Young Y. Wang 1, 2 Paul (Chung Pui) Cheng 2 Daniel W. Chan 1, 2 1 Department of Pathology, Johns Hopkins Medical Institutions, Baltimore, MD, USA 2 Tumor Marker Laboratory, Johns Hopkins Singapore, Singapore Science Park II, Singapore A simple affinity spin tube filter method for removing high-abundant common proteins or enriching low-abundant biomarkers for serum proteomic analysis Although it is possible to identify new proteins from crude cell extracts using proteom- ics technology, it is often difficult to elucidate low-abundant biomarkers in the pres- ence of a large amount of high-abundant proteins in serum. We have developed a simple and rapid method using an affinity spin tube filter to remove high-abundant common proteins and enrich the low-abundant biomarkers. The affinity spin tube filter contains protein G, coupled with antibodies against either high-abundant proteins or specific proteins of interest. After incubating with serum, the flow-through or the elute was collected and analyzed by two-dimensional gel electrophoresis. By using this affinity spin tube filter, the possibilities of identifying new biomarkers are shown. This technique could be used for large-scale sample preparation for high-throughput proteomic analysis. Keywords: Biomarker / Serum / Spin tube filter / Two-dimensional gel electrophoresis PRO 0348 1 Introduction Proteomics has the potential to identify novel cancer bio- markers from tumor tissue and biological fluids [1]. One of the challenges of identifying as many serum proteins as possible is the difficulty of finding lower abundance proteins in the presence of higher abundance proteins. The differences in concentration among serum proteins may be over a thousand to a million times. Therefore, the strategy to effectively remove high-abundant proteins and enrich low-abundant proteins of interest is very important [2–5]. In 2-DE, the ability to visualize lower abundant biomarkers in serum is often obscured by the presence of higher abundant proteins. For example, HSA and immunoglobulin G (IgG) are two high-abundant common proteins in serum. By removing them, over 75% of the total proteins are cleared, thereby enabling the detection of the remaining proteins present in much lower concentrations [6]. However, the removal of HSA was not successful using centrifugal ultrafiltration [7]. Other approaches, such as Cibracon Blue, remove not only the high-abundant proteins but also proteins of interest. Affin- ity column chromatography has been shown to improve the HSA and IgG removal efficiency [6], while specific antibodies have also been shown to enrich low-abundant proteins of interest on other platform [8]. However, this simple affinity spin tube filter method using high-affinity antibodies combined with protein G has not been reported. Here we describe a method for the removal of high-abundant HSA and IgG or enriching specific proteins of interest in serum samples. Examples are shown in 2-DE for the improvement in the differential expression of serum proteins between disease and healthy status and in the identification of the circulating forms of fatty acid synthase (FAS). This method could have widespread uti- lity for the preparation of large number of samples in the discovery of serum biomarkers. 2 Materials and methods 2.1 Materials The Spin-X  centrifuge tube filters were purchased from Corning (Corning, NY, USA). HiTrap Protein G HP, HiTrap Blue and Immobiline  DryStrips were purchased from Amersham Biosciences (Uppsala, Sweden). The bicin- choninic acid protein assay kits (BCA) were purchased from Pierce (Rockford, IL, USA). 2.2 Antibodies Rabbit antihuman albumin was purchased from Dako (Glostrup, Denmark). Monoclonal antihuman FAS anti- body was produced from the ZR-75-1 breast cancer cell Correspondence: Dr. Daniel W. Chan, Dept. of Pathology, Johns Hopkins Medical Institutions, 600 N. Wolfe St., Meyer B121, Baltimore, MD 21287, USA E-mail: dchan@jhmi.edu Fax: +1-410-955-0767 Abbreviations: FAS, fatty acid synthase; Ig, immunoglobulin Proteomics 2003, 3, 243–248 243  2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 0173-0835/03/0303–243 $17.501.50/0