Notes & Tips Assessment of an ad hoc procedure for isolation and characterization of human albuminome Domenica Scumaci, Marco Gaspari, Milena Saccomanno, Giuseppe Argirò, Barbara Quaresima, Concetta M. Faniello, Pietrantonio Ricci, Francesco Costanzo, Giovanni Cuda ⇑ Department of Experimental and Clinical Medicine, Magna Graecia University, School of Medicine, Germaneto University Campus, 88100 Catanzaro, Italy article info Article history: Received 2 May 2011 Received in revised form 21 June 2011 Accepted 23 June 2011 Available online 30 June 2011 Keywords: Albuminome Biomarkers Plasma Proteomics abstract The dynamic range of plasma protein abundance, ranging from milligrams to picograms per milliliter, makes characterization of this proteome nearly impossible with current analytical methods. Plasma pre- processing by high-abundance protein depletion may concomitantly remove important diagnostic infor- mation. This article describes an original chromatographic procedure to isolate proteins bound to human serum albumin (HSA). Using HSA as an ‘‘affinity agent’’, we significantly improved the detection and iden- tification of HSA ligands by two-dimensional liquid chromatography tandem mass spectrometry (2D LC– MS/MS). Some of the characterized species were not previously reported in published blood databases. Albumin-binding proteins may be classified as belonging to several putative functional categories and span a wide variety of predicted physiological functions. Ó 2011 Elsevier Inc. All rights reserved. One of the major driving forces in clinical proteomics is the dis- covery of biomarkers, that is, proteins that change in concentration or state in association with a specific biological process or disease. Plasma contains 60–80 mg of proteins per milliliter in addition to various small molecules, including salts, lipids, amino acids, and sugars. The major protein constituents of plasma include albumin, immunoglobulins, transferrin, haptoglobin, and lipoproteins. The presence of abundant proteins may interfere with the identifica- tion and quantification of the less represented proteins, whose concentration is at least 7–8 orders of magnitude less than the most abundant ones [1,2]. Traditional proteomic approaches seem inadequate to detect low-abundance plasma proteins without effective removal (or sep- aration) of high-abundance proteins. On the other hand, it is possi- ble that the depletion of high-abundance proteins may cause the loss of many potentially interesting low-molecular-weight (LMW) 1 biomarkers [3]. The LMW proteome includes small peptides as well as fragments of large proteins. Within normal and diseased tissue, proteolysis-in- duced events, such as stromal–epithelial interactions, immune cell MHC (major histocompatibility complex) presentation, and apopto- sis, may generate peptides that passively diffuse into the circula- tion. It is known that LMW biomarkers are rapidly cleared throughout the kidney. As a consequence, such rapid physiological excretion might significantly reduce the concentration of low- molecular-mass biomarkers to a level below detection. The abun- dant high-molecular-weight carrier proteins are well above the cut- off for kidney clearance; therefore, their half-life is many orders of magnitude larger than that of small molecules. LMW biomarkers are usually bound to high-molecular-weight carriers, which in turn increase their hydrophilicity and extend their half-life in the blood- stream. Thus, circulating carrier proteins become a reservoir for the accumulation of bound low-mass biomarkers. For this reason, dis- carding these proteins prior to the analysis of the native serum or plasma is akin to ‘‘throwing out the baby with the bathwater’’. The most important and studied plasma ‘‘cargo’’ protein is hu- man serum albumin (HSA). HSA binds to endogenous important species such as hormones, cytokines, and lipoproteins as well as to exogenous compounds such as drugs [2]. The purpose of this investigation was to establish new prepara- tive methods to isolate albumin partners in order to increase the dynamic range available for the identification and characterization of potential plasma biomarkers. Human blood plasma was obtained from 8 healthy anonymous donors and was collected in accordance with HUPO (Human Prote- ome Organization) plasma proteome guidelines. The sample was centrifuged within 2 h of collection at 1300g for 10 min, aliquoted into silicon tubes, and stored at À80 °C until use [4]. An ad hoc chromatographic procedure was set to isolate pro- teins binding to albumin. The method is based on the use of HSA as an ‘‘affinity agent’’ for low-abundance species with the aim to enrich the concentration of HSA ligands and to provide a more 0003-2697/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2011.06.032 ⇑ Corresponding author. Fax: +39 0961 3694090. E-mail address: cuda@unicz.it (G. Cuda). 1 Abbreviations used: LMW, low-molecular-weight; HSA, human serum albumin; 2D LC–MS/MS, two-dimensional liquid chromatography–tandem mass spectrometry; PBC, sodium phosphate buffer; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Analytical Biochemistry 418 (2011) 161–163 Contents lists available at ScienceDirect Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio