia, G. Drouin, J. Orthop. Res. 7 (2) (1989) 243. [4] K.L. Sung, L. Yang, D.E. Whittemore, Y. Shi, G. Jin, A.H. Hsieh, W.H. Akeson, L.A. Sung, Proc. Natl. Acad. Sci. U. S. A. 20 (93) (1996) 9182. [5] C.E. Evans, I.A. Trail, J. Hand Surg. [Br] 26 (4) (2001) 307.