Proteomic Analysis of Human Serum by Two-Dimensional Differential

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Proteomic Analysis of Human Serum by Two-Dimensional Differential Gel Electrophoresis after Depletion of High-Abundant Proteins Brett A. Chromy,† Arlene D. Gonzales,† Julie Perkins, Megan W. Choi, Michele H. Corzett, Brian C. Chang, Christopher H. Corzett, and Sandra L. McCutchen-Maloney* Biology and Biotechnology Research Program, Biodefense Division, Lawrence Livermore National Laboratory, 7000 East Avenue, L-452, Livermore, California 94550 Received April 13, 2004

Two-dimensional differential gel electrophoresis (2-D DIGE) was used to analyze human serum following the removal of albumin and five other high-abundant serum proteins. After protein removal, serum was analyzed by SDS-PAGE as a preliminary screen, and significant differences between four highabundant protein removal methods were observed. Antibody-based albumin removal and highabundant protein removal methods were found to be efficient and specific. To further characterize serum after protein removal, 2-D DIGE was employed, enabling multiplexed analysis of serum through the use of three fluorescent protein dyes. Comparison between crude serum and serum after removal of high-abundant proteins clearly illustrates an increase in the number of lower abundant protein spots observed. Approximately 850 protein spots were detected in crude serum whereas over 1500 protein spots were exposed following removal of six high-abundant proteins, representing a 76% increase in protein spot detection. Several proteins that showed a 2-fold increase in intensity after depletion of high-abundant proteins, as well as proteins that were depleted during abundant protein removal methods, were further characterized by mass spectrometry. This series of experiments demonstrates that high-abundant protein removal, combined with 2-D DIGE, is a practical approach for enriching and characterizing lower abundant proteins in human serum. Consequently, this methodology offers advances in proteomic characterization, and therefore, in the identification of biomarkers from human serum. Keywords: 2-D DIGE • serum • albumin removal • high-abundant proteins • biomarkers • proteomics

Introduction Proteomic characterization of human serum for identification of disease-specific biomarkers promises to be a powerful diagnostic tool for defining the onset, progression and prognosis of human diseases.1 Serum provides a rich sample for diagnostic analyses because of the expression and release of proteins (potential biomarkers) into the bloodstream in response to specific physiological states such as bacterial infections, cancer, and Alzheimer’s disease to name a few. Therefore, serum offers a medium to define differential expression characteristics specific to those physiological states. While the easily obtainable nature and the high protein content of serum deem it a valuable specimen for biomarker determination,2 there are still numerous hurdles to overcome when analyzing human serum, one of the most complex proteomes known. Significantly, 10 or more orders of magnitude in concentration separate albumin, the most abundant protein in serum, from the scarcest of proteins.1 In addition, many serum proteins * To whom correspondence should be addressed: Sandra L. McCutchenMaloney. Tel.: 925-423-5065. Fax: 925-422-2282. E-mail: [email protected]. † Authors contributed equally to this work.

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have similar molecular weight and overall charge, making protein separation difficult. Therefore, biomarkers for disease, that may be present at extremely low concentrations in serum, could be hidden by more abundant proteins possessing similar biophysical characteristics.3 As such, the reliable proteomic characterization of serum and identification of biomarkers would be dramatically improved by reducing the complexity of the serum proteome. In the case of human serum, albumin constitutes anywhere from 55% to 75% of the total protein content and consequently, is an overwhelming signal in separation and detection assays.1 Even following albumin removal, serum still remains a complex protein mixture with the inclusion of five other high-abundant proteinssIgG, IgA, transferrin, haptoglobin, and antitrypsin. Collectively, these six abundant proteins constitute over 85% of the human serum proteome.4 Therefore, removal of these proteins represents a fundamental improvement toward characterization of the serum proteome. Classically, Cibracon Blue5 and protein A/G chromatography methods6 have been used to deplete serum of albumin and the immunoglobulins. However, an increasing number of 10.1021/pr049921p CCC: $27.50

