Sains Malaysiana 50(11)(2021): 3297-3302

http://doi.org/10.17576/jsm-2021-5011-13

 

 

Trichloroacetic Acid/Acetone Precipitation Method to Optimize Canine Synovial Fluid for One and Two-Dimensional Gel Electrophoresis Studies

(Kaedah Pemendakan Asid Trikloroasetik/Aseton untuk Mengoptimumkan Cecair Sinovial Kanin dalam Kajian Elektroforesis Gel Satu dan Dua Dimensi)

 

WEI MIAO TAN1, SENG FONG LAU1*, SHARIFAH ZAKIAH SYED SULAIMAN1, NOR SHAHIRA SOLEHAH UMRAN1, MOKRISH AJAT1, ROZAIHAN MANSOR1, PUTERI AZAZIAH MEGAT ABD-RANI1, INTAN NURFATIHA SHAFIE1, ANGELA MIN HWEI NG2 & NORASFALIZA BINTI RAHMAD3

 

1Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43300 UPM Serdang, Selangor Darul Ehsan, Malaysia

 

2Tissue Engineering Centre, Universiti Kebangsaan Malaysia Medical Centre, 56000 Cheras, Kuala Lumpur, Federal Territory, Malaysia

 

3Agro-Biotechnology Institute Malaysia, National Institute of Biotechnology Malaysia, 43300 Serdang, Selangor Darul Ehsan, Malaysia

 

Diserahkan: 7 Disember 2020/Diterima: 28 Februari 2021

 

ABSTRACT

The challenge associated with the use of synovial fluid for osteoarthritic proteome studies is the optimization step, which involves the depletion of high abundant proteins from the samples. The objective of this study was to develop a cost efficient and effective method to remove albumin from canine synovial fluid for proteome studies. Pooled synovial fluid samples were obtained from clinically healthy dogs (n=5), with no radiographic features of osteoarthritis. The acetone precipitation method and 10% w/v of trichloroacetic acid/acetone were chosen to deplete the albumin from canine synovial fluid and the outcome from the different methods were compared using one dimensional and two-dimensional gel electrophoresis studies. The results showed that the 10% w/v TCA/acetone precipitation method removed highly abundant proteins from synovial fluid for gel electrophoresis studies compared to the acetone precipitation method. The 10% w/v TCA/acetone precipitation method provides an effective method to remove albumin from the synovial fluid using gel electrophoresis, especially two-dimensional gel electrophoresis. The accessibility and cost of TCA and acetone make this method of precipitation a simple and cost-effective technique in preparing a canine synovial fluid for two-dimensional gel electrophoresis analysis.

 

Keywords: Albumin; biomarkers; gel electrophoresis; osteoarthritis; synovial fluid

 

ABSTRAK

Satu cabaran dalam kajian proteomik berkaitan osteoartritis dengan menggunakan cecair sinovium adalah langkah pengoptimuman yang melibatkan pengurangan protein berkelimpahan tinggi daripada sampel. Objektif penyelidikan ini adalah untuk mengkaji kaedah yang lebih berkesan dan jimat bagi mengeluarkan albumin daripada cecair sinovium kanin untuk kajian proteomik. Sampel cecair sinovium yang dikumpulkan telah diperoleh daripada anjing yang dikenal pasti sihat secara klinikal (n=5), serta tidak mempunyai sebarang ciri-ciri osteoartritis berdasarkan keputusan radiografi. Kaedah mendakan aseton dan 10% w/v asid trikloroasetik/aseton telah dipilih untuk mengeluarkan albumin daripada sampel cecair sinovium kanin dan hasil daripada kaedah berbeza ini telah dibandingkan dengan menggunakan kajian elektroforesis gel satu dimensi dan dua dimensi. Hasil kajian menunjukkan kaedah 10% w/v TCA/aseton berjaya mengeluarkan protein berkelimpahan tinggi daripada elektroforesis gel berbanding kaedah mendakan aseton. Kaedah 10% w/v TCA/aseton telah berjaya menghasilkan satu kaedah yang berkesan untuk mengeluarkan albumin daripada cecair sinovium, terutamanya untuk kajian elektroforesis gel dua dimensi. Kebolehcapaian dan kos TCA dan aseton membuatkan kaedah mendakan ini lebih ringkas dan jimat dalam penyediaan cecair sinovium kanin untuk analisis elektroforesis gel dua dimensi.

 

Kata kunci: Albumin; cecair sinovium; elektroforesis gel; osteoartritis; penanda biologi

 

RUJUKAN

Bennike, T., Ayturk, U., Haslauer, C.M., Froehlich, J.W., Proffen, B.L., Barnaby, O., Birkelund, S., Murray, M.M., Warman, M.L., Stensballe, A. & Steen, H. 2014. A normative study of the synovial fluid proteome from healthy porcine knee joints. Journal of Proteome Research 13(10): 4377-4387.

Chen, C.P., Hsu, C.C., Yeh, W.L., Lin, H.C., Hsieh, S.Y., Lin, S.C., Chen, T.T., Chen, M.J. & Tang, S.F. 2011. Optimizing human synovial fluid preparation for two-dimensional gel electrophoresis. Proteome Science 9(1): 1-10.

Chen, R.L., Sage, E.A., Dunn, M.J., Wait, R. & Preston, J.E. 2006. Optimising ovine cerebrospinal fluid preparation for two‐dimensional gel electrophoresis. Proteomics 6(10): 3170-3175.

Chen, Y.Y., Lin, S.Y., Yeh, Y.Y., Hsiao, H.H., Wu, C.Y., Chen, S.T. & Wang, A.H.J. 2005. A modified protein precipitation procedure for efficient removal of albumin from serum. Electrophoresis 26(11): 2117-2127.

Cretu, D., Diamandis, E.P. & Chandran, V. 2013. Delineating the synovial fluid proteome: Recent advancements and ongoing challenges in biomarker research. Critical Reviews in Clinical Laboratory Sciences 50(2): 51-63.

Fam, H., Bryant, J.T. & Kontopoulou, M. 2007. Rheological properties of synovial fluids. Biorheology 44(2): 59-74.

Fernández-Costa, C., Calamia, V., Fernández-Puente, P., Capelo-Martínez, J.L., Ruiz-Romero, C. & Blanco, F.J. 2012. Sequential depletion of human serum for the search of osteoarthritis biomarkers. Proteome Science 10(1): 1-12.

Iolascon, G., Gimigliano, F., Moretti, A., De Sire, A., Migliore, A., Brandi, M.L. & Piscitelli, P. 2017. Early osteoarthritis: How to define, diagnose, and manage. A systematic review. European Geriatric Medicine 8(5-6): 383-396.

Kay, R., Barton, C., Ratcliffe, L., Matharoo‐Ball, B., Brown, P., Roberts, J., Teale, P. & Creaser, C. 2008. Enrichment of low molecular weight serum proteins using acetonitrile precipitation for mass spectrometry based proteomic analysis. Rapid Communications in Mass Spectrometry: An International Journal Devoted to the Rapid Dissemination of Up-to-the-Minute Research in Mass Spectrometry 22(20): 3255-3260.

Mahendran, S.M., Oikonomopoulou, K., Diamandis, E.P. & Chandran, V. 2017. Synovial fluid proteomics in the pursuit of arthritis mediators: An evolving field of novel biomarker discovery. Critical Reviews in Clinical Laboratory Sciences 54(7-8): 495-505

Miller, I., Schlosser, S., Palazzolo, L., Veronesi, M.C., Eberini, I. & Gianazza, E. 2020. Some more about dogs: Proteomics of neglected biological fluids. Journal of Proteomics 218: 103724.

Peffers, M.J., Smagul, A. & Anderson, J.R. 2019. Proteomic analysis of synovial fluid: Current and potential uses to improve clinical outcomes. Expert Review of Proteomics 16(4): 287-302.

Ruiz-Romero, C., Calamia, V., Carreira, V., Mateos, J., Fernández, P. & Blanco, F.J. 2010. Strategies to optimize two-dimensional gel electrophoresis analysis of the human joint proteome. Talanta 80(4): 1552-1560.

Stewart, R.C., Nelson, B.B., Kawcak, C.E., Freedman, J.D., Snyder, B.D., Goodrich, L.R. & Grinstaff, M.W. 2019. Contrast‐enhanced computed tomography scoring system for distinguishing early osteoarthritis disease states: A feasibility study. Journal of Orthopaedic Research 37(10): 2138-2148.

Tiderius, C.J., Hawezi, Z.K., Olsson, L.E. & Dahlberg, L.E. 2020. Pre-contrast T1 and cartilage thickness as confounding factors in dGEMRIC when evaluating human cartilage adaptation to physical activity. BMC Medical Imaging 20(1): 1-6.

van Spil, W.E. & Szilagyi, I.A. 2019. Osteoarthritis year in review 2019: Biomarkers (biochemical markers). Osteoarthritis and Cartilage 28(3): 296-315.

van Tiel, J., Kotek, G., Reijman, M., Bos, P.K., Bron, E.E., Klein, S., Nasserinejad, K., van Osch, G.J., Verhaar, J.A., Krestin, G.P. & Weinans, H. 2016. Is T1ρ mapping an alternative to delayed gadolinium-enhanced MR imaging of cartilage in the assessment of sulphated glycosaminoglycan content in human osteoarthritic knees? An in vivo validation study. Radiology 279(2): 523-531.

Wang, Y., Teichtahl, A.J. & Cicuttini, F.M. 2016. Osteoarthritis year in review 2015: imaging. Osteoarthritis and Cartilage 24(1): 49-57.

Warder, S.E., Tucker, L.A., Strelitzer, T.J., McKeegan, E.M., Meuth, J.L., Jung, P.M., Saraf, A., Singh, B., Lai-Zhang, J., Gagne, G. & Rogers, J.C. 2009. Reducing agent-mediated precipitation of high-abundance plasma proteins. Analytical Biochemistry 387(2): 184-193.

Yamagiwa, H., Sarkar, G., Charlesworth, M.C., McCormick, D.J. & Bolander, M.E. 2003. Two-dimensional gel electrophoresis of synovial fluid: Method for detecting candidate protein markers for osteoarthritis. Journal of Orthopaedic Science 8(4): 482-490.

 

*Pengarang untuk surat-menyurat; email: lausengfong@upm.edu.my

   

 

 

sebelumnya