Impact of the depletion of high-abundance proteins in blood plasma/serum on the proteome of these media in growing farm animals
The aim of the study was to use hexapeptide combinatorial libraries to evaluate the influence of high-abundance protein depletion on low-abundance protein enrichment, protein concentration, and 2D gel quality in plasma and serum proteomes of growing piglets and calves. Depleted and nondepleted samples of plasma/serum were resolved using 2D electrophoresis (2DE). Bioinformatic analysis of the 2D gels showed an increased number of detected spots on each gel after depletion of excess high-abundance proteins in calf plasma (+26.46%) and in piglet serum (+49.40%). Relative expression of selected low-abundance protein spots in the blood plasma/serum samples was higher after high-abundance protein depletion. Both media showed decreased expressions of high-abundance protein spot clusters on 2D gels after treatment. The results of this study confirm both the high efficiency of high-abundance protein depletion and the increase in concentrations of low-abundance proteins on 2D gels after treatment. The method of depletion is reproducible and it may be used as an alternative to immunodepletion for preparing blood plasma/serum samples from young, growing animals for 2DE. Increased content of the low-abundance proteins on the 2D gels enables complete analysis of plasma/serum proteome and thereby allows for the discovery of novel biomarkers that might be useful in veterinary medicine.
Impact of the depletion of high-abundance proteins in blood plasma/serum on the proteome of these media in growing farm animals
The aim of the study was to use hexapeptide combinatorial libraries to evaluate the influence of high-abundance protein depletion on low-abundance protein enrichment, protein concentration, and 2D gel quality in plasma and serum proteomes of growing piglets and calves. Depleted and nondepleted samples of plasma/serum were resolved using 2D electrophoresis (2DE). Bioinformatic analysis of the 2D gels showed an increased number of detected spots on each gel after depletion of excess high-abundance proteins in calf plasma (+26.46%) and in piglet serum (+49.40%). Relative expression of selected low-abundance protein spots in the blood plasma/serum samples was higher after high-abundance protein depletion. Both media showed decreased expressions of high-abundance protein spot clusters on 2D gels after treatment. The results of this study confirm both the high efficiency of high-abundance protein depletion and the increase in concentrations of low-abundance proteins on 2D gels after treatment. The method of depletion is reproducible and it may be used as an alternative to immunodepletion for preparing blood plasma/serum samples from young, growing animals for 2DE. Increased content of the low-abundance proteins on the 2D gels enables complete analysis of plasma/serum proteome and thereby allows for the discovery of novel biomarkers that might be useful in veterinary medicine.
___
- Miller I, Wait R, Sipos W, Gemeiner M. A proteomic reference map for pig serum proteins as a prerequisite for diagnostic applications. Res Vet Sci 2009; 86: 362–367. 2. Hu S, Loo JA, Wong DT. Human body fluid proteome analysis. Proteomics 2006; 6: 6326–6353.
- Karczmarski J, Mikula M, Henning E, Rubel T, Otrowski J. Perspectives of mass spectrometry-based disease biomarkers discovery in plasma or serum peptidome. Postępy Nauk Medycznych 2009; 2: 149–156 (article in Polish with English abstract).
- Herosimczyk A, Dejeans N, Sayd T, Ożgo M, Skrzypczak WF, Mazur A. Plasma proteome analysis: 2D gels and chips. J Physiol Pharmacol 2006; 57: 81–93.
- Bendixen E, Danielsen M, Hollung K, Gianazza E, Miller I. Farm animal proteomics—a review. J Proteomics 2011; 74: 282–293. 6. D’Ambrosio C, Arena S, Talamo F, Ledda L, Renzone G, Ferrara L, Scaloni A. Comparative proteomic analysis of mammalian animal tissues and body fluids: bovine proteome database. J Chromatogr B 2005; 815: 157–168.
- Huang Y, Olson DJ, Gordon JR, Middleton DM, Simko E. Plasma protein profiles of neonatal pigs before and after suckling. Can J Vet Res 2012; 76: 1–7.
- Skrzypczak WF, Ożgo M, Herosimczyk A, Lepczyński A. Defining the blood plasma protein repertoire of seven day old dairy calves - a preliminary study. J Physiol Pharmacol 2011; 62: 313–319.
- Wait R, Miller I, Eberini I, Cairoli F, Veronesi C, Battocchio M, Gemeiner M, Gianazza E. Strategies for proteomic with incompletely characterized genomes: the proteome of Bos taurus serum. Electrophoresis 2002; 23: 3418–3427.
- Herosimczyk A, Lepczynski A, Ozgo M, Dratwa-Chałupnik A, Michalek K, Skrzypczak WF. Blood plasma protein and lipid profile changes in calves during the first week of life. Pol J Vet Sci 2013; 16: 425–434.
- Xia C, Zhang HY, Wu L, Xu C, Zheng JS, Yan YJ, Yang LJ, Shu S. Proteomic analysis of plasma from cows affected with milk fever using two-dimensional differential in-gel electrophoresis and mass spectrometry. Res Vet Sci 2012; 93: 857–861.
- Chen YY, Lin SY, Yeh YY, Hsiao HH, Wu CY, Chen ST, Wang AHJ. A modified protein precipitation procedure for efficient removal of albumin from serum. Electrophoresis 2005; 26: 2117–2127.
- Chevalier F. Highlights on the capacities of “Gel-based” proteomics. Proteome Sci 2010; 8: 23.
- Liumbruno G, D’Alessandro A, Grazzini G, Zolla L. Blood- related proteomics. J Proteomics 2010; 73: 483–507.
- Herosimczyk A, Lepczyński A, Dratwa-Chałupnik A, Kurpińska A, Klonowska A, Skrzypczak WF. Age-related changes of selected blood biochemical indicators in dairy calves during their first week of life. Folia Biol (Kraków) 2011; 59: 25–30.
- Kim MR, Kim CW. Human blood plasma preparation for two- di mensional gel electrophoresis. J Chromatogr B 2007; 849: 203–210.
- Kossowska B, Dudka I, Gancarz R, Antonowicz-Juchniewicz J. Proteomic analysis of protein profiles in some pathological stages of the human organism. Postępy Higieny i Medycyny Doświadczalnej 2009; 63: 549–563 (article in Polish with English abstract).
- Li L, Sun C, Freeby S, Yee D, Kieffer-Jaquinod S, Guerrier L, Boschetti E, Lomas L. Protein sample treatment with peptide ligand library: coverage and consistency. J Proteomics Bioinform 2009; 2: 485–094.
- Hagiwara T, Saito Y, Nakamura Y, Tomonaga T, Murakami Y, Kondo T. Combined use of a solid-phase hexapeptide ligand library with liquid chromatography and two-dimensional difference gel electrophoresis for intact plasma proteomics. Int J Proteomics 2011; 2011: 739615.
- Jaszczołt M, Boczkaj G, Grygoryshyn K, Kamiński M. Immunochromatography: a review and characterization of immunosorbents and methods of immobilization. Camera Separatoria 2011; 3: 69–85 (article in Polish with English abstract).
- Nisnevitch M, Firer MA. The solid phase in affinity chromatography: strategies for antibody attachment. J Biochem Biophys Methods 2001; 49: 467–480.
- Qian WJ, Kaleta DT, Petritis BO, Jiang H, Liu T, Zhang X, Mottaz HM, Varnum SM, Camp DG, Huang L et al. Enhanced detection of low abundance human plasma proteins using a tandem IgY12-SuperMix immunoaffinity separation strategy. Mol Cell Proteomics 2008; 7: 1963–1973.
- Echan LA, Tang HY, Ali-Khan N, Lee K, Speicher DW. Depletion of multiple high-abundance proteins improves protein profiling capa cities of human serum and plasma. Proteomics 2005; 5: 3292–3303.
- Boschetti E, Righetti PG. Hexapeptide combinatorial ligand libraries: the march for the detection of the low abundance proteome continues. Biotechniques 2008; 44: 663–665.
- Guerrier L, Righetti PG, Boschetti E. Reduction of dynamic protein concentration range of biological extracts for the discovery of low abundance proteins by means of hexapeptide ligand library. Nat Protoc 2008; 3: 883–890.
- Sennels L, Salek M, Lomas L, Boschetti E, Righetti PG, Rappsilber J. Proteomic analysis of human blood serum using peptide library beads. J Prot Res 2007; 6: 4055–4062.
- Pink M, Verma N, Rettenmeier AW, Schmitz-Spanke S. CBB staining protocol with higher sensitivity and mass spectrometric compatibility. Electrophoresis 2010; 31: 593– 598.
- Marco A, Rovira G, Bassols A. Use of ProteoMiner in veterinary research. Proteomica 2010; 5: 168–170.
- Marco-Ramell A, Bassols A. Enrichment of low-abundance proteins from bovine and porcine serum samples for proteomic studies. Res Vet Sci 2010; 89: 340–343.
- Rasaputra KS, Liyanage R, Lay JO, Erf GF, Okimato R, Rash NC. Changes in serum protein profiles of chickens with tibial dyschondroplasia. Open Proteomics J 2012; 5: 1–7.