 2004 American Chemical Society

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2-D DIGE Analysis of Human Serum

methods for the removal of other high-abundant proteins from serum are becoming commercially available, making serum analysis a more routine laboratory procedure.4 Albumin is a carrier/transport protein that binds other important entities in blood;7 and consequently, the removal of albumin from a serum sample could also remove other physiologically important species. Therefore, in choosing a method for highabundant protein removal, the efficacy of albumin removal as well as the amount of additional proteins that are nonspecifically removed should be considered. Several reports in the literature have addressed the issue of serum complexity and proteomic analyses. For example, Pieper and co-workers8 used immuno-affinity subtraction chromatography to remove 10 proteins from human blood plasma. Following protein depletion, Coomasie blue stained 2-dimensional electrophoresis (2-DE) gels revealed approximately 650 protein spots compared with only 220 spots visible in a sample of crude serum. Silver staining of the protein-depleted sample revealed an even larger number, 950 spots. Chan and coworkers9 used an affinity spin tube filter method to remove albumin and IgG to enrich for low-abundant cancer biomarkers in serum. Over 250 potential biomarkers for breast cancer were identified in this study. Finally, Steel and co-workers10 also used an immuno-affinity resin to remove albumin and IgG from human serum samples in order to simplify the serum proteome. These examples relied on traditional 2-DE, and while 2-DE is limited in the ability to detect low abundant proteins, it remains a valuable method for the separation of a complex mixture of proteins.11 Notably, the introduction of fluorescent dyes for 2-DE analysis12,13 has offered many improvements over traditional silver staining and Coomassie blue staining techniques. For example, fluorescent stains such as SYPRO ruby, Deep purple, and LightningFast,12,14 which are used for postelectrophoretic staining and bind noncovalently, provide a greater dynamic range of protein detection. Also of note, 2-D differential gel electrophoresis (DIGE) was developed for multiplex proteomic analysis based on the spectrally resolvable fluorescent dyes Cy2, Cy3, and Cy5.15 The Cy dyes are covalently attached to proteins via lysine residues prior to electrophoresis. Since the dyes are molecular weight and charge matched, and the positive charges originally associated with the free lysine residues in the protein sample are replaced with quaternary amino groups in the dye molecules, labeled proteins migrate to the correct isoelectric point and vary only slightly in size from their original state. As such, up to three proteomic samples can be electrophoresed on the same gel alleviating the difficulties of gel-to-gel comparisons. This multiplex capability improves the reliability of comparative experiments by increasing the statistical significance of differential expression. In addition, the Cy2 dye can be used to label a pooled, internal standard allowing more consistent and accurate gel-to-gel comparisons in larger sample sets.15 There have been numerous examples of the application of 2-D DIGE technology to detect proteomic differences,16-23 including an example of 2-D DIGE used to examine plasma glycoproteins24 after lectin affinity purification of glycosylated proteins. Since albumin is not glycosylated, this effectively removed albumin from plasma. However, only a subset of the plasma proteins is glycosylated and this study detected 737 protein spots in human plasma. In the study presented here, 2-D DIGE was utilized to characterize human serum after removal of six abundant serum

proteins. Four different approaches were taken to remove the high-abundant proteins from serum. After which, serum, before and after depletion of high-abundant proteins, was analyzed by SDS-PAGE as a preliminary screen for protein removal efficacy. Finally, 2-D DIGE was used to examine serum, after successful removal of high-abundant proteins, defining over 1500 protein spots. This result represents a significant improvement in proteomic characterization of human serum.

Materials and Methods Serum Samples. Blood samples were taken from two healthy volunteers with informed consent under Institutional Review Board approval from Lawrence Livermore National Laboratory. Samples were collected in 2.5 mL BD vacutainer SST glass serum tubes (BD Biosciences). The blood samples were stored upright at 4 °C until they were spun at 2500 rpm at 4 °C for 30 min. The separated serum was aliquoted and stored at -80 °C until further analysis. Albumin Removal. The serum samples were returned to 4 °C. Using a VivaScience Albumin Removal Kit (Q Mini H), a 50 µL aliquot of serum was diluted in 250 µL albumin buffer (kit reagent) before loading onto the spin column. The protocol was followed as described in the Vivapure instruction manual. Flow-through contained the albumin-depleted serum fraction. The column wash fraction was combined with this flowthrough fraction. The combined albumin-depleted sample was not concentrated. Bound albumin was eluted from the column with 25 mM Tris, 1 M NaCl, pH 8.0 buffer. Using a Millipore Montage Albumin Deplete Kit, 50 µL of serum was diluted with 150 µL of equilibration buffer (kit reagent) before application to the column. The protocol was followed as described in the Montage Albumin Deplete Kit user guide. Flow-through contained the albumin-depleted serum fraction. Albumin was recovered from the column with the 40 mM Tris acetate pH 7.0, 10% sodium dodecyl sulfate. Using an ABI affinity POROS anti-albumin 0.2 mL column, 7 µL of serum was diluted 10-fold with PBS, pH 7.2, to final volume of 70 µL and applied to the column. The equilibration was carried out according to the kit instruction manual. The column was manually washed four times with 100 µL of PBS, pH 7.2, and the albumin-depleted fraction was collected. Albumin was eluted from the column with 1 mL of 12 mM HCl. Using an Agilent Multiple Affinity Removal System, 30 µL of serum was diluted 5-fold in Agilent buffer A, filtered through a 0.22 µm spin tube at 16 000 rpm at room temperature, and injected onto a 4.6 × 100 mm column at room temperature with a flow of 0.5 mL/min Agilent buffer A on a Shimadzu VP HPLC system. Serum devoid of high-abundant proteins was collected between 2 and 4 min. After 10 min with buffer A, the flow was changed to Agilent buffer B at 1 mL/min. The six bound proteins were eluted from the column between 13 and 14.5 min. After 17 min, the column was regenerated with buffer A. Of the four protein removal methods used, each manufacturer recommended different loading volumes as dictated by the capacity of the columns. These differences also allow qualitative comparison of depletion efficacy by subsequent SDS-PAGE analysis. Protein concentrations were used to adjust amounts used in the following 2-D DIGE experiments. SDS-PAGE Analysis. Crude serum, serum depleted of albumin, and the captured albumin fraction (2.5 µg or 5 µg) (Millipore, Vivascience and ABI columns) were run on a 10% Tricine mini gel (Invitrogen) for 105 min at 90 V with TrisJournal of Proteome Research • Vol. 3, No. 6, 2004 1121

research articles trycine buffer (Invitrogen). Crude serum, serum depleted of six abundant proteins, and the captured six abundant proteins (5 µg) (Agilent column) were run on a 4-12% Bis-Tris gel for 90 min at 150 V. All gels were fixed with 40% methanol, 7% acetic acid overnight and then stained with SYPRO ruby (Bio-Rad) for 4 h, followed by destaining with 10% methanol/7% acetic acid for 1 h. Gel images were obtained with a Typhoon Imager 9410. 2-DE SYPRO Ruby Stained Gels. Crude serum and serum depleted of albumin (25 µg) (ABI column) were separated on 7 cm 3-10 nL IPG strips (Amersham). Rehydration buffer consisted of 7 M urea, 2 M thiourea, 4% CHAPS, 1% Pharmalyte and 1.2% destreak (Amersham). Prior to SDS-PAGE, IPG strips were equilibrated with a dithiothreitol (10 mg/mL) SDS equilibration solution followed by a treatment with iodoacetamide (25 mg/mL) SDS equilibration solution as described in the Amersham Ettan DIGE protocol. The first dimension IPG strips were run on an Amersham IPGphor until 8610 Vh was reached. The IPG strips were washed with SDS-PAGE running buffer. Second dimension PAGE followed using 10% Bis-Tris gels (Invitrogen) was run at a constant 150 V with MES buffer until the bromophenol blue dye front reached the end of the gels. Gels were run in triplicate and stained with SYPRO ruby (BioRad) overnight after fixing in 10% methanol, 7% acetic acid for 30 min. The gels were destained for 30 min (10% methanol, 7% acetic acid) and rinsed in water prior to visualization with an AlphaInnotech CCD camera with the appropriate SYPRO ruby filters. Precision prestained standards (Bio-Rad) were used as molecular weight markers. Protein Labeling for 2-D DIGE. Serum samples were cleaned prior to labeling using a 2-D Cleanup Kit (Amersham). The protein pellets were resuspended in ice-cold 20 mM Tris, 7 M Urea, 2 M thiourea, 4% CHAPS (Anatrace) pH 8.5 buffer. 40-50 µg of protein samples were labeled with excess Cy dyes. 50 µg was used in the case of albumin-depleted serum, and either 40 µg or 50 µg was used for serum depleted of the six abundant proteins. Two amounts of protein were initially used for the sample depleted of the six abundant proteins in order to optimize detection of protein spots and gel running conditions. When comparing spot numbers between crude serum, albumin depleted serum, and serum depleted of the six high-abundant proteins, 50 µg was used for each sample. Crude serum was labeled with 400 pmol Cy3. Serum depleted of albumin (ABI column) was labeled with 400 pmol Cy5. A mixture of crude serum and albumin-depleted serum was labeled with 400 pmol of Cy2 for the pooled standard. The fraction containing the six abundant proteins removed from serum (Agilent column) was labeled with 200 pmol of Cy3. Serum depleted of the six abundant proteins (Agilent column) was labeled with 200 pmol Cy5. IsoElectric focusing (IEF) pI protein standards (SERVA) were labeled with 200 pmol Cy2. All reaction mixtures were left for 30 min in the dark on ice. The labeling reactions were terminated by the addition of 1 µL of 10 mM lysine. 2-D DIGE. The labeled extracts were loaded on 24 cm 3-10NL IPG strips (Amersham). Rehydration buffer consisted of 7 M urea, 2 M thiourea, 4% CHAPS, 1% Pharmalyte and 1.2% destreak (Amersham). The first dimension IPG strips were run on an Amersham IPGphor until 65860 Vh was reached. Prior to SDS-PAGE, IPG strips were equilibrated with a dithiothreitol (DTT) (10 mg/mL) SDS equilibration solution followed by treatment with iodoacetamide (25 mg/mL) SDS equilibration solution as described in the Amersham Ettan DIGE protocol. 1122

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The IPG strips were washed with SDS-PAGE running buffer. 12.5% acrylamide gels cast using low-fluorescent glass were used for the second dimension PAGE and the gels were run on an Amersham Ettan Dalt 6 at 2.5 W per gel for 30 min followed by 100 W until the bromophenol blue dye front reached the end of the gels. Gels were run in triplicate. 2-DE Image Analysis. Gels were scanned with the Amersham Typhoon Imager 9410. Gel images were analyzed using Amersham DeCyder 5.0 software using the automated spot detection algorithm of the DIA (Differential In-gel Analysis) module. For multiplex analysis, the Cy2 dye was excited at 490 nm and emission spectra obtained at 510 nm, the Cy3 dye was excited at 550 nm and emission spectra obtained at 570 nm, and the Cy5 dye was excited at 650 nm and emission spectra obtained at 670 nm. Protein Digestion and Mass Spectrometry. Protein spot digestion and mass spectrometry were performed by Proteomic Research Services (PRS, Ann Arbor, MI). Differentially expressed protein spots were subjected to robotic in-gel digestion using trypsin (ProGest) following reduction with DTT and alkylation with iodoacetamide. A portion of the resulting digest supernatant was used for matrix assisted laser ionization desorptionmass spectrometry (MALDI-MS) analysis. Spotting was performed robotically (ProMS) with ZipTips; peptides were eluted from the C18 material with matrix (R-cyano 4-hydroxy cinnamic acid) in 60% acetonitrile, 0.2% TFA. MALDI-MS data was acquired on an Applied Biosystems Voyager DE-STR instrument and the observed m/z values were submitted to ProFound for peptide mass fingerprint searching using the NCBInr database. Those samples that proved inconclusive following MALDI-MS were analyzed by LC/MS/MS on a Micromass Q-Tof2 using a 75 µm C18 column at a flow-rate of 200 nL/min. The MS/MS data were used for database search using MASCOT. See Table 1 for protein identification and MS methods used.

Results and Discussion The approach used to deplete serum of high-abundant proteins for subsequent proteomic 2-D DIGE analysis is outlined in Scheme 1. While our preliminary use of Cibracon Blue and protein A/G purification methods showed modest protein removal for albumin and IgG, the protein removal was not sufficient for subsequent protein analyses as high-abundant proteins were still present. Therefore, crude serum was first subjected to one of three alternative albumin removal systems, and a high-abundant protein removal system. Two of the albumin removal kits utilized ion exchange-based techniques. One of the albumin removal kits and the six abundant protein removal kit used antibody-based affinity methods. Following high-abundant protein removal from serum, the serum samples were subjected to SDS-PAGE as a preliminary screen for efficacy and specificity of removal methods. Serum, effectively depleted of high-abundant proteins, was then characterized by 2-D DIGE. Serum SDS-PAGE Analysis After Depletion of HighAbundant Proteins. After using the two ion exchange albumin removal methods, serum depleted of albumin clearly still contained a relatively large amount of albumin (Figure 1, Lanes 4 and 6). Also, the albumin fraction removed with the ion exchange columns (Lanes 3 and 5) contained several protein bands in addition to albumin with over 20 protein bands clearly visible in each of the albumin fractions. The presence of these additional proteins may be a consequence of nonspecific retention on the ion exchange columns during separation. However, specific binding of proteins to albumin may also

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2-D DIGE Analysis of Human Serum Table 1. Proteins Identified in High-abundant Protein Depletion Fraction and in Serum after High-abundant Protein Depletion spot no.

protein name

accession no.

MS method

1a 1b 1c 1d 1e 1f 1g 1h 2a 2b 2c 2d 3a 3b 4a 4b 5 6a 6b 6c 7a 7b 7c 8 9 10 11 12 13 14 15 16 17 18 19

serum albumin precursor serum albumin precursor serum albumin precursor serum albumin precursor serum albumin precursor serum albumin precursor serum albumin precursor serum albumin precursor serotransferrin precursor serotransferrin precursor serotransferrin precursor serotransferrin precursor IgA heavy chain constant region IgA heavy chain constant region IgG heavy chain constant region IgG heavy chain constant region Ig κ light chain VLJ R-1-antitrypsin precursor R-1-antitrypsin precursor R-1-antitrypsin Haptoglobin precursor Haptoglobin precursor Haptoglobin precursor H factor 1 (complement) complement factor B precursor R-1B-glycoprotein precursor vitamin D-binding protein hemopexin R-2-glycoprotein 1, zinc complement component 3 complement component C4Aγ AMBP protein precursor serum amyloid component apolipoprotein A-I precursor transthyretin

IPI00022434 IPI00022434 IPI00022434 IPI00022434 IPI00022434 IPI00022434 IPI00022434 IPI00022434 IPI00022463 IPI00022463 IPI00022463 IPI00022463 IPI00336074 IPI00336074 IPI00382937 IPI00382937 IPI00419424 IPI00305457 IPI00305457 IPI00305457 IPI00019571 IPI00019571 IPI00019571 IPI00029739 IPI00019591 IPI00022895 IPI00298853 IPI00022488 IPI00166729 IPI00164623 IPI00375506 IPI00022426 IPI00022391 IPI00021841 IPI00022432

MALDI MALDI MALDI MALDI LC/MS/MS MALDI MALDI MALDI MALDI LC/MS/MS MALDI LC/MS/MS LC/MS/MS LC/MS/MS MALDI MALDI LC/MS/MS MALDI MALDI MALDI MALDI LC/MS/MS LC/MS/MS MALDI LC/MS/MS MALDI MALDI LC/MS/MS MALDI LC/MS/MS LC/MS/MS LC/MS/MS MALDI MALDI MALDI

Proteins 1-7 identified in the high-abundant fraction. Proteins 8-19 identified in serum after high-abundant protein depletion.

be a contributing factor, as albumin is well-known as a carrier and transport protein in serum.7 These results are consistent with other published studies that also report large numbers of proteins carried over in albumin fractions.9,25-27 Significantly, serum with albumin removed using the antibody-based albumin removal column was essentially devoid of albumin (Figure 1, Lane 8), and the albumin fraction contained predominantly albumin (Lane 9), as determined by band intensity. The removal of six high-abundant proteins from serum using an antibody-based approach was comparable to antibodybased albumin removal, and in addition, effectively removed five other high-abundant proteins, IgG, IgA, transferrin, haptoglobin, and antitrypsin. Serum after high-abundant protein removal (Figure 1, Lane 11) shows clear depletion of six protein bands. The high-abundant proteins removed from serum are shown in Lane 12. There were 10 detectable bands in this sample, of which all have been shown to originate from the six abundant proteins in serum as determined by mass spectrometry. Identification of the additional protein bands revealed fragments or oligomers of the high-abundant proteins. While nonspecific removal of proteins in addition to the highabundant proteins is possible, this was not detected in our studies based on the proteins identified. These results clearly illustrate the advantages of using specific, antibody-based methods for the removal of high-abundant proteins from serum.

Scheme 1. Diagram of High-Abundant Protein Depletion and 2-D DIGE Analysis of Seruma

a High-abundant proteins were removed from serum using four commercially available methods. Serum following highabundant protein depletion was examined by SDS-PAGE as a preliminary screen for efficacy and specificity of protein removal. Serum effectively depleted of albumin, serum effectively depleted of six high-abundant proteins, and crude serum were characterized using 2-D DIGE.

2-DE and SYPRO Ruby Staining. To further characterize the effect of antibody-based albumin removal on serum, crude serum and serum depleted of albumin were separated by 2-DE and visualized by SYPRO ruby staining. The number of individual proteins visible following albumin removal (Figure 2b) was increased, particularly in the 50-75 kDa range, where in crude serum (Figure 2a), the proteins were masked by the overwhelming albumin signal. However, when comparing these individual SYPRO ruby stained gels, difficulties associated with the comparative analysis of individual 2-D gels were encountered. Migration of the spots along the pH gradient was not consistent between different gels. In addition, gel-to-gel variations in the first dimension and second dimension also decreased the ability to reliably compare serum samples. Even using commercially manufactured gels, some differences in protein migration along the molecular weight gradient were detected. Finally, the post-electrophoretic staining of the gels resulted in spot diffusion within the gels, additionally complicating proteomic comparisons. 2-D DIGE Analysis of Serum After Albumin Removal. To alleviate the comparative difficulties between 2-DE gels, 2-D DIGE was employed to analyze serum after albumin removal. Since the antibody-based albumin removal method was most effective in this study, this sample was compared with a crude serum sample. Figure 3a shows the 2-D DIGE image of crude serum. Figure 3b shows the 2-D DIGE image of serum after albumin removal. To quantitatively compare the two samples, Journal of Proteome Research • Vol. 3, No. 6, 2004 1123

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Figure 1. SDS-PAGE of serum samples before and after high-abundant protein removal. Lane 1: Mark12 protein marker. Lane 2: Crude serum. Lane 3: Albumin fraction (Millipore albumin removal kit). Lane 4: Albumin-depleted serum (Millipore albumin removal kit). Lane 5: Albumin fraction (Vivascience albumin removal kit). Lane 6: Albumin-depleted serum (Vivascience albumin removal kit). Lane 7: Crude serum. Lane 8: Albumin-depleted serum (ABI albumin removal kit). Lane 9: Albumin fraction (ABI albumin removal kit). Lane 10: Crude serum. Lane 11: Serum depleted of high-abundant proteins (Agilent multiple affinity removal column). Lane 12: High-abundant protein fraction (Agilent multiple affinity removal column). Approximately 10 µg of protein was loaded in lanes 3 and 10; approximately 5 µg of protein was loaded in lanes 2, 4, 5, and 6; and approximately 2.5 µg of protein was loaded in lanes 7, 8, 9, 11, and 12. Different amounts of protein were loaded in order to visualize the high-abundant proteins and any carryover of additional nonspecific proteins during protein removal. The high-abundant proteins identified after removal from serum are labeled to the right, and molecular weights of the Mark12 standards are shown to the left.

