Ultradepletion of Human Plasma using Chicken Antibodies: A Proof of

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Ultradepletion of Human Plasma using Chicken Antibodies: A Proof of Concept Study Sock-Hwee Tan, Abidali Mohamedali, Amit Kapur,† and Mark S. Baker* Department of Chemistry and Biomolecular Sciences and Australian Proteome Analysis Facility, Macquarie University, Sydney, NSW, 2109, Australia S Supporting Information *

ABSTRACT: Human plasma arguably represents the most comprehensive version of the human proteome. Despite its immense theoretical discovery potential, plasma has many high and medium abundance proteins that mask low abundance protein disease biomarkers of relevance, making the discovery of novel diagnostic markers particularly difficult. Some form of protein depletion and/or fractionation is essential in order to detect markers of low abundance. Here, we describe a “proof of concept” two-pronged approach to immunodeplete abundant proteins from human plasma. The method, called API (Abundant Protein Immunodepletion), involves the fractionation of plasma using dual ion exchange columns (protein repetitive orthogonal offline fractionation (PROOF)) to simplify the proteome, the production of polyclonal IgY against each fraction and finally using the purified antibodies in a immunodepletion column. We explored the use of this product for immunodepletion of human plasma and identified a total of 165 nonredundant proteins after depletion. Of these, 38 proteins that were not previously identified in nondepleted plasma were now detected. It is envisaged that further optimization of the method as well as its cyclic implementation (by reinjecting depleted plasma into chickens for second round of antibody production) can make this technology highly robust, extremely cost-effective, and ideal for high throughput biomarker discovery. KEYWORDS: ultradepletion, complex protein fractionation, biomarker discovery, immunodepletion, high abundance protein depletion, chicken antibody, shotgun proteomics

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identified include those for Alzheimer’s disease,7 myocardial infarction8 and coronary heart disease.9,10 Given that cancers like lung, colon and breast cancer remain the top few leading causes of all registered deaths globally today,11 the search for clinically useful biomarkers remains the main focus of many biomedical proteomics efforts. Furthermore, despite the potential to identify biomarkers from plasma, the discovery has been very particularly challenging owing to the immense complexity and the wide dynamic range of protein concentration (greater than 10 orders of magnitude)12 found in plasma. Currently, there is no single technology that can resolve the entire plasma proteome. Although current shotgun proteomic approaches have the ability to detect and identify proteins in the femto-attomole range, simultaneous detection of these proteins in the backdrop of the most abundant proteins is simply impossible.13 The reason for this is that high abundant proteins always mask signals from lower abundance proteins in either gel or nongel (MS) based methodologies. Therefore, to comprehensively characterize the plasma proteome, extensive fractionation is inevitable. It has been reported in the litrature that by combining various separation strategies, like multi-

INTRODUCTION Human plasma contains perhaps one of the most complex human-derived proteomes and has been routinely sampled in all healthcare settings for therapeutic and/or diagnostic medicine purposes.1,2 It contains a large variety of proteins including liver-derived proteins (high abundance), tissue leakage proteins (medium abundance) and many types of receptor ligands such as cytokines, chemokines and interleukins (low abundance).2 The diversity of the plasma proteome has the ability to reflect changes in cell function temporally and spatially and most importantly during human disease states. Although high abundant plasma proteins such as the acute phase response proteins (e.g., α1 protease inhibitor and other serpins, serum amyloid A, C-reactive protein, haptoglobin among a myriad of others) could potentially be used as markers of cancer,3 it is often proteins of lower abundance that promise diagnostic outcomes in measurable terms of sensitivity and specificity. For an example, three widely known lower abundance proteins, CA19-9, CA125 and prostate specific antigen (PSA), have at some time all been used to assess disease risk, predict prognosis, monitor response to treatment and disease regression in pancreatic (in addition to AFP and CEA),4 ovarian5 and prostate cancer,6 respectively. Other plasma-derived disease biomarkers that have already been © XXXX American Chemical Society

Received: August 2, 2012

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remove a large number of highly and moderately abundant plasma proteins.

