Hong-Lei Huang 1 Taras Stasyk 1 Sandra Morandell 1 Maurice Mogg 2 Martin Schreiber 2 Isabel Feuerstein 3 Christian W. Huck 3 Guenther Stecher 3 Guenther K. Bonn 3 Lukas A. Huber 1 1 Biocenter, Division of Cell Biology, Innsbruck Medical University, Innsbruck, Austria 2 Division of Special Gynecology, Medical University of Vienna, Vienna, Austria 3 Institute of Analytical Chemistry and Radiochemistry, Leopold-Franzens University, Innsbruck, Austria Enrichment of low-abundant serum proteins by albumin/immunoglobulin G immunoaffinity depletion under partly denaturing conditions We present a simple protocol for affinity depletion to remove the two most abundant serum proteins, albumin and immunoglobulin G (IgG). Under native conditions, albu- min/IgG were efficiently removed and several proteins were enriched as shown by two-dimensional electrophoresis (2-DE). Besides that, partly denaturing conditions were established by adding 5 or 20% acetonitrile (ACN) in order to disrupt the binding of low-molecular-weight (LMW) proteins to the carrier proteins albumin/IgG. 2-DE results showed that the total number of detected LMW proteins increased under denaturing conditions when compared to native conditions. Interestingly, the pres- ence of 5% ACN in serum revealed better enrichment of LMW proteins when com- pared to 20% ACN condition. Seven randomly distributed spots in albumin/IgG depleted serum samples under 5% ACN condition were picked from the 2-DE gels and identified by mass spectrometry (MS). The intensity of five LMW protein spots increased under denaturing conditions when compared to native conditions. Three of the seven identified spots (serum amyloid P, vitamin D-binding protein, and trans- thyretin) belong to a group of relatively low-abundant proteins, which make up only 1% of all serum proteins. The method presented here improves the resolution of the serum proteome by increasing the number of visualized spots on 2-D gels and allowing the detection and MS identification of LMW proteins and proteins of lower abundance. Keywords: Denaturing condition / Immunoaffinity depletion / Low-abundant protein / Low-mo- lecular-weight protein / Serum DOI 10.1002/elps.200500167 1 Introduction Serum proteins may serve as indicators of disease and constitute a rich source for biomarker discovery [1, 2]. The protein content of human serum is composed of millions of proteins from almost every type of cell and tissue within the body [3]. Unfortunately, more than ten orders of magnitude dynamic range of protein abun- dance exceeds the analytical capabilities of standard procedures (e.g., 2-DE combined with MS), making the detection of lower abundance serum proteins extremely challenging [4, 5]. The reduction of sample complexity is thus an essential first step in the analysis of the serum proteome. Affinity methods have been developed to remove highly-abundant proteins, such as albumin and immunoglobulins, from serum prior to further analysis [5]. Removal of these proteins alone clears about 75% of the total proteins present in serum, thereby allowing detec- tion of the remaining proteins that are present in far lower concentration [6]. One of the fundamental drawbacks of serum protein depletion methodologies is that many important low-mo- lecular-weight (LMW) proteins or peptides might be con- comitantly removed by the sample preparation process [7]. It is known now that albumin can function as a carrier and transporter of proteins within the blood and binds physiologically important species such as hormones, cytokines, and lipoproteins [8]. Since the affinity methods used to deplete high-abundant serum proteins target globular proteins under native conditions, these methods are also likely removing those proteins or peptides bound to the target proteins. In this study, a method for the removal of high-abundant proteins from serum without the concomitant loss of LMW components has been devel- Correspondence: ProfessorDr. Lukas A. Huber, Biocenter, Divi- sion of Cell Biology, Innsbruck Medical University, Fritz-Pregl- Strasse 3, 6020-Innsbruck, Austria E-mail: Lukas.A.Huber@uibk.ac.at Fax: 143-(0)512-507-2873 Abbreviations: DOC, Na-deoxycholate; IgG, immunoglobulin G; LMW, low molecular weight Electrophoresis 2005, 26, 2843–2849 2843 Supplementary material for this article is available on the WWW under www.electrophoresis-journal.de.  2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Proteomics and 2-DE