Enrichment of low-abundance proteins from bovine and porcine serum samples for proteomic studies Anna Marco-Ramell, Anna Bassols * Departament de Bioquímica i Biologia Molecular, Facultat de Veterinària, Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallès, Spain article info Article history: Accepted 8 March 2010 Keywords: Proteomics Bovine Porcine Serum Electrophoresis DIGE abstract One of the main applications of serum proteomics is the identification of new biomarkers for animal dis- ease or animal production. However, potential obstacles to these studies are the poor performance of affinity serum depletion methods based on human antigens when using animal samples, and loss of minor serum components bound to albumin and other proteins. In the present study, we have analyzed the efficiency and reproducibility of the ProteoMiner Ò beads with bovine and porcine serum samples, and compared to a traditional immunoaffinity-based albumin and IgG depletion system specific for human samples. The ProteoMiner kit is based on the use of a combinatorial peptide binding library and intends to enrich low-abundance proteins. Ó 2010 Elsevier Ltd. All rights reserved. Proteomics is a rapidly emerging technology with a large range of applications, including veterinary sciences. The identification of new biomarkers for diagnosis or for monitoring the general status of an individual is one of the main potential applications for pro- teomics and already new potential markers have been identified in human medicine. In this kind of analysis, serum is potentially the most valuable biological sample, because it contains thousands of different proteins and peptides and it is the most easily accessi- ble, noninvasive, and widely collected sample (Adkins et al., 2002). Unfortunately, the protein content of serum is dominated by a handful of high-abundance proteins, with their estimated concen- tration exceeding the low-abundance proteins highly by 10 orders of magnitude (Anderson and Anderson, 2002). To detect these proteins present in low levels using currently available technologies, it is advisable to remove the most abundant proteins first. Many strategies have been developed for the selec- tive removal of albumin and other high-abundance proteins to facilitate the analysis of other, less abundant proteins in serum (Fang and Zhang, 2008). Albumin and other major proteins can be removed by immunoaffinity approaches, IgY microbeads (Sep- pro Ò ) or other commercial reagents (Fang and Zhang, 2008). Among the protein–ligand affinity-based approaches are the re- moval of IgG by using immobilized protein A or protein G (Fang and Zhang, 2008), the Affibody Ò ligands (Gronwall et al., 2007), the use of lectin affinity columns to capture or enrich serum glyco- proteins (Hirabayashi, 2004) or the depletion of some plasma pro- teins by heparin chromatography (Lei et al., 2008). Furthermore, it is well known that albumin and other proteins may also act as transport proteins and thus are likely to bind many species of interest, such as peptide hormones, cytokines, and che- mokines (Sahab et al., 2007). As such, the affinity-based depletion systems may also eliminate some other proteins bound to those highly-copied proteins simultaneously, in a process of co-depletion (Granger et al., 2005). Thus, it has been described that during hu- man serum albumin depletion, another 815 species were co-de- pleted and that, when capturing IgGs, another 2091 species were co-depleted (Boschetti and Righetti, 2008). Recently, a new method for enriching low-abundance proteins has been commercially available. This technology is known under the trade name of Prote- oMiner Ò and it is based on the use of a combinatorial peptide bind- ing library, which affinity-captures and amplifies the low- abundance proteome (Boschetti and Righetti, 2008). Proteomics constitutes an interesting approach to veterinary medicine and animal production (Doherty et al., 2008). In this sense, porcine and bovine livestock are the most interesting spe- cies due to their economical interest. Surprisingly, there are very few applications of serum proteomics to veterinary science. The bovine serum 2-DE map has been published (Wait et al., 2002; Tal- amo et al., 2003; D’Ambrosio et al., 2005) and some applications into the physiopathological changes described (Cairoli et al., 2006). The serum pig proteome has only been recently reported (Miller et al., 2008). Many of the most used methodologies for depletion of abundant proteins are based in an immunological approach and, thus, they have a high specificity for human or laboratory animal proteins (Echan et al., 2005). The ProteoMiner technology, since it is not based on an immunological approach, should be species independent, and thus of potential application 0034-5288/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2010.03.019 * Corresponding author. Tel.: +34 93 581 1042; fax: +34 93 581 2006. E-mail address: anna.bassols@uab.cat (A. Bassols). Research in Veterinary Science 89 (2010) 340–343 Contents lists available at ScienceDirect Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc