Hexadecanedionic Acid−Sepharose 4B: A New Tool for Preparation of

Hexadecanedionic Acid-Sepharose 4B: A New Tool for Preparation of Albumin-Depleted Plasma Vukic Soskic,* Gerhard Schwall, Elke Nyakatura, Slobodan Poznanovic, Werner Stegmann, and Andre Schrattenholz ProteoSys AG, Carl-Zeiss-Strasse 51, 55129 Mainz, Germany Received July 30, 2006

Abstract: Serum and plasma are the major sources of human material for clinical molecular diagnostics and drug discovery. However, due to the high abundance of some proteins, of which serum albumin (SA) is most prominent, lower-abundance proteins often remain undetectable in proteomic analysis of these body fluids. We have used hexadecanedionic acid (HDA) immobilized to Sepharose 4B to develop an affinity resin that is effective in the removal of SA from plasma. Two-dimensional gel analysis of the SA-depleted samples shows a significant enhancement of the low-abundance proteins and highly specific capture of serum albumin. The HDA resin shows better performance in terms of specificity than dye-based resins. Keywords: Plasma•proteomics•hexadecanedionic acid•affinity separation•two-dimensional separation•mass spectrometry•human plasma albumin

Experimental Section

Introduction Human plasma and serum play important roles in clinical medicine as a rich source of molecular information about physiological status of patients, mainly due to their easy accessibility. Plasma and serum analysis is primary focused on the identification of enzymatic activities and the concentration of certain protein biomarkers, as well as qualitative and quantitative determination of low molecular weight metabolites.1,2 Although the number of known protein biomarkers is large, there are ongoing efforts yo identify new ones and to increase sensitivity of current assays. Unfortunately, there are restrictions of current analytical methods by the limited dynamic range of detection technologies, ranging from 1 to roughly 3 orders of magnitude in dye-based staining procedures and mass spectrometry. In this context, the presence of a few high-abundance proteinssserum albumin (SA), which makes up 40-60% of total serum protein; immunoglobulins of IgG class (between 8% and 28%); and fibrinogen and transferrin, each representing 3-6%s creates a severe background hurdle for the identification of novel protein biomarkers from these important clinical sources. For instance, plasma proteins resolved on two-dimensional polyacrylamide gel electrophoresis (2D PAGE) become crowded from 45 to 80 kDa and pI range * Corresponding author: phone +49-6131-5019247; fax +49-6131-5019211; e-mail [email protected]. 10.1021/pr060387q CCC: $33.50

of 4.5-6. The removal of highly abundant proteins such as SA from serum and plasma is absolutely necessary for the detection of low-abundance proteins by 2D PAGE or other multidimensional protein separation methods.5 Therefore, a methodology is needed that effectively fractionates the highly abundant and less abundant proteins. Currently there are several methods used for SA removal. Cibacron Blue-based affinity chromatography has been widely used but lacks specificity.3-5 Anti-albumin antibodies are also used in immunoaffinity systems but are expensive and have potential of introducing proteins from affinity supports into the sample, potentially interfering with subsequent mass spectromertry.6-8 In this report, a novel method for the highly specific removal of albumin from human plasma is described. Hexadecanedionic acid (HDA), which is covalently linked to Sepharose 4B, is used for depletion of SA from plasma samples. With the platform described herein, very specific and high-throughput depletion of albumin from human plasma is achieved.

