Multicolumn Separation Platform for Simultaneous Depletion and

Multicolumn Separation Platform for Simultaneous Depletion and Prefractionation Prior to 2-DE for Facilitating In-Depth Serum Proteomics Profiling Yazen Jmeian and Ziad El Rassi* Department of Chemistry, Oklahoma State University, Stillwater, Oklahoma 74078-3071 Received May 5, 2009

In this report, we describe an integrated fluidic platform composed of tandem affinity columns for the depletion of high-abundance proteins from human serum and on-line fractionation/concentration of medium- and low-abundance proteins by tandem immobilized metal-ion affinity chromatography (IMAC) columns and reversed phase (RP) column for in-depth proteomics analysis. The depletion columns were based on monolithic polymethacrylate with surface immobilized protein A, protein G′, and antibodies for depleting the top 8 high-abundance proteins. The IMAC fractionation/concentration columns consisted of monolithic stationary phases with surface bound iminodiacetic acid (IDA) chelated with Zn2+, Ni2+ and Cu2+, while the RP column was packed with nonpolar polymer beads. The integrated multicolumn fluidic platform was very effective in reducing simultaneously both the dynamic range differences among the protein constituents of serum and the complexity of the proteomics samples, thus, facilitating the in-depth proteomics analysis by 2-DE followed by MALDI-TOF and LC-MS/MS. In fact, the number of detected spots was ∼1450 using SYPRO fluorescent stain from which 384 spots were subsequently detected by Coomassie Blue. Since the investigation was simply a proof of concept, 295 proteins were readily identified in some selected spots by MALDI-TOF and LC-MS/MS. Keywords: Multidimensional LC • Affinity LC • 2-DE • Immobilized Metal Ion Affinity Chromatography • Proteomics

1. Introduction Depletion of high-abundance proteins is required for indepth serum proteomics.1-3 In fact, depleting the top 8 highabundance proteins removes ∼85% of serum protein total mass, thus, reducing the dynamic range differences between serum proteins.4 However, even after depleting the top 8 highabundance proteins, serum samples are still complex due to the wide dynamic range between the medium-abundance and low-abundance proteins as well as the very large number of low-abundance proteins.5,6 It is well-known that the depletion process usually results in dilution.7,8 Potential protein biomarkers are usually present at low concentrations and the depletion process will result in further dilution. Thus, a concentration step is usually required.3 In addition, serum fractionation following the depletion of high-abundance proteins is required to reduce the complexity of the serum proteins. New developments in the analytical and preparative methods that increase the proteomic depth have been reviewed recently.3,9-11 For example, the prefractionation of serum and plasma proteins by reversed-phase chromatography (RPC) has been described in a few studies.12-14 In one study, plasma proteins were depleted from 14 high-abundance proteins and simultaneously desalted and fractionated on a macroporous * To whom correspondence should be addressed. Professor Ziad El Rassi, Department of Chemistry, Oklahoma State University, Stillwater, OK 74075. Tel.: (405) 744-5931. Fax: (405) 744-1235. E-mail: [email protected].

4592 Journal of Proteome Research 2009, 8, 4592–4603 Published on Web 08/11/2009

reversed-phase C18 column.13 Prefractionation of glycoproteins by lectin affinity was also described.2,15,16 For example, albumin and IgG depleted and nondepleted plasma samples were prefractionated on agarose-based multilectin affinity columns, including concanavalin A (Con A), wheat germ agglutinin (WGA) and jacalin.2 Faca et al.1 employed an orthogonal 2D chromatographic system based on anion exchange and RPC. Stalder et al.17 described a 2D approach for the fractionation of depleted serum, where proteins were first fractionated according to their pI values by Off-Gel IEF, followed by the RP-fractionation of each pI region. Proteins collected from the RP-column were digested and further analyzed by LC-MS/MS. A proteomic strategy where the proteins of depleted serum were separated by free-flow electrophoresis (FFE) into different pI zones followed by the rapid RP separation of each zone was described by Moritz el al.18 Also, a novel multidimensional fractionation strategy, known as protein array pixelation, was reported.6,19 On the basis of the above overview, although significant advances have been made in protein depletion and fractionation strategies, further progress is required to reduce the complexity of the analytical proteomics problem and in turn facilitate in-depth proteomics analysis. Thus, the need for selective fractionation/concentration steps for medium- and low-abundance proteins subsequent to selective depletion of high-abundance proteins. Our investigation addressed this need by designing a multicolumn fluidic platform in which 10.1021/pr900399q CCC: $40.75

 2009 American Chemical Society

Multicolumn Separation Platform multi fractionation is performed on-line with depletion using affinity interactions. In two recent studies,8,20-23 we have introduced tandem affinity columns based on protein A, protein G′ and antibodies for the depletion of high-abundance proteins from human serum and the on-line capturing/concentration of mediumand low-abundance proteins by reversed phase column used in tandem with depletion columns. In the present investigation, we have expanded the system to include other affinity columns, namely, immobilized metal-ion affinity chromatography (IMAC) for the selective capturing and fractionation of medium- and low-abundance proteins. Porath and co-workers introduced IMAC as a powerful fractionation technique for serum proteins in 1975.24 IMAC is based on protein interaction with a transition metal (e.g., Cu2+, Zn2+, Ni2+, etc.) bound to a chelator such as iminodiacetic acid (IDA) immobilized on the surface of a suitable support such as agarose, silica, and so forth.25 In general, it was found that surface imidazolyl, thiol and indolyl groups are the main protein side chains to interact with the immobilized Cu2+, Ni2+ or Zn2+.26,27 Later, it was also shown by Sulkowski and collaborators that proteins may be fractionated according to the surface histidine (His) available for interaction with the chelated metal.28,29 In principle, IMAC of protein on IDA-Cu2+, IDA-Ni2+ or IDA-Zn2+ can be considered as His affinity chromatography.28-30 That is, (i) one His group at the protein surface will result in its retention on IDA-Cu2+, (ii) at least two His residues available on the protein surface are required for the retention on IDA-Ni2+, (iii) two vicinal His (i.e., the two His are located on an R-helical segment separated by two or three amino acids) are required for the retention on IDA-Zn2+, and (iv) the presence of more than two His residues on the protein surface will result in its retention on IDA-Zn2+ 28. In addition to His, the presence of tryptophan and cysteine may contribute to the protein retention on the immobilized metal affinity column. However, it was found that the presence of tryptophan on the protein surface will not change the previous His pattern significantly.30 In addition, tryptophan is rarely found on the protein surface.31 Also, the presence of a free thiol group on the protein surface may scavenge a transition metal and this will not adversely affect the His affinity chromatography on IMAC.30 Besides this wide range of selectivity offered by the nature of immobilized metal, IMAC proved very useful in the fractionation of membrane proteins, for example, the human erythrocyte sialoglycoproteins, the glycophorins.32 As will be shown in this report, IMAC was an efficient and selective fractionation approach when used in an integrated fluidic platform in tandem with an RP column and on-line with tandem affinity-based depletion columns. This integrated fluidic platform was very instrumental in reducing both the dynamic range differences between serum proteins and the complexity of the remaining proteins in a single chromatographic run. One important feature of the platform is its ability for on-line concentration since multiple injections can be performed before the accumulated medium- and low-abundance proteins on the IMAC and RP columns are eluted in a small volume, thus, increasing the concentration of the selectively captured low- and medium-abundance proteins.

2. Experimental Section 2.1. Instrumentation. The liquid chromatograph was assembled from an XTS Micro-LC binary pump system with either a 100 µL or a 1 mL loop from Micro-Tech Scientific (Vista,