dimensional chromatography, with abundant protein depletion together with analyses through 2DGE and MS, an additional factor of 1 or two logs of separation power can be achieved.14−16 Immunodepletion of high abundance proteins has been widely used as the first step toward plasma proteome analysis and the use of antibody affinity columns for the depletion of high abundance proteins has been found to be highly efficient, specific and reproducible.17 At present, there are three main depletion strategies commercially available: (a) the multiple affinity removal system (MARS) commercialized by Agilent Technologies, (b) the ProteoPrep immunodepletion kit from Sigma and (c) an IgY-based immunoaffinity resins Seppro developed by Geneway Biosciences and SuperMix technology now available through license to Sigma (registered under patent number AU20090035849). There are approximately 210 proteins that have been described to be closely associated with the top six abundant serum proteins18 and therefore depleted with immunoaffinity columns. For instance, albumin is well-known to be particularly “sticky” and has a physiological role as a carrier protein for bilirubin and various sex hormones (e.g., sex hormone binding globulin). As such, immunodepletion of albumin alone could result in the consequential removal of a significant number of low abundance proteins, including potential disease biomarkers. Furthermore, the inherent nature of mammalian antibodies suggests potential cross reactivity and a further pull-down of nonspecific proteins. The use of resins to remove albumin has also been reported to codeplete various low abundance cytokines from human plasma.19 Other limitations of affinity based prefractionation approaches include the high cost of immunodepletion columns made from separate mixed monoclonal or polyclonal antibodies, significant development time,20 their relatively low loading capacities, the short half-lives of columns21 and the difficulty in working up methodologies for the high throughput necessary for statistically robust clinical biomarker studies. Chromatography methods are usually used as a second step in plasma proteome analysis. For example, ion exchange chromatography (IEC)22−24 and size exclusion chromatography (SEC)23 have been employed to reduce plasma complexity prior to MS analysis. Gradiflow technology, a type of 2D liquid enrichment system that uses membrane-based preparative electrophoresis,25,26 has been used as a depletion tool to remove highly abundant proteins from human plasma.27,28 More recently, Fitzgerald and Walsh reported the use of the MicroFlow MF10 to separate plasma by molecular weight and charge under native conditions.29 Despite using multidimensional strategies, the dynamic range in protein concentration of the resulting eluate remains very high and hinders the ability to discover clinically useful biomarkers. Furthermore, the state of the proteins usually does not allow seamless downstream applications in a high throughput fashion. In this study, we evaluate a robust and costefficient immunodepletion strategy, termed Abundant Protein Immunodepletion (API) to drill deeper into the human plasma proteome. This strategy (registered under patent numbers US 2011/008900A1, AU 2002951240) involves prefractionation of human plasma using a tandem dual ion exchange method (protein repetitive orthogonal offline fractionation (PROOF)), immunization of chickens with different plasma fractions, immobilization of purified egg yolk polyclonal antibodies onto solid phase support, combination of these solid phase supports and finally the depletion of unfractionated human plasma to

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MATERIALS AND METHODS

Plasma Sample

The human EDTA plasma samples were obtained from pools of plasma from 30 healthy (15 male, 15 female) anonymous donors generously donated with informed consent by the Australian Red Cross Blood Service, Sydney, NSW, Australia. Protein Repetitive Orthogonal Offline Fractionation (PROOF) of Human Pooled Plasma

A HiLoad 26/10 Q Sepharose HP (Strong Anion Exchange SAX) column and a HiLoad 26/10 SP Sepharose HP (Strong Cation Exchange SCX) column (GE Healthcare, Uppsala, Sweden) were connected in tandem onto a high performance liquid chromatography system (AKTA basic 100 F-HPLC; GE Healthcare) with a valve located between the columns. Both columns were initially washed and equilibrated with 8 column volumes (CV) of buffer A (10 mM tris, 10 mM disodium orthophosphate, pH 7.4). One hundred milliliters of diluted plasma (diluted 1:100 with buffer A) was loaded at a flow rate of 8.0 mL/min initially onto the SAX column to bind anionic proteins. The unbound cationic proteins were then introduced onto the SCX column. Unbound (i.e., uncharged) proteins that did not bind to either SCX or SAX columns were collected and labeled as the flow-through fraction (FT). By judicious use of the valve to bypass selected columns, elution of bound proteins was performed first from the isolated SAX column and subsequently from the SCX column. Elution of proteins from both columns was carried out by stepwise 1 M NaCl gradients in 10 mM Tris, 10 mM disodium orthophosphate, pH 7.4 (buffer B) with steps of 5, 10, 20 and 100% for the SAX column and 5 and 100% for the SCX column. The soluble protein contents of the seven different PROOF human plasma IEC fractions were quantified using the FluoroProfile (Sigma) protein quantification kit with BSA as a standard and stored at −80 °C until used. One-Dimensional Gel Electrophoresis

Approximately 10 μg total protein was separated on a 10% bistris precast gradient gel with MOPS running buffer (Invitrogen, Carlsbad. CA) as per the manufacturer’s instruction. The gels were then fixed, stained overnight with Flamingo Pink (BioRad, Hercules, CA) and imaged on the Typhoon Trio Variable Mode Laser Imager (GE Healthcare, Uppsala, Sweden) with photo multiplier tube (PMT) voltage set to 5 V below saturation of the most intense spot. Two-Dimensional Gel Electrophoresis

Each of the PROOF-fractionated human plasma samples were buffer exchanged with sample solubilization buffer (7 M urea, 2 M thiourea and 4% (w/v) CHAPS) using an Amicon Ultra-15 centrifugal filter unit (5 KDa NMWL) (Millipore). The proteins were then reduced with 5 mM tributyl phosphine (TBP) and alkylated with 10 mM acrylamide and 100 μg protein loaded onto pH 5−8 linear gradient 11 cm ReadyStrip IPG Strips (BioRad). Isoelectric focusing was carried out using a GE Healthcare Ettan IPGPhor2 until a total of 110 kVh was reached. The strips were then equilibrated in an equilibration buffer (6 M urea, 3% (w/v) SDS, 20% (v/v) glycerol, 375 mM Tris-HCl (pH 8.8), 5 mM TBP and 10 mM acrylamide) twice for 10 min with gentle rocking. The strips were then placed on the top of 8−16% Criterion precast gels (BioRad) and run at B

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mM NaCl, pH 8.3) using an Amicon Ultra-15 centrifugal filter unit (3 kDa NMWL). NHS-activated Sepharose 4 fast flow gel was packed into a XK50/30 (GE Healthcare, Uppsala, Sweden) column and washed with 15 column volumes (CV) of ice cold 1 mM HCl. The plasma sample was then added to the NHS activated gel bed and incubated overnight at 4 °C with gentle rocking in the dark. After coupling, nonreactive groups were deactivated with 5 CV of blocking buffer (500 mM ethanolamine, 500 mM NaCl, pH 8.3) for 30 min at room temperature. The plasma protein-NHS gel bed was then washed with 3 CV of Tris buffer (100 mM Tris-HCl, pH 8) followed by 3 CV of acetate buffer (100 mM acetate buffer, 500 mM NaCl, pH 4.0). This process was repeated twice and the gel bed finally equilibrated with 5 CV of PBS, pH 7.4. The IgY solution was diafiltered with three volumes of PBS and applied to the equilibrated plasma-NHS gel bed. Unbound IgYs were washed with 5 CV of PBS, pH 7.4 and collected. The specific antibodies then eluted with 5 CV of elution buffer (100 mM glycine, 500 mM NaCl, pH 2.5) and neutralized immediately with 1.0 M Tris, pH 7.5. Specific IgY was immediately buffer exchanged with 3 volumes of PBS to remove any traces of glycine and the IgY concentration measured at 280 nm using an extinction coefficient for IgY of 1.33.32

10 mA/gel until the bromophenol blue dye ran to the bottom of gels. Gels were fixed and scanned as described above. Western Blotting

Polyclonal antibody binding to various PROOF fraction proteins was assessed using a traditional Western blot method. Separated proteins on the 1DGE were electrophoretically transferred onto 0.45 μm Immobilon-P PVDF membranes (Millipore) using a XCell Blot Modules (Invitrogen) with Towbin buffer30 (192 mM glycine, 25 mM Tris, 20% methanol, pH 8.3) at 25 V for 2 h. After the transfer, the PVDF membranes were immediately incubated in blocking buffer (2% (w/v) skim milk in 0.1% (v/v) Tween-Tris buffered saline (TTBS)) buffer for 1 h at room temperature with gentle shaking. The blots were washed 4−5 times with T-TBS and incubated with primary antibody (purified antibody from eggs of chickens that were inoculated with the seven ion-exchange plasma fractions) in antibody dilution buffer (5% (w/v) skim milk, in T-TBS) at 0.5 μg/mL overnight at 4 °C with gentle shaking. The blots were washed four times (10 min each) with T-TBS, followed by incubation with a rabbit antichicken/turkey antibody conjugated to HRP (Invitrogen) diluted in antibody dilution buffer at 1:20 000 for 1.5 h at room temperature with gentle shaking. After four washes with T-TBS (10 min each), the immunoreactivity was visualized using an Immobilon Western chemiluminescent HRP Substrate (Millipore) on Kodak BioMax films according to the manufacturer’s instructions.

Preparation of Affinity-purified IgY Antibody Column (API)

Immobilisation of specific antiplasma protein purified IgYs onto UltraLink Hydrazide Gel (Pierce) was performed as per manufacturer’s instructions. HPLC purified antiplasma chicken polyclonal IgYs derived from the seven PROOF fractions were mixed in a 1:1:1:1:1:1:1 ratio and the resulting combination of solid phase supports buffer exchanged with 0.1 M sodium phosphate, pH 7.0. The antibodies were then oxidized with sodium meta-periodate (5 mM) and incubated for 15 min at room temperature. The oxidized IgY was further desalted with 100 mM sodium phosphate pH 7.0, using Amicon Ultra-15 centrifugal filter unit (30 kDa NMWL). A 20 mL volume of gel slurry was placed into a disposable column (BioRad) and drained after settling for 15 min and equilibrated with 5 gel bed volumes of 100 mM sodium phosphate, pH 7.0. The oxidized IgY was then added to the gel (∼1 mL of oxidized protein per ml of gel) and incubated overnight at room temperature. The column was then washed with one gel-bed volume of 100 mM sodium phosphate, pH 7.0. The resulting Ultralink-IgY immunodepletion media was then transferred into a Xk26/20 column (GE Healthcare, Uppsala, Sweden) and washed with 5 gel-bed volumes of 1 M NaCl (wash solution) followed by 5 gel bed volumes of PBS containing 0.05% sodium azide.

Chicken Immunization

Thirty-five ISA Brown chickens (21 week old Gallus gallus domesticus) were randomly allocated to be immunized with each of the seven plasma PROOF fractions. Antigen suspensions composed of plasma fractions emulsified with equal volumes of Freund’s incomplete adjuvant (FIA) were used for 4 subsequent immunizations. In brief, 1 mg of each plasma fraction was injected intramuscularly in the thigh at four sites as an initial injection (day 0) and 0.5 mg for each booster dose was given on days 14, 21, and 28. Eggs were collected daily and yolks were stored at −20 °C in 0.01% sodium azide until needed. All animal procedures were performed under protocols approved by the Macquarie University Animal Ethics Committee (2005/012). Purification and Concentration of Subsequent IgY from Eggs and Analysis by Western Blotting

Approximately 6 L of frozen egg yolks were diluted with 9 volumes of distilled water as per Akita and Nakai.31 Diluted egg yolk was homogenized at 600 rpm for 30 min using an electric motor stirrer and then incubated at 4 °C overnight. The aqueous phase was filtered through a high quality 50 kDa. Besides this, the “albumin” band appeared to be more intensely stained F

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alpha-2-macroglobulin, fibrinogen beta chain, fibrinogen gamma chain, Ig alpha-1 chain C region, serotransferrin and serum albumin were found in all fractions (FT, Q and S). This was not surprising given that these proteins are among the most abundant proteins in human plasma. In addition, this provided evidence that the relative abundance of a protein in a mixture did affect antibody responses generated (Supporting Information Table A).

the lower abundance proteins. The 2D gel images of FT, Q3, and Q4 fractions showed the presence of both high and low molecular weight proteins. However, again, the high molecular weight protein spots dominated the gel. Additionally, vertical smears were seen at the acidic end of Q3 and Q4 which implied the presence of proteins with pI less than 5. On the other hand, the gel images of the S1 and S2 fractions showed an equal distribution of both high and low molecular weight proteins. This may result from a significant depletion of albumin in the Q fractions, hence subsequently revealing lower abundance cationic proteins. Furthermore, vertical smears seen at the basic end of S1 and S2 imply the presence of proteins with pI > 8.

API Human Plasma Abundant Protein Ultradepletion

Antibodies generated against the seven PROOF fractions were tested to be active as shown. The antibodies were immobilized onto UltraLink Hydrazide (Pierce) beads for chromatography analysis. The conditions employed to oxidize antibodies played a crucial role in their activity and coupling efficiency. Excessive and prolonged oxidation diminished antibody activity, while insufficient oxidation decreased coupling efficiency. We tested different oxidation conditions and determined that oxidizing IgY antibodies with 5 mM sodium periodate for 15 min was the most effective (data not shown) protocol. For ultradepletion studies, unfractionated human plasma was first passed through a Sigma ProteoPrep anti-HSA/IgG column to ensure removal of as much albumin and immunoglobulin as possible (Figure 5) prior to API. The ProteoPrep column was

Immunoreactivity of Antigen-specific IgY

The efficacy of immune responses against plasma proteins fractions was evaluated by Western blotting. Purified IgY was shown to be immunoreactive against all antigens with which chickens were immunized. As can be seen (Figure 4A), multiple

Figure 4. Western blotting analysis of the seven human plasma fractions acquired from PROOF IEC. (A) The blot was probed using polyclonal antibodies to respective fractions. Lane 1, FT fraction; Lane 2, Q1 fraction; Lane 3, Q2 fraction; Lane 4, Q3 fraction; Lane 5, Q4 fraction; Lane 6, S1 fraction; Lane 7, S2 fraction. (B) Proteins identified by excising bands from the gel that were seen on the 1D blot. All proteins were identified using GPM with a 1% FDR. A total of 69 proteins were identified with only 6 common proteins. Arrows indicate proteins that were more immunogenic.

Figure 5. (Left) Bound (B) and flow through (FT) fractions from the ProteoPrep column to deplete albumin and IgG. (Right) Plasma depleted of serum albumin and IgG which was then subsequently depleted through the API column. Gels were loaded with 10 μg of protein and stained with Flamingo Pink (Bio-Rad).

bands were detected in all lanes with a pattern closely resembling that of the 1DGE patterns for each plasma fraction (Figure 3A). The most notable exception was lane 4 (Q2 fraction), where only one band between 50 kDa and 75 kDa was observed (corresponding to serum albumin). This data was in accordance with the 2D and 1D gel images of the Q2 fraction (Figure 3) whereby “albumin” appeared to be most intensely stained. It was further observed that some proteins appeared more immunogenic than others, such as some in the FT fraction (lane 1) and S2 fraction (lane 7) indicated with arrows. In order to identify proteins in the antigen mixtures that elicited an antibody response, the corresponding bands seen on the 1D blot (Figure 4B) were excised from the gel (Figure 3A), digested using trypsin and subjected to MS analysis. The differences in the nonredundant protein identifications between the fractions were compared. Of the total 69 protein identifications, only six (6) proteins, namely

relatively efficient at pulling down serum albumin and IgG although significant “carryover” was observed. The resulting flowthrough was passed through the API column leading to two distinct fractions; namely, the API flow through and the API bound fraction. Proteins bound to the API column were then eluted under low pH conditions (glycine pH 2.5) and analyzed by 1DGE. A large number of proteins were detected in the API bound fraction, demonstrating the potential for the removal of multiple highly abundant proteins (i.e., ultradepletion). However, depletion efficiency was never seen to reach 100%. The API flow through pattern though did not resemble closely the ProteoPrep flow through plasma sample suggesting a significant depletion (segregation) of the proteome. Evaluation of Peptide IPG-IEF for Protein Identification

Peptide IPG-IEF was chosen as the first-dimension technique for “shotgun” proteomics to evaluate the efficiency of the API G

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Figure 6. (A) Distribution of tryptic peptides throughout BR range pI 3−10 IPG strips. (B) Number of total protein identifications from unfractionated human plasma, API depleted human plasma and API bound plasma proteins. Venn diagrams representing the number of protein identifications compiled from duplicate experiments from BR, NR and MR IPG strips. Proteins identified from (C) unfractionated human plasma showing 132 common proteins, (D) API depleted human plasma showing 120 common proteins, and (E) API bound human plasma showing 115 common proteins.

different IPG strips; broad range (pI 3−10; BR), narrow range (pI 4−7; NR) and micro range (pI 3.5−4.5; MR). Only proteins that met the predefined selection criteria were considered as positively identified. Comparisons between BR, NR and MR strips showed minimal variation in protein identifications (Figure 6B), suggesting the use of more than one IPG strips might not be useful for increasing total protein assignments. It appeared that the BR IPG strip performed optimally for highly complex samples containing large amounts of highly abundant proteins. Meanwhile, MR IPG strips appeared better at separating peptides derived from less complex samples such as depleted human plasma. To determine differences in protein identifications between IPG strips, proteins for each set of IPG strips were compiled and compared (Figure 6C−E). Using a set of stringent selection criteria, a total of 164, 165, and 165 nonredundant proteins were identified from ND, API depleted and API bound human plasma preparations respectively. Of the total, 164 protein identifications found in ND human plasma, 132 proteins (80.5%) were common to all three sets of IPG strips and only one protein (0.6%) was unique to each IPG strip. Similarly, the analysis of API depleted human plasma demonstrated a total of 165 protein identifications with 120 (72%) found in all three

ultradepletion methodology. The distribution of tryptic peptides by isoelectric focusing (IEF) using broad range (pI 3−10; BR) IPG strips was evaluated by calculating the number of peptides per fraction as an average of duplicate experiments for nondepleted and ProteoPrep+API-depleted human plasma (Figure 6A). The peptide distribution throughout the 24 fractions demonstrated 2 and 3 separation peaks for ND and API-depleted human plasma respectively. Generally, over 70% of all peptides were clustered in the acidic to neutral regions (fractions 1−14) of IPG strips. This has been attributed to the tryptic digestion which produces more acidic peptides.39,40 Further analysis revealed that 49% and 31% of peptides were found between pI 3.5−5.0 (fractions 3−8) in ND and APIdepleted human plasma respectively, in line with previous studies.40−42 However, the increased number of acidic peptides in the ND human plasma could possibly suggest that highly abundant proteins are more subject to modifications such as acetylation or deamidation which renders a higher negative charge.39 In the basic end of the pH gradient, there was a large difference in the number of peptides observed between ND human plasma and API-depleted plasma. The total number of nonredundant protein identifications from human plasma preparations was assessed using three H

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Figure 7. Quantitative distribution of proteins identified from API bound human plasma. Normalized spectral abundance factors (NSAF) were calculated for each protein. The immunoglobulins occupied over 50% the total protein identifications with ALB at 23.3%. As was expected from immune response studies, some medium and low abundance proteins were also captured albeit in low concentrations.

Comparison of ND and API-depleted Human Plasma for Total Nonredundant Protein Identifications

IPG strips and no unique proteins found in BR IPG strip (Figure 6D). A total of 165 protein identifications were found in API bound human plasma with 115 (69%) identifed in all three IPG strips with 30 proteins (18%) common to at least two IPG strips. These results show the use of BR IPG strips was superfluous. However, using a combination of NR and MR IPG strips could increase overall protein identifications by approximately 20% particularly for depleted human plasma.

