Centrifugal ultrafiltration as tool for low abundance proteins isolation
October 6, 2011
Centrifugal ultrafiltration as tool for low abundance proteins isolation
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Title: | Centrifugal ultrafiltration as tool for low abundance proteins isolation |
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Article_Title: | Centrifugal ultrafiltration as tool for low abundance proteins isolation |
Authors: | Ioana Lancrajan, Doru Ardelean, Svetlana Kogalniceanu |
Affiliation: | „Vasile Goldiş” Western University of Arad, Faculty of Life Sciences, Romania |
Abstract: | Because of the large amount of albumin and others “high abundance proteins” (HAP), the “dynamic range” and heterogeneity of proteins, the separation, isolation, characterization and quantifying of “low abundance proteins” LAP from serum, most of them important diagnostically marchers, is technically difficult. The aim this study is to test a simple and cheap method to reduce the serum protein complexity in order to identify and quantify LAP, particularly growth hormone. Ultrafiltration was used as preparative method to remove HAP from serum and enrich the sample in LAP. Denaturation seems to be essential before centrifugal ultrafiltration (Georgiou, 2001/ Tirumalai, 2003/ Harper, 2004). Filtration was performed under denaturing conditions and recovery of hGH calculated. The higher recovery rate (46%) was attempted when the samples was denatured before centrifugal ultrafiltration in buffer containing RapiGest, compared with denaturation in SDS (6,2% recovery). The results are helpful for procedures of quantitative determination of LAP e.g. hGH from serum. |
Keywords: | ultrafiltration, denaturation, recovery, low abundance protein, quantify |
References: | Adkins JN, Varnum SM, Auberry KJ, Moore RJ, Angell NH, Richard D. Smith, Springer DL and Pounds JG 2002, Toward a human blood serum proteome. Analysis by multidimensional separation coupled with mass spectrometry, Molecular & Cellular Proteomics 1(12): 947-955 Anderson NL and Anderson NG, 2002, The human plasma proteome. History, character, and diagnostic prospects, Molecular & Cellular Proteomics 1.11, Rewiews/Perspectives, 845-867 Boguszewski CL, Hynsjo L, Johannsson G, Bengtsson BA, Carlsson LM. 1996, Eur J 22-kD growth hormone exclusion assay: a new approach to measurement of non-22-kD growth hormone isoforms in human blood, Endocrinol. Baumann G, 1999, Growth Hormone Heterogenity in Human Pituitary and Plasma, Horm. Res.; 51 (suppl 1): 2-6 Chan K, Lucas DA, Hise DM, Schaefer CF, Xiao Z, Janini GM, Buetow KH, IssaqHJ, Veenstra T and Conrads TP, 2004, Analysis of the human serum proteome, Clinical Proteomics, 1(2) 179-185. Georgiou HM, Rice GE, Baker MS 2001, Proteomic analysis of human plasma: failure of centrifugal ultrafiltration to remove albumin and other high molecular weight proteins, Proteomics 1(12):1503-6 Harper RG, Workman SR, Schuetzner S, Timperman AT, Sutton JN, 2004, Low-molecular-weight human serum proteome using ultrafiltration, isoelectric focusing, and mass spectrometry, Electrophoresis, 25, 1299-1306 Kuhn E, Wu J, Karl J, Liao H, Zolg W and Guild B., 2004, Quantification of C-reactive protein in the serum of patients with rheumatoid arthritis using multiple reaction monitoring mass spectrometry and 13C-labeled peptide standards, Proteomics, 1175-1186 Rose K, Bougueleret L, Baussant T, Bohm G, Botti P, Colinge J, Cusin I, Gaertner H, Gleizes