research articles CA), a solvent delivery system Model CM3500, two metering pumps Model III CM from Milton Roy, LDC division (Riviera Beach, FL), and a Model 200 UV-vis variable wavelength detector from Linear Instruments (Reno, NV). Chromatograms were recorded with PowerChrom software version 2.5.4 from eDAQ (Denistone East, NSW, Australia). The first dimension of the two-dimensional (2-DE) gel electrophoresis experiments were performed on a Multiphor II IEF system from GE Healthcare (Uppsala, Sweden), while the second dimension was performed on PROTEAN II XL module for 18.3 × 19.3 cm gels from Bio-Rad Laboratories (Hercules, CA). Fluorescent gel images were taken with Typhoon Trio Plus from GE Healthcare (Uppsala, Sweden). All mass spectra were obtained using a Voyager DE PRO MALDITOF mass spectrometer (PerSeptive Biosystems, Foster City, CA) and a hybrid LTQ-Orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA). 2.2. Reagents and Materials. Pooled human serum was purchased from Innovative Research (Southfield, MI). Protein A (from Staphylococcus aureus), protein G′ (from Streptococcus sp.), the IgG fraction of anti-human albumin (rabbit), the IgG fraction of anti-human haptoglobin (rabbit), the IgG fraction anti-human R2-macroglobulin (rabbit), anti-human R1-antitrypsin fractionated antiserum (rabbit), anti-human transferrin fractionated antiserum (goat), R-cyano-4-hydroxycinnamic acid and iodoacetamide were purchased from Sigma Chemical Co. (St. Louis, MO). Glycidyl methacrylate (GMA), ethylene glycol dimethacrylate (EDMA), 3-(trimethoxysilyl)propyl methacrylate, 2,2′-azobis(isobutyronitrile) (AIBN) and 1-dodecanol were purchased from Aldrich Co. (Milwaukee, WI). Cyclohexanol, HPLC grade acetone, HPLC grade acetonitrile (ACN), zinc sulfate, cupric chloride, nickelous nitrate and sodium dodecyl sulfate (SDS) were obtained from Fisher Scientific (Fair Lawn, NJ). Glycine, tris (hydroxymethyl)-aminomethane (Tris), acrylamide, bromophenol blue, Bio-Safe Coomassie, SYPRO ruby protein gel stain, ReadStrip IPG strip 17 cm pH 4-7, Bio-Lyte 3/10, dithiothreitol (DTT), and 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS) were from Bio-Rad Laboratories (Hercules, CA). Iminodiacetic acid (IDA) was a gift from W. R. Grace (Nashua, NH). Porcine sequencing grade modified trypsin was purchased from Promega (Madison, WI). Trisglycine gels 10-20% Tris-HCl (18.3 cm W × 19.3 cm L) 1 mm thickness were purchased from Jule Inc. (Milford, CT). Poly(styrene/divinylbenzene) reversed-phase (RP) medium with particle size of 20 µm and pore size of 300 Å (PLRP-S, 20 µm, 300 Å) was purchased from Polymer Laboratories (Amherst, MA). 2.3. Affinity Columns and Reversed-Phase Column Preparation. In situ polymerization of the monolithic separation media and the subsequent protein immobilization onto the surface of the monoliths of protein A, protein G′ and polyclonal antibodies to yield affinity columns were performed according to previously published work.8,20-23 In summary, a well mixed and degassed polymerization mixture of 18% GMA, 12% EDMA, 59.5% cyclohexanol and 10.5% dodecanol containing 1.0% (w/ w) AIBN with respect to the monomers was introduced into a 25.0 cm × 4.6 mm i.d. stainless steel column that functions as a mold. The column ends were plugged and the column was heated at 50 °C for 24 h. The resulting monolithic column was washed extensively with ACN and then with water, followed by either (i) 0.12 M HNO3 and heated at 80 °C for 3 h in order to form a diol monolithic surface or (ii) 0.9 M IDA in 2.0 M potassium carbonate and heated at 80 °C for 24 h to form an Journal of Proteome Research • Vol. 8, No. 10, 2009 4593

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IDA-modified surface. Both the diol-column and IDA-column were then rinsed with H2O. The modified monolithic support was transferred from the 25.0 cm column to the shorter columns (10, 5, or 3 cm) by connecting the two columns with a 1/4 in.-union and running water through the columns at a flow rate of 3.0 mL/min until the modified monolithic support is transferred. The immobilization of protein A, protein G′ and antibodies is published elsewhere.8 To immobilize the metal on the IDA-monolithic surface, 10 column vol. of 5.0 mg/mL metal solution was pushed through the column. The columns were washed with 10 column vol. of water, 5 column vol. of loading mobile phase and 5 column vol. of eluting mobile phase (see section 2.4.2). The RP column was prepared by dry packing a 3 cm × 4.6 mm i.d. stainless steel column as described previously.8 2.4. Chromatographic Conditions. 2.4.1. Albumin Depletion. Albumin depletion was performed as described in our previous work.8 A longer anti-HSA column (100 mm × 4.6 mm i.d.) was used for the depletion to increase the throughput of the depletion. A total of 100 µL of 1:5 diluted serum was injected onto the column that was previously equilibrated with 10 bed vol. of the binding mobile phase made of 0.05 M Na2HPO4, 0.05 M NaCl, pH 7.4. The pass-through (i.e., albumin depleted fraction) was collected, and thereafter, the mobile phase was changed to 0.05 M NaH2PO4, pH 2.2, to strip off the retained HSA from the column. The column was equilibrated with 10 column vol. of loading mobile phase to prepare it for the next injection. The experiments were conducted at ambient temperature at a flow rate of 0.8 mL/min and the baseline was monitored at λ ) 280 nm and peak areas were recorded. 2.4.2. Simultaneous Second Stage Depletion and Protein Fractionation. The simultaneous second stage depletion and protein fractionation was achieved by injecting the albumindepleted sample onto a series of 3.0 cm × 4.6 mm i.d. depletion columns (connected in the order: protein G′, protein A, antihuman R1-antitrypsin, anti-human transferrin, anti-human haptoglobin and anti-human R2-macroglobulin) that are connected to a series of 4.60 mm i.d. fractionation columns (connected by the following length and order: 5.0 cm, IDAZn2+; 5.0 cm, IDA-Ni2+; 5.0 cm, IDA-Cu2+; and 3.0 cm RPcolumn) through a 2-position switching valve (labeled SV) and a dual-stem 3-way valve (number 1) as shown in Figure 1. The RP column was first washed with 10 bed vol. of water to remove ACN in which the RP column was initially stored or eluted. In one position (first position) of the switching valve (SV), the flow passes through the tandem affinity depletion columns as well as the tandem fractionation columns. In the second position of the switching valve, the mobile phase flows through the tandem column directly to the detector bypassing the fractionation affinity columns. Also, dual-stem 3-way valves (numbered from 2 to 5) from SSI (State College, PA) were inserted in the system as shown in Figure 1 to control the direction of the flow. These 3-way valves have 3 positions. The mobile phase that enters the 3-way valve will exit from the top tubing when the 3-way valve is in position 1. In position 2, the mobile phase that enters the 3-way valve will exit from both the top and the bottom tubing. In this position, the top and the bottom exits are connected. That is, if the entrance tubing is blocked, a flow that enters from the top tubing will exit from the bottom tubing and vice versa. When the 3-way valve is in position 3, the flow that enters the valve will exit from the bottom tubing. When the 3-way valves (1-5) are in position 1 and the 2-position switching valve (SV) is in position 1, the mobile 4594