Shotgun peptide IPG IEF proteomics identified a total of 164 and 165 nonredundant proteins from nondepleted and ProteoPrep+API depleted human plasma correspondingly (Supporting Information Tables C and D) . Pie chart representations of the distribution of the top 20 proteins found in ND and API-depleted human plasma are shown in Figures 8A−B. Despite the removal of many abundant proteins, the allocation of proteins in both samples remained similar, with albumin occupying a large proportion of total protein mass. Despite this problematic “bleed through” issue found with all immunodepletion strategies, the presence of complement factor B and complement factor H in the top 20 proteins in the ProteoPrep+API-depleted human plasma demonstrated how previously “hidden” proteins could be enriched and identified post ultradepletion. In order to compare differences in nonredundant protein identifications between the two plasma samples, proteins for each set of samples were compiled and compared (Figure 8C). Of these protein identifications, 127 proteins were common to both samples, while only 37 and 38 proteins were unique to unfractionated and API depleted human plasma respectively. In the unfractionated human plasma, it was noted that 22 out of the 37 proteins were not detectable in the ProteoPrep+APIbound plasma. It is possible that these proteins are removed completely along with serum albumin and IgG by the ProteoPrep HSA/IgG depletion, and therefore were not present in ProteoPrep+API-depleted plasma sample. In addition, these proteins usually have a low total peptide count (20 abundant proteins is required to significantly increase the depth of plasma proteomics, as well as to discover potentially lower abundance disease-specific plasma biomarkers. API is an ideal ultradepletion column if used in conjunction with another depletion systems like MARS-14 because it has the ability to capture as much as 165 high and medium abundance proteins from human plasma. Like has been previously observed, depletion efficiencies of 100% were not achieved in this study as trace amount of target proteins were still be present in flow through fractions as well as being bound to “sticky” proteins. For example, albumin peptides also remained detectable after MARS-7/1454 and IgY12 depletion.15 Due to the diversity of antibodies in the API column, it may be useful after a first round of depletion. One of the major concerns of immunodepletion is the concomitant removal of untargeted proteins. Studies directed to address this issue show that nontargeted removal of proteins from depletion columns occurs and that the number of nonspecific proteins binding varies. For example, 3859 and 460 proteins have been found to bind nonspecifically to Seppro IgY column. Similar studies showed an additional removal of 24, 20, and 19 proteins nonspecifically from MARS-6, MARS-7 and MARS-14 depletions columns respectively.54,61 Recently, Yadav et al. have identified 45, 53, and 61 proteins nonspecifically removed by MARS-6, MARS-14 and ProteoPrep20 depletion systems, respectively.62 Therefore, it is important to analyze the bound fractions to avoid missing valuable information. In this study, a total of 234 nonredundant proteins were identified from the API-bound and API-depleted plasma samples. Since the API column is composed of a mixture of antibodies generated against seven fractions human plasma, the immunoreactivity of these proteins in the host might depend on many factors such as protein abundance and immunogenicity. In some instances, antibodies might be limiting for some proteins and yet plentiful for others. Further optimization of antibody mixing ratios of the API column may further enhance the depletion of high- and medium-abundance proteins. Additionally, a cyclic implementation of this technology by reinjecting depleted plasma into chickens for second round of antibody production will allow human biomarker discovery projects to see “far more deeply” into the human plasma proteome. This “depth” is essential for the discovery of efficacious and reproducible markers of disease states and with this technology, the potential to carry out high throughput screening and analysis for biomarkers is significantly improved.