A, Heller M, Jimenez S, Johnson A, Kussmann M, Menin L, Menzel C, 2004, Industrial-scale proteomics: from liters of plasma to chemically synthesized proteins, Proteomics 4(7):2125-50. Pieper R, Gatlin CL, Makusky AJ, Russo PS, Schatz CR, Miller SS, Su Q, McGrath AM, Estock MA, Parmar PP, Roy P, Truntzer C, Maucort-Boulch D, Jouve D, and Molinari N 2011, Protein mass spectra data analysis for clinical biomarker discovery: a global review Brief Bioinform 12(2): 176-186 Sacher, R.A., and McPherson, R. A. 2000, Widmann`s Clinical Interpretation of Laboratory Tests, F.A. Davis Co., Philadelphia, PA, Sonksen PH 2001, Insulin, growth hormone and sport, Journal of Endocrinology 170, 13-25 Tirumalai RS, King C. Chan, DaRue A. Prieto, Haleem J. Issaq, Thomas P. Conrads, and Timothy D. Veenstra 2003, Characterisation of the low molecular weight human serum proteome, Molecular & Cellular Proteomics 2.10, 1096-1103 Zhao M, Huang ST, Zhou J, Wang F, Esquer-Blasco R, Anderson NL, Taylor J, Steiner S, 2003, The human serum proteome: display of nearly 3700 chromatographically separated protein spots on two dimensional electrophoresis gels and identification of distinct proteins, Proteomics 3 (7): 1345-64. Wang HX, W Qian, HM Mottaz, T Clauss, DJ Anderson, RJ Moore, DG Camp II, AH Khan, DM Sforza, M Pallavicini, DJ Smith, and RD Smith. 2005, Development and Evaluation of a Micro- and Nanoscale Proteomic Sample Preparation Method, Journal of Proteome Research 4(6):2397-2403. Won-A Joo, Do-Youn Lee, and Chan-Wha Kim 2003, Development of an effective sample preparation method for the proteome analysis of body fluids using 2-D Gel electrophoresys, Biosci. Biotechnol. Biochem., 67(7), 1574-1577. |
Read_full_article: | pdf/21-2011/21-3-2011/SU21-3-2011-Lancrajan.pdf |
Correspondence: | Ioana Lancrajan, „Vasile Goldiş” Western University of Arad, Faculty of Life Sciences, Rebreanu st. 91-93; email: Lancrajan _ioana@yahoo.com |
Read full article | |
Article Title: | Centrifugal ultrafiltration as tool for low abundance proteins isolation |
Authors: | Ioana Lancrajan, Doru Ardelean, Svetlana Kogalniceanu |
Affiliation: | „Vasile Goldiş” Western University of Arad, Faculty of Life Sciences, Romania |
Abstract: | Because of the large amount of albumin and others “high abundance proteins” (HAP), the “dynamic range” and heterogeneity of proteins, the separation, isolation, characterization and quantifying of “low abundance proteins” LAP from serum, most of them important diagnostically marchers, is technically difficult. The aim this study is to test a simple and cheap method to reduce the serum protein complexity in order to identify and quantify LAP, particularly growth hormone. Ultrafiltration was used as preparative method to remove HAP from serum and enrich the sample in LAP. Denaturation seems to be essential before centrifugal ultrafiltration (Georgiou, 2001/ Tirumalai, 2003/ Harper, 2004). Filtration was performed under denaturing conditions and recovery of hGH calculated. The higher recovery rate (46%) was attempted when the samples was denatured before centrifugal ultrafiltration in buffer containing RapiGest, compared with denaturation in SDS (6,2% recovery). The results are helpful for procedures of quantitative determination of LAP e.g. hGH from serum. |
Keywords: | ultrafiltration, denaturation, recovery, low abundance protein, quantify |
References: | Adkins JN, Varnum SM, Auberry KJ, Moore RJ, Angell NH, Richard D. Smith, Springer DL and Pounds JG 2002, Toward a human blood serum proteome. Analysis by multidimensional separation coupled with mass spectrometry, Molecular & Cellular Proteomics 1(12): 947-955 Anderson NL and Anderson NG, 2002, The human plasma proteome. History, character, and diagnostic prospects, Molecular & Cellular Proteomics 1.11, Rewiews/Perspectives, 845-867 Boguszewski CL, Hynsjo L, Johannsson G, Bengtsson BA, Carlsson LM. 1996, Eur J 22-kD growth hormone exclusion assay: a new approach to measurement of non-22-kD growth hormone isoforms in human blood, Endocrinol. Baumann G, 1999, Growth Hormone Heterogenity in Human Pituitary and Plasma, Horm. Res.; 51 (suppl 1): 2-6 Chan K, Lucas DA, Hise DM, Schaefer CF, Xiao Z, Janini GM, Buetow KH, IssaqHJ, Veenstra T and Conrads TP, 2004, Analysis of the human serum proteome, Clinical Proteomics, 1(2) 179-185. Georgiou HM, Rice GE, Baker MS 2001, Proteomic analysis of human plasma: failure of centrifugal ultrafiltration to remove albumin and other high molecular weight proteins, Proteomics 1(12):1503-6 Harper RG, Workman SR, Schuetzner S, Timperman AT, Sutton JN, 2004, Low-molecular-weight human serum proteome using ultrafiltration, isoelectric focusing, and mass spectrometry, Electrophoresis, 25, 1299-1306 Kuhn E, Wu J, Karl J, Liao H, Zolg W and Guild B., 2004, Quantification of C-reactive protein in the serum of patients with rheumatoid arthritis using multiple reaction monitoring mass spectrometry and 13C-labeled peptide standards, Proteomics, 1175-1186 Rose K, Bougueleret L, Baussant T, Bohm G, Botti P, Colinge J, Cusin I, Gaertner H, Gleizes A, Heller M, Jimenez S, Johnson A, Kussmann M, Menin L, Menzel C, 2004, Industrial-scale proteomics: from liters of plasma to chemically synthesized proteins, Proteomics 4(7):2125-50. Pieper R, Gatlin CL, Makusky AJ, Russo PS, Schatz CR, Miller SS, Su Q, McGrath AM, Estock MA, Parmar PP, Roy P, Truntzer C, Maucort-Boulch D, Jouve D, and Molinari N 2011, Protein mass spectra data analysis for clinical biomarker discovery: a global review Brief Bioinform 12(2): 176-186 Sacher, R.A., and McPherson, R. A. 2000, Widmann`s Clinical Interpretation of Laboratory Tests, F.A. Davis Co., Philadelphia, PA, Sonksen PH 2001, Insulin, growth hormone and sport, Journal of Endocrinology 170, 13-25 Tirumalai RS, King C. Chan, DaRue A. Prieto, Haleem J. Issaq, Thomas P. Conrads, and Timothy D. Veenstra 2003, Characterisation of the low molecular weight human serum proteome, Molecular & Cellular Proteomics 2.10, 1096-1103 Zhao M, Huang ST, Zhou J, Wang F, Esquer-Blasco R, Anderson NL, Taylor J, Steiner S, 2003, The human serum proteome: display of nearly 3700 chromatographically separated protein spots on two dimensional electrophoresis gels and identification of distinct proteins, Proteomics 3 (7): 1345-64. Wang HX, W Qian, HM Mottaz, T Clauss, DJ Anderson, RJ Moore, DG Camp II, AH Khan, DM Sforza, M Pallavicini, DJ Smith, and RD Smith. 2005, Development and Evaluation of a Micro- and Nanoscale Proteomic Sample Preparation Method, Journal of Proteome Research 4(6):2397-2403. Won-A Joo, Do-Youn Lee, and Chan-Wha Kim 2003, Development of an effective sample preparation method for the proteome analysis of body fluids using 2-D Gel electrophoresys, Biosci. Biotechnol. Biochem., 67(7), 1574-1577. |
*Correspondence: | Ioana Lancrajan, „Vasile Goldiş” Western University of Arad, Faculty of Life Sciences, Rebreanu st. 91-93; email: Lancrajan _ioana@yahoo.com |