 2006 American Chemical Society

Materials. Bio-Rad Micro Bio-Spin chromatography columns (732-6204) ware used in SA depletion experiments. Onedimensional PAGE was run with a Bio-Rad Protean II minigel system (Bio-Rad, Mu¨nchen, Germany). Two-dimensional PAGE was run with IPG strips, IPGphor, and Hoefer IsoDalt system (Amersham-Biosciences, Freiburg, Germany). Protein spots were excised from 2D PAGE gels by an Investigator ProPic picking robot (Genomic Solutions Ltd., Huntington, U.K.). Ingel digestions were performed by an Investigator ProGest digestion robot (Genomic Solutions Ltd., Huntington, U.K.). The matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) measurements were carried out on a Bruker Autoflex time-of-flight mass spectrometer (Bruker Daltonics, Bremen, Germany). Cibacron Blue resin was purchased from Pierce Biotechnology Inc. (Rockford, IL) as a part of SwellGel Blue albumin removal kit. Preparation of HDA-Sepharose 4B. Suction-dried Sepharose 4B resin (100 g) was transferred to a 1000 mL flask containing 100 mL of 2 M NaOH, 5.0 mL of epichlorohydrine, and 100 mL of deionized water. The flask was rolled on a roller at ambient temperature for 3 h, when a new portion of 20 mL of epichlorohydrine was added and the reaction was left to proceed for another 21 h. The resin was than washed on a Bu ¨ chner funnel with 2 L of deionized water and transferred to a 1000 mL flask containing 200 mL of 1 M NH4OH. The flask Journal of Proteome Research 2006, 5, 3453-3458

3453

Published on Web 11/11/2006

technical notes

Albumin-Depleted Plasma from HDA-Sepharose 4B

was transferred to a roller and rolled at ambient temperature overnight. The resin was than again washed on a Bu ¨ chner funnel, with 2 L of deionized water followed by 500 mL of 50% 2-propanol. The resin was than resuspended in 250 mL of 50% 2-propanol that contained 1.5 g of HDA and 3.6 mL of N-methylmorpholine. Coupling reagent 4-(4,6-dimethoxy-1,3,5triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM, 1.4 g) was added to the reaction mixture as freshly prepared methanol solution in seven equal portions (200 mg of DMT-MM/5 mL of methanol) in 60 min intervals; after the last portion was added, the rolling of the flask was continued for another 17 h. The course of reaction was monitored by the trinitrobenzenesulfonic acid (TNBSA) test for free amino groups. Thereafter, to block free unreacted amino groups, 100 µL of acetic anhydride was added and rolling was continued for another 2 h. The resin was subsequently washed with 2000 mL of 50% 2-propanol and 500 mL of water and resuspended in 200 mL of 5% NaHCO3, and the suspension was transferred to a flask and incubated under shaking at room temperature overnight. Finally, it was washed with 2000 mL of water, resuspended in 30% 2-propanol, and kept at 4 °C until further usage. Acidimetric titration of HDA-Sepharose 4B gave a density of about 4.2 µmol/mL of settled resin. Human Plasma Samples. Human heparin plasma was acquired from a healthy donor. Human blood was obtained by venipuncture and collected into tubes containing heparin sulfate as anticoagulant. The blood was further centrifuged at 1000g for 10 min at room temperature immediately after collection. The resultant plasma was stored in small aliquots at -80 °C. They were thawed only once shortly before used. The protein concentration was determined by BCA assay.9 Albumin Depletion Procedure. Human plasma was diluted with 4 volumes of binding buffer (50 mM Tris-HCl, 200 mM NaCl, 7 M urea, and 0.05% CHAPS, pH 8.0) before use. Prior to use, the HDA-Sepharose 4B suspension was washed with 10 volumes of water and 5 volumes of binding buffer and resuspended in the same buffer to give a 10% suspension. That suspension (1 mL) was placed into a Bio-Rad spin column and the buffer was drained. The outlet of the spin column was then closed and 200 µL of diluted plasma was added. The column was incubated for 30 min on a roller, and subsequently the column was spun at 2000g for 1 min to yield the albumindepleted fraction. For control of material bound to the HDA-Sepharose 4B beads, the column was washed 3 times with 1.5 mL of 50 mM Tris-HCl, pH 7.4. Subsequently, the outlet of the spin column was closed, followed by addition of 400 µL of extraction buffer (125 mM Tris-HCl, 4% SDS, and 20% glycerol, pH 6.8) and incubation at 95 °C for 5 min. The elute obtained was collected by centrifugation for further analysis. One-Dimensional SDS-PAGE. Standard SDS-PAGE discontinuous Laemmli gels10 on 12% separation gels with 4% stacking gels were used. To verify the efficient albumin removal, 1 µg of protein was loaded per gel lane. After electrophoresis, gels were stained with silver according to Shevchenko et al.11 Two-Dimensional SDS-PAGE. First dimension was run on IPG strips, pH range 4-7, and IPGphor system. Separation in the second dimension was performed on 10% separation gels as described by Cahill et al.12 In-Gel Digestions and MALDI-TOF-MS Measurements. Sample preparation and MALDI-TOF-MS measurements were performed as described by in our previous publication.13 Database Searching. For identification of the proteins, the 3454