In order to analyze the extent of depletion, we used peptide IPG-IEF as an alternative to strong cation exchange chromatography for separation of peptides prior to mass spectrometry analysis to determine the efficacy of fractionation of proteins eluted or bound to the API column. The use of peptide IPG-IEF has proven to be efficacious for the analysis of complex proteomes, for an example, Joel et al. reported 1549 protein identifications from rat liver membrane preparations.39 Similarly, a study undertaken by McQuade et al. identified 2292 proteins from human embryonic stem cell membrane enriched fraction33 using this method. Eriksson et al. also reported a total of 3704 protein identification from lung cancer microsomal preparations.42 Apart from membrane preparations, studies using peptide IPG-IEF of top 7 and top 14 immunodepleted plasma preparations have resulted in the identification of 216 and 208 human plasma proteins respectively.54 In our study, we identified an average of 150 nonredundant proteins from human plasma preparations (i.e., nondepleted and API depleted) and showed that using more than one IPG strip (narrow range, pI 4−7, and micro range, pI 3.5−4.5) increased the number of total protein identifications by approximately 20%. The novel immunoaffinity strategy described here allows binding/removal of 165 proteins, including immunoglobulins, albumin, alpha-2-macroglobulin, fibrinogen, apolipoprotein A-I and haptoglobin. These proteins were among the most abundant proteins suggesting that relative abundance likely plays a role in chicken IgY antibody responses. However, some exceptions may occur. For an example, although alpha-1antitrypsin is present in the plasma at high concentrations 0.9− 1.75g/L,55 it has a relatively low NSAF value of 0.6% in the API-bound plasma sample. This demonstrated that immunogenicity possibly has an influence on the antibody responses as this serpin is known to be highly conserved (both sequence and structurally) among viruses, plants, birds and mammals.56 Ninety four bound proteins were also identified in the APIdepleted human plasma. High levels of protein bleed through are not surprising given that the API column contains an undefined combination of polyclonal chicken antibodies generated against undefined mixtures of human plasma proteins. A similar immunodepletion system using a combination of IgY14 and SuperMix columns has successfully captured 57 proteins,15 of which 52 matched with proteins identified in this study. Analysis of API depleted human plasma detected a total of 165 nonredundant proteins from all three IPG strips. Unfortunately, 77% (127) of these proteins were also identified in the ND plasma preparation. A majority of these overlapping proteins however, did not show noticeable differences in NSAF values. This implies that the amount of antibodies available for these proteins might be limiting. Nevertheless, the API column did show some level of depletion of highly abundant proteins like hemoglobin subunit beta, complement C3, alpha-1-acid glycoprotein 1, alpha-1-acid glycoprotein 2 and alpha-2macroglobulin. The distribution of proteins in human plasma changed dramatically pre- and postdepletion. For example, partial removal of immunoglobulins from human plasma increased the NSAF values of serotransferrin from 2.5 to 7.6%. Additionally, previously unidentified proteins such as complement factor B, complement factor H and protein AMBP were now able to be detected and were found in the new top 20 protein list. In all, 31 proteins were uniquely identified post API ultradepletion of which 14 are not found in the human plasma L

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CONCLUSIONS The present paper introduces a highly reproducible tandem separation methodology (PROOF) to fractionate any complex protein samples. It also provides the proof of principle for an immunodepletion strategy, which uses chicken egg yolk antibodies raised against complex mixtures of human plasma proteins. Chicken has again proven to be an ideal host to produce enormous amount of antibodies at relatively low cost. The combination of existing immunodepletion columns, such as MARS-14, API column coupled with the advances in mass spectrometry instrumentation such as AB SCIEX TripleTOF 5600 system, would significantly increase the discovery of low copy number biomarkers that exist in human plasma.

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ASSOCIATED CONTENT

S Supporting Information *

Table A. Nonredundant list of proteins that elicited an IgY response. Number of protein identification in FT, Q1, Q2, Q3, Q4, S1 and S2 fractions were 12, 9, 4, 23, 33, 24 and 41, respectively. Out of these proteins, only six were identified in all fractions. Table B. Nonredundant list of proteins captured by the API column; 165 proteins were identified using 3 different IPG strips; pH 3.5−4.5 (NR), pH 4−7 (MR) and pH 3−10 (BR). Table C. Nonredundant list of proteins identified from Nondepleted plasma; 164 proteins were identified using 3 different IPG strips; pH 3.5−4.5 (NR), pH 4−7 (MR) and pH 3−10 (BR). Table D. Nonredundant list of proteins identification from ProteoPrep followed by API depleted plasma; 166 proteins were identified using 3 different IPG strips; pH 3.5−4.5 (NR), pH 4−7 (MR) and pH 3−10 (BR). Table E. Proteins that were found not to have significantly different NSAF values in nondepleted and API depleted plasma. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +61 2 9850 8211. Fax: +61 2 9850 8313. E-mail: mark. [email protected]. Present Address †

Wollongong Hospital, South Eastern Sydney and Illawarra Area Health Service, Wollongong, 2500 NSW, Australia Notes

The authors declare no competing financial interest.

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REFERENCES

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