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phase passes through IDA-Zn , IDA-Ni2+, IDA-Cu2+ and the RP-column, consecutively. The system, including both the depletion and the fractionation tandem columns, was first equilibrated with 10 column vol. of the 0.05 M Na2HPO4, 0.05 M NaCl, pH 7.4, and the 2-position switching valve and the 3-way valves were in position 1. Thereafter, 0.9 mL of the albumin-depleted serum was injected onto the system at a flow rate of 0.5 mL/min. After 15.0 min of washing with the binding mobile phase, the 2-position switching valve (SV) was then switched to the second position, the 3-way valve number 1 was switched to position 3 and the mobile phase passing through the affinity columns was changed to the eluting buffer made of 0.05 M Na2HPO4, pH 2.2, at 0.5 mL/min for 15 min to desorb the bound proteins from the depletion columns. After that, the mobile phase was switched back to the binding mobile phase for 15 min followed by repositioning the valves labeled SV and 1 to the first position at exactly the same flow rate (see Figure 1). The continuous depletion/fractionation cycle was repeated 10 times in order to accumulate sufficient amount of proteins on each of the fractionation tandem columns (i.e., IDA-Zn2+, IDA-Ni2+, IDACu2+ and RP columns). The 3-way valve number 1 is kept at position 3 all the time during the elution of the retained proteins for the IMAC/RP columns. To elute the retained proteins from the IDA-Zn2+ column, the 3-way valve number 2 was in position 1, the 3-way valve number 3 was switched to position 2 and the other 3-way valves numbers 4 and 5 were switched to position 3. The column was first washed with 0.05 M Na2HPO4, 0.05 M NaCl, pH 7.0, for 5.0 min and then with 0.05 M Na2HPO4, 0.05 M NaCl, 100 mM imidazole, pH 7.0, to elute the retained proteins at 0.5 mL/min. In this position, the flow will pass through the IDA-Zn2+ column and bypass the other IMAC/RP columns. Figure 2A is a typical chromatogram of the collected fraction from the IDA-Zn2+ column. The retained fraction was collected and stored for further experiments. To elute the retained proteins from the IDA-Ni2+ column, the 3-way valves numbers 2 and 5 were in position 3, while the 3-way valves numbers 3 and 4 were switched to position 1 and 2, respectively. In this position, the flow bypasses IDA-Zn2+ column to reach the IDA-Ni2+ column. The effluent of the IDA-Ni2+ column bypasses the other columns and reaches the detector. The same chromatographic conditions were used to elute and collect the retained proteins and a typical chromatogram is shown in Figure 2B. To elute the retained proteins from the IDA-Cu2+ column, the 3-way valves numbers 2 and 3 were in position 3, while the 3-way valve number 4 was in position 1. Also, the 3-way valve number 5 was switched to position 2. In this position, the flow bypasses both IDA-Zn2+ and IDA-Ni2+ columns. The effluent of the IDACu2+ column bypasses the RP-column and reaches the detector. The same chromatographic conditions were used to elute and collect the retained proteins and a typical chromatogram is shown in Figure 2C. Finally, to collect the retained protein from the RP-column, the 3-way valves numbers 2, 3, and 4 were in position 3, while the 3-way valve number 5 was in position 1. The RP-column was first washed with water for 5 min to remove the salt followed by 80:20 (v/v) ACN/H2O at 0.1% TFA to elute the retained proteins. The retained proteins from the RP column were collected, evaporated to dryness with a SpeedVac from Savant Instruments, Inc. (Holbrook, NY) and stored at - 20 °C for 2-DE experiments. 2.5. Two-Dimensional Gel Electrophoresis (2-DE). Collected fractions from the IMAC columns were inserted in