Journal of Proteome Research • Vol. 5, No. 12, 2006

Figure 1. Determination of capacity of HDA-Sepharose 4B affinity resin: 1D PAGE overview of plasma SA depletion by HDA-Sepharose. Lane 1, molecular weight markers; lane 2, plasma (3 µg of protein); lanes 3-6, SA-depleted samples (1 µg of protein/lane). Increasing amounts of plasma were depleted over 100 µL of HDA-Sepharose 4B: lane 3, 20 µL of plasma; lane 4, 40 µL of plasma; lane 5, 80 µL of plasma; and lane 6, 120 µL of plasma.

peptide masses extracted from the mass spectra were searched against the NCBI nonredundant protein database (www.ncbi.nlm.nih.gov) by use of MASCOT software, version 1.9 (Matrix Science, London; detailed description can be found at www.matrixscience.com) as described previously.14

Results HDA was chosen because it resembles the structure of longchain fatty acids, because it can relatively easy be coupled to primary amino group-containing resins, and because it is available from commercial suppliers. Successful coupling was achieved with amino-Sepharose 4B and DMT-MM15 as coupling agent. To achieve monofunctionalization, HDA was used in about 10-fold excess to the available primary amino group on the resin. The capacity of the resin was tested by performing removal of SA from different volumes of plasma with a constant volume of resin (Figure 1). Increasing volumes of human plasma (from 40 to 120 µL) were incubated with 100 µL of the HDA resin in a column format (see Experimental Section). Proteins that did not bind to the column (the flowthrough fraction) were prepared and examined by 1D PAGE. As assessed by visual inspection, the 100 µL column was able to remove SA from volumes of serum up to 60 µL but became slightly overloaded with 80 µL or more of total plasma. Total protein recovery was checked by the bicinchoninic acid (BCA) protein assay and compared to the recovery obtained with commercial Cibacron Blue resin (Table 1). Overall recovery of proteins in the flowthrough and eluted fractions from our HDA column and Cibacron Blue resin is close to 100% ((10%), but nonspecific retention of low-abundance proteins that are not even evident in the 2D gels cannot be ruled out. To evaluate the potential difference in specificity between the commercial Cibacron Blue resin and the HDA-Sepharose 4B, human serum was subjected to albumin depletion in parallel on HDA-Sepharose 4B and Cibacron Blue resins. The flowthrough fraction and proteins specifically bound by the resins were examined by 2D PAGE (Figure 2 for albumindepleted fractions and Figure 3 for resin-bound fractions). After

technical notes

Soskic et al.