Figure 2. 2-DE with SYPRO ruby staining of crude serum and serum depleted of albumin. (a) 25 µg of crude serum and (b) 25 µg of serum after albumin removal (ABI antibody-based removal) separated by 2-DE on 10% Bis-Tris gels are shown. Gels were stained with SYPRO ruby. The large, dark albumin protein spot (a) was effectively removed (b).

the gel images were evaluated using the DIA module of DeCyder software, which normalizes the protein spot ratios between two images based on the Cy2 labeled pooled sample.23 Because the pooled sample consisted of crude serum and serum depleted of albumin and was the same in each gel, this experiment was designed to enable direct spot matching and comparisons between multiple gels. The 2-D DIGE comparison of serum before and after albumin removal showed a significant difference in the number and the intensity of protein spots detected, indicating that albumin removal exposed numerous lower abundant proteins that were not detected in the presence of albumin. As such, serum, with and without albumin, represent significantly different proteomic samples, presenting a unique challenge for the DeCyder software, which was designed to detect proteomic differences in ‘like’ samples. Therefore, in this experiment, even though samples were separated by 2-D DIGE in multiplex format (three different samples per gel), samples labeled with the different Cy dyes were not co-detected, but rather, were obtained individually using DeCyder. Notably, the Cy2 dye is used in 2-D DIGE experiments to allow gel-to-gel comparisons, rather than 1124

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sample-to-sample comparisons. Future studies comparing clinical serum samples, where all samples have been depleted of the six high-abundant proteins, will utilize the pooled standard for normalization of gel-to-gel comparison and will benefit from methods described here. From the gel analysis, an average of 866 protein spots (C. V. ) 2.4%, triplicate gels) in crude serum were detected by 2-D DIGE. Significantly, serum after albumin removal exposed an average of 1229 protein spots (C. V. ) 3.2%, triplicate gels), representing a 42% increase in protein detection capability due solely to albumin removal. An overlay of the gel images for crude serum and serum depleted of albumin shown in Figure 3c, illustrates that albumin removal allowed the detection of over 350 lower abundant protein spots that were not detected in the presence of albumin. 2-D DIGE Analysis of Serum After Removal of Six HighAbundant Proteins. To characterize serum after removal of albumin, IgG, IgA, transferrin, haptoglobin, and antitrypsin, the 2-D DIGE experimental design was adjusted based on the observation that serum with and without albumin must be analyzed individually by DeCyder. Figure 4a shows the 2-D DIGE image of the six high-abundant proteins removed from serum, and Figure 4b shows the 2-D DIGE image of serum after high-abundant protein removal. The green protein spots in the image overlay shown in Figure 4c are IEF pI protein standards included in this gel. The number of protein spots for each sample was determined using DeCyder software, and the differences between serum before and after high-abundant protein removal were considerable. An average of 1527 protein spots (C. V. ) 4.4%, triplicate gels) were detected in serum after the removal of the high-abundant proteins, representing a 76% increase in protein detection capability with over 660 additional lower abundant protein spots detected. The increased number of proteins found in this study demonstrates that 2-D DIGE, in combination with removal of abundant proteins to reduce the complexity of serum, represents an advance in proteomic characterization of serum. As a consequence of the effects seen in comparing significantly different proteomes (discussed above for albumin removal), the internal pooled standard was not used for the

2-D DIGE Analysis of Human Serum

Figure 3. 2-D DIGE of crude serum and serum depleted of albumin. (a) 50 µg of crude serum, gel image generated from excitation of Cy3 dye. (b) 50 µg of albumin-depleted serum (ABI antibody-based removal), gel image generated from excitation of Cy5 dye. (c) Image overlay of crude (Cy3) and albumindepleted (Cy5) images. Blue spots present in panel (c) represent proteins that are enhanced as a consequence of the removal of albumin. Red spots present in panel (c) correspond to albumin spots.

high-abundant protein removal experiment. This experiment focused on evaluating efficacy of high-abundant protein removal in serum analysis, and although the pooled standard was eliminated, it remains an important feature of 2-D DIGE and will be used in future comparative proteomic studies focused on patient-to-patient variability and on serum biomarker identification. Identification of Proteins. To confirm the identities of the proteins depleted using the antibody-based high-abundant protein removal system, protein spots were excised from gels