Table 1. Total Protein Recovery from HDA-Sepharose 4B and Cibacron Blue Resin fractions

total protein (µg)

fraction of total plasma (%)

plasma albumin-depleted fraction, HDA-Sepharose albumin-depleted fraction, Cibacron blue resin SDS-extracted fraction, HDA-Sepharose SDS-extracted fraction, Cibacron blue resin total recovery, HDA-Sepharose total recovery, Cibacron blue resin

2800 1173.5 872.4 1635.4 1870.2 2808.9 2742.6

100 41.90 31.05 58.41 66.89 100.31 97.94

Figure 2. Comparison of SA plasma depletion methods: 2D PAGE of SA-depleted fraction obtained from HDA-Sepharose 4B and Cibacron Blue resin. (A) HDA-Sepharose 4B-depleted sample; (B) Cibacron Blue resin-depleted sample.

separation of serum proteins on the affinity column, the flowthrough fraction shows essentially no remaining SA (Figure 2A) while the SA is recovered in the fraction eluted from the column (Figure 3A). In 2D gels, SA comes as a large, poorly resolved, and clearly overloaded spot (Figure 3A,B), and it is accompanied by a few nonalbumin proteins. These accompanying proteins are more prevalent in samples prepared with Cibacron Blue resin, revealing its reduced specificity for albumin in comparison to the HDA-Sepharose 4B. Further identification of the numerous minor spots found in the eluted fraction was achieved by mass spectrometry (Figure 3A). Individual spots excised from the gel were digested with trypsin and analyzed by MALDI-TOF peptide mass fingerprinting. A

Figure 3. Comparison of specificity between SA plasma depletion methods: 2D PAGE of proteins eluted from HDA-Sepharose 4B and Cibacron Blue resin. (A) HDA-Sepharose 4B-bound proteins; (B) Cibacron Blue resin-bound proteins. The identified proteins are listed by their numbers in Table 2.

list of identified proteins is shown in Table 2. All of the spots identified are consistent with their migration in the 2D gel as compared with published data (Swiss 2D PAGE). The data obtained from some spots were insufficient for protein identification. Most of the analyzed proteins belong to abundant nonalbumin proteins from plasma. Two-dimensional gels of plasma and albumin-depleted plasma are shown in Figure 4C,D; a small part of the gels are enlarged in Figure 4 panels A and B, respectively, to demonstrate the improvement of specificity with corresponding protein patterns of 2D gels. Low-abundance proteins that were Journal of Proteome Research • Vol. 5, No. 12, 2006 3455

3456

Journal of Proteome Research • Vol. 5, No. 12, 2006

5.7 5.4 6.4 6.4 6.5 6.0 5.0 5.0 5.2 5.3 5.4 5.6 5.7 6.4 6.4 6.5 6.7 6.9 6.8 5.5

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

150 000 133 000 90 000 78 000 78 000 69 000 56 000 56 000 56 000 53 000 53 000 51 000 51 000 50 000 60 000 54 000 54 000 57 000 53 000 24 000

molecular mass (Da)

gi|66932947 gi|4557485 gi|4557385 gi|4557871 gi|4557871 gi|55669910 gi|1942629 gi|1942629 gi|1942629 gi|455970 gi|455970 gi|223170 gi|223170 gi|27692693 gi|4389275 gi|223002 gi|223002 gi|76826840 gi|17939658 gi|90108666

AccNo (NCBInr)

130 110 164 332 247 347 304 314 277 332 331 100 92 184 106 149 143 127 164 257

PMF score

8.0 × 1.8 × 10-6 1.0 × 10-11 2.7 × 10-24 2.5 × 10-20 3.2 × 10-30 1.1 × 10-25 1.1 × 10-23 1.7 × 10-20 1.3 × 10-28 2.7 × 10-27 8.0 × 10-6 1.1 × 10-4 3.4 × 10-14 1.1 × 10-5 4.4 × 10-9 1.8 × 10-8 1.8 × 10-7 8.0 × 10-12 1.6 × 10-18

10-8

expectation values

6.0 5.5 6.7 7.0 7.0 5.6 5.4 5.4 5.4 5.2 5.2 5.7 5.7 7.3 5.9 7.9 7.9 8.4 6.9 5.3

pI

164 614 122 983 191 827 84 083 84 083 67 154 44 280 44 280 44 280 54 513 54 513 46 823 46 823 48 641 67 988 51 358 51 358 56 577 52 984 28 061

molecular mass (Da)

theoretical

R2-macroglobulin ceruloplasmin [Homo sapiens] complement component 3 precursor; acylation-stimulating protein cleavage product transferrin [H. sapiens] transferrin [H. sapiens] serum albumin [H. sapiens] R1-antitrypsin [H. sapiens] R1-antitrypsin [H. sapiens] R1-antitrypsin [H. sapiens] vitamin D binding protein [H. sapiens] vitamin D binding protein [H. sapiens] fibrinogen γ-chain [H. sapiens] fibrinogen γ-chain [H. sapiens] ALB protein [H. sapiens] human serum albumin, complex with myristic Acid and triiodobenzoic acid fibrinogen -β-chain [H. sapiens] fibrinogen β-chain [H. sapiens] fibrinogen β-chain, preproprotein similar to immunoglobulin heavy constant γ3 chain C, apolipoprotein A-I

description

a All proteins identified after the analysis of spots (Figure 3A) are listed. For each spot, pI, molecular mass, coordinates (in-gel and sequence-based), NCBI Accession Number, peptide mass fingerprint (PMF) score, MASCOT expectation values, and protein name are given. Significance threshold score for PMF was p < 0.05 for all proteins reported.

pI

spot no.

experimental

Table 2. List of Proteins Found upon 2D PAGE of Proteins Eluted from HDA-Sepharose 4Ba

Albumin-Depleted Plasma from HDA-Sepharose 4B

technical notes

technical notes

Soskic et al.

Figure 4. Enhanced spot detection resulting from serum albumin depletion: 2D PAGE of plasma and HDA-Sepharose 4B SA-depleted plasma. (A) Enlarged view of the indicated region of gel C; (B) enlarged view of the indicated region of gel D; (C) total plasma; (D) HDA-Sepharose 4B-depleted plasma.

previously hidden can now be resolved (see boxed regions, Figure 4A,B). It is evident that higher protein loads are now possible and thus access to interesting classes of lowerabundance proteins that now can be analyzed.

Discussion The rapid progress of detection and fractionation technologies for proteomic analyses is providing increasingly sensitive methods for protein profiling of body fluids. In particular, together with appropriate pooling strategies for pattern control,16,17 extensive fractionation even from scarce amounts of samples becomes an option. However, in the serum/plasma proteome the domination of the protein pattern by a small number of highly abundant proteins is obscuring the desired biomarker information.1 Removal of high-abundance proteins from a sample can be beneficial in order to enhance detection of other components of a sample or to increase sensitivity of assays utilizing the sample. Serum and plasma are valuable sources of material for the monitoring of disease markers, but albumin and IgG account for more than 70% of the total protein. Without proper depletion methodologies to specifically remove these proteins, many low-abundance proteins are not detectable with even the most sensitive mass spectrometers. The linear dynamic range of plasma protein concentrations, lies between 1010 and 1012 and makes these samples the most complex and challenging. Here we describe a technique for removing albumin, by far the most abundant serum protein, in a way that is efficient and specific enough to be suitable for use with any type of subsequent proteomic technologies. There are currently two methods for serum albumin removal. One is using immobilized triazine dyes, such as Cibacron Blue,3,4 which has been reported to lack specificity18 and thus may remove interesting lowabundance proteins. It was considered to be a general method for SA removal across all mammalian species. The second

method is using anti-albumin antibodies in immunoaffinity systems is expensive, not of general use, and has potential of introducing proteins from affinity supports into the sample.7,8 Our research involves the examination of alternative highaffinity ligands that overcome the limitations of Cibacron Blue and immunoglobulins. Long-chain fatty acids come as obvious candidates due to their high affinities for SA.19,20 According to published data, it is necessary to have a carboxyl group of the acid in free dissociable form to achieve high affinity; therefore, coupling to the matrix must keep the carboxyl group unengaged. A screening of commercial availability of appropriate fatty acid-like compounds that can easily be coupled to solid supports led to hexadecanedionic acid (HDA). One carboxylic group of HDA was used for coupling through amide bound to the solid matrix while the other was kept free. Among the several primary amino groups containing resins (silica-based, activated glass, Fractogel, and Sepharose 4B) and coupling methods (EDC and DMT-MM) under different experimental conditions (e.g., pH, ionic strength, and detergents), Sepharose 4B and DMT-MM turned out to be the most favorable and economic choice (result of the tests are not shown). Under optimal conditions presented in this paper, the performance of HDA-Sepharose 4B is markedly better than dye-based resins in terms of both the efficiency and specificity of albumin removal. At the same time it is free from disadvantages of immunoaffinity-based resins where high costs, species specificity, and the possibility of sample contamination are limiting factors. The fact that binding and transport of free fatty acids is one of the important functional aspects of SA makes it likely that this method will work with the serum and plasma proteome from different animal species. Some of our preliminary results confirm that notion. This makes the HDASepharose 4B method for SA depletion an even more attractive choice. Journal of Proteome Research • Vol. 5, No. 12, 2006 3457

technical notes

Albumin-Depleted Plasma from HDA-Sepharose 4B

Acknowledgment. This work has been supported by a grant from the European FP6 STREP program (LSHG-CT2005: NEUPROCF, Contract 512044). We thank Dr. Simone Schillo for help with MALDI-TOF measurements and MS data analysis. References (1) Anderson, N. L.; Anderson, N. G. The human plasma proteome: History, character, and diagnostic prospects. Mol. Cell. Proteomics 2002, 1 (11), 845-867. (2) Zolotarjova, N.; Martosella, J.; Nicol, G.; Bailey, J.; Boyes, B. E.; Barrett, W. C. Differences among techniques for high-abundant protein depletion. Proteomics 2005, 5 (13), 3304-3313. (3) Travis, J.; Pannell, R. Selective removal of albumin from plasma by affinity chromatography. Clin. Chim. Acta 1973, 49 (1), 4952. (4) Travis, J.; Bowen, J.; Tewksbury, D.; Johnson, D.; Pannell, R. Isolation of albumin from whole human plasma and fractionation of albumin-depleted plasma. Biochem. J. 1976, 157 (2), 301-306. (5) Raymackers, J.; Daniels, A.; DeBrabandere, V.; Missiaen, C.; Dauwe, M.; Verhaert, P.; Vanmecheien, E.; Meheus, L. Identification of two-dimensionally separated human cerebrospinal fluid proteins by N-terminal sequencing, matrix-assisted laser desorption/ionization-mass spectrometry, nanoliquid chromatographyelectrospray ionization-time of flight-mass spectrometry, and tandem mass spectrometry. Electrophoresis 2000, 21 (11), 22662283. (6) Steel, L. F.; Trotter, M. G.; Nakajima, P. B.; Mattu, T. S.; Gonye, G.; Block, T. Efficient and Specific Removal of Albumin from Human Serum Samples. Mol. Cell. Proteomics 2003, 2 (4), 262270. (7) Hinerfeld, D.; Innamorati, D.; Pirro, J.; Tam, S. W. Serum/Plasma depletion with chicken immunoglobulin Y antibodies for proteomic analysis from multiple Mammalian species. J. Biomol. Tech. 2004, 15 (3), 184-190. (8) Gong, Y.; Li, X.; Yang, B.; Ying, W.; Li, D.; Zhang, Y.; Dai, S.; Cai, Y.; Wang, J.; He, F.; Qian, X. Different Immunoaffinity Fractionation Strategies to Characterize the Human Plasma Proteome. J. Proteome Res. 2006, 5 (6), 1379-1387. (9) Hinson, D. L.; Webber, R. J. Miniaturization of the BCA protein assay. BioTechniques 1988, 6 (14), 16-19. (10) Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970, 227 (5159), 680-685. (11) Shevchenko, A.; Wilm, M.; Vorm, O.; Mann, M. Mass Spectrometric Sequencing of Proteins from Silver-Stained Polyacrylamide Gels. Anal. Chem. 1996, 68 (5), 850-858.

3458

Journal of Proteome Research • Vol. 5, No. 12, 2006

(12) Cahill, M. A.; Wozny, W.; Schwall, G.; Schroer, K.; Holzer, K.; Poznanovic, S.; Hunziger, C.; Vogt, J. A.; Stegmann, W.; Matthies, H.; Schrattenholz, A. Analysis of relative isotopologue abundances for quantitative profiling of complex protein mixtures labelled with the acrylamide/D3-acrylamide alkylation tag system. Rapid Commun. Mass. Spectrom. 2003, 17 (12), 1283-1290. (13) Sommer, S.; Hunzinger, C.; Schillo, S.; Klemm, M.; Biefang-Arndt, K.; Schwall, G.; Putter, S.; Hoelzer, K.; Schroer, K.; Stegmann, W.; Schrattenholz, A. Molecular analysis of homocysteic acid-induced neuronal stress. J. Proteome Res. 2004, 3 (4), 572-581. (14) Hunzinger, C.; Wozny, W.; Schwall, G. P; Poznanovic, S.; Stegmann, W.; Zengerling, H.; Schoepf, R.; Groebe, K.; Cahill, M. A.; Osiewacz, H. D.; Jagemann, N.; Bloch, M.; Dencher, N. A.; Krause, F.; Schrattenholz, A. Comparative profiling of the mammalian mitochondrial proteome: multiple aconitase-2 isoforms including N-formylkynurenine modifications as part of a protein biomarker signature for reactive oxidative species. J. Proteome Res. 2006, 5 (6), 625-633. (15) Kunishima, M.; Kawachi, C.; Hioki, K.; Terao, K.; Tani, S. Formation of carboxamides by direct condensation of carboxylic acids and amines in alcohols using a new alcohol- and water soluble condensing agent: DMT-MM, Tetrahedron 2001, 57 (8), 1551-1558. (16) Schrattenholz, A. Proteomics: How to control highly dynamic patterns of millions of molecules and interpret changes correctly? Drug Discovery TodaysTechnol. 2004, 1 (1), 1-8. (17) Neubauer, H.; Clare, S. E.; Kurek, R.; Fehm, T.; Wallwiener, D.; Sotlar, K.; Nordheim, A.; Wozny, W.; Schwall, G. P.; Poznanovic, S.; Sastri, C.; Hunzinger, C.; Stegmann, W.; Schrattenholz, A.; Cahill, M. A. Breast cancer proteomics by laser capture microdissection, sample pooling, 54-cm IPG IEF, and differential iodine radioisotope detection. Electrophoresis 2006, 27 (9), 1840-1852. (18) Bailey, J.; Zhang, K.; Zolotarjova, N.; Nicol, G.; Szafranski, C. Removing high-abundance proteins from serum. Genet. Eng. News 2003, 23 (19), 32-36. (19) Simard, J. R.; Zunszain, P. A.; Ha, C. E.; Yang, J. S.; Bhagavan, N. V.; Petitpas, I.; Curry, S.; Hamilton, J. A. Locating high-affinity fatty acid-binding sites on albumin by X-ray crystallography and NMR spectroscopy. Proc. Natl. Acad. Sci. U.S.A. 2005, 102 (50), 17958-17963. (20) Zunszain, P. A.; Ghuman, J.; Komatsu, T.; Tsuchida, E.; Curry, S. Crystal structural analysis of human serum albumin complexed with hemin and fatty acid. BMC Struct. Biol. 2003, 3 (6), 1-9.

PR060387Q