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Figure 4. 2-D DIGE of serum after removal of high-abundant proteins. (a) 40 µg of fraction containing the six high-abundant proteins, gel image generated from excitation of Cy3 dye. (b) 40 µg of serum after removal of high-abundant proteins (Agilent antibody-based removal), gel image generated from excitation of Cy5 dye. (c) Image generated from overlaying three images: six high-abundant proteins (red), serum after removal of highabundant proteins (blue), and the IEF pI protein standards (green). The three samples show very little overlap based on distinct color of protein spots.

and subsequently identified by mass spectrometry and database search. Protein identities are shown on a 2-DE gel image of crude serum in Figure 5 and listed in Table 1. These identities confirm that the six high-abundant proteins were removed using the antibody-based removal method (Figure 5a). Further, after examining all other visible proteins bands on the SDSPAGE gel of in the fraction containing the six high-abundant proteins, only those six high-abundant proteins were identified (Figure 1). Although it is possible that some nonspecific protein Journal of Proteome Research • Vol. 3, No. 6, 2004 1125

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Conclusions Proteomic results described in this study represent significant progress in the characterization of human serum. Enhancement of lower abundant proteins in serum by removal of the overwhelming signal from high-abundant proteins increased protein detection capabilities by 76%, as determined by the total number of proteins spots detected by 2-D DIGE. The results presented highlight two antibody-based protein removal methods, which were more effective and specific for high-abundant protein removal than two ion-exchange methods also evaluated. 2-D DIGE analysis of human serum after high-abundant protein removal demonstrated a significant improvement in detection of serum proteins. Serum after albumin removal revealed over 1200 protein spots, and serum after removal of six high-abundant proteins exposed over 1500 protein spots. Methods described here for the removal of highabundant proteins, followed by 2-D DIGE proteomic analysis, strengthen the ability to characterize human serum, and consequently should contribute to the potential to define serum biomarkers for early detection of diseases as well as for monitoring disease progression and prognosissthe ultimate goal of serum proteomics.

Acknowledgment. The authors thank Rod Balhorn for the use of his Shimadtzu HPLC system. This work was funded by the Department of Energy (Chemical and Biological National Security Program), and the Department of Homeland Security (Biological Countermeasures Program) and Lawrence Livermore National Laboratory (Laboratory Directed Research and Development Program). This work was performed under the auspices of the U.S. Department of Energy by University of California Lawrence Livermore National Laboratory under contract No. W-7405-Eng-48. (UCRL-JRNL-202515). Figure 5. Proteins identified by mass spectrometry. 2-DE gel images of the depleted six high-abundant protein fraction (a), spots 1-7; and serum after depletion of the six high-abundant proteins (b), spots 8-19. (a) 50 µg of high-abundant proteins removed using the antibody-based removal system were identified by MS. 1: Albumin; 2: Transferrin; 3: IgA; 4: IgG heavy chain; 5: Ig light chain; 6: Antitrypsin; 7: Haptoglobin. (b) 50 µg of serum of after depletion of high-abundant protein showing a sample set of proteins with at least a 2-fold increase in intensity following high-abundant protein removal that were identified by MS. 8: Complement factor H; 9: Complement factor B precursor; 10: R-1-B-glycoprotein; 11: Vitamin D-binding protein; 12: Hemopexin; 13: R-2-glycoprotein 1; 14: Complement factor 3 precursor; 15: Complement C4Aγ; 16: AMBP protein precursor; 17: Serum amyloid protein P component (SAP); 18: ApoA-I; 19: Transthyretin. See Table 1 for more information regarding protein identification.

depletion occurs, this was not observed in our study. Additionally, a sample set of proteins that were shown to have at least a 2-fold increase in intensity following high-abundant protein removal were identified using MS and are shown in Figure 5b, of which several have been previously defined as biomarkers for diseases including Vitamin D binding protein as a biomarker for prostate cancer, haptoglobin as a marker for lung cancer, and serum amyloid component and transthyretin as biomarkers for neurodegeneration.28-31 The ability to detect and identify more proteins in human serum after the removal of high-abundant proteins is an important step toward enabling improved biomarker discovery, and methods described here address an important limitation in serum proteomics. 1126

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