Multicolumn Separation Platform

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Figure 1. Chromatographic setup for the depletion of high-abundance proteins and subsequent on-line fractionation/concentration of medium- and low-abundance proteins. First, the 2-position switching valve (SV) and the 3-way valve number 1 are in the first position, which ensures that the binding mobile phase (0.05 M Na2HPO4, 0.05 M NaCl, pH 7.4) flows through the tandem columns and the IMAC/RP columns. Also, the 3-way valves (2-5) are in position 1. In this step, the sample is injected onto the columns to deplete high-abundance proteins and fractionate/concentrate the medium- and low-abundance proteins on the IMAC/RP columns. In a second step, the valve (SV) is switched to the second position to allow the eluting mobile phase (0.05 M NaH2PO4, pH 2.2) to flow through the tandem affinity columns and the detector while bypassing the IMAC/RP columns. The first two steps are repeated 10 times to accumulate sufficient amount of the low- and medium-abundance proteins on the IMAC/RP columns. The 3-way valve number 1 is kept at position 3 all the time during the elution of the retained proteins from the IMAC/RP columns. To elute the retained proteins from the IDA-Zn2+ column, the 3-way valve number 3 was switched to position 2 and the other 3-way valves numbers 4 and 5 were switched to position 3. The column was first washed with 0.05 M Na2HPO4, 0.05 M NaCl, pH 7.0, for 5.0 min and then with 0.05 M Na2HPO4, 0.05 M NaCl, 100 mM imidazole, pH 7.0, to elute the retained proteins at 0.5 mL/min. In this position, the flow will pass through the IDAZn2+ column and bypass the other columns. To elute the retained proteins from the IDA-Ni2+ column, the 3-way valves numbers 2 and 5 were in position 3, while the 3-way valves numbers 3 and 4 were switched to position 1 and 2, respectively. In this position the flow bypass IDA-Zn2+ column to reach the IDA-Ni2+. The outlet of the IDA-Ni2+ column bypasses the other columns and reaches the detector. The same chromatographic conditions as in the case of IDA-Zn2+ column were used to collect the retained proteins on the IDA-Ni2+. To elute the retained proteins from the IDA-Cu2+ column, the 3-way valves numbers 2 and 3 were in position 3, while the 3-way valve number 4 was in position 1. Also the 3-way valve number 5 was switched to position 2. In this position, the flow bypasses both IDA-Zn2+ and IDA-Ni2+ columns. The outlet of the IDA-Cu2+ column bypasses the RP-column and reaches the detector. Again, the same chromatographic conditions as in the case of IDA-Zn2+ and ID-Ni2+ columns were used to collect the retained proteins from IDA-Cu2+ column. Finally, to collect the retained proteins from the RP-column, the 3-way valves numbers 2, 3, and 4 were in position 3, while the 3-way valve number 5 was in position 1. The RP-column was first washed with water for 5 min to remove the salt followed by 80:20 (v/v) ACN/H2O at 0.1% TFA to elute the retained proteins.

Sectra/Por dialysis bags from Spectrum Laboratories, Inc. (Houston, TX) and dialyzed against water according to the manufacturer’s recommendation at 4 °C for 24 h. The dialyzed samples were evaporated to dryness and stored at -20 °C for 2-DE experiments. The samples were redissolved in 300 µL of the rehydration solution made of 7 M urea, 2 M thiourea, 4% (w/v) CHAPS, and 0.8% (w/v) Bio-Lyte 3/10. The first-dimension, second-dimension, staining, protein digestion and MALDITOF procedures were as described previously.8 Thereafter, the tryptic digest of some selected spots were analyzed by LC-MS/ MS as described in the next section. In addition, the remaining of those selected tryptic digests were combined with the digests of all excised spots after 2DE separation of the effluents of each column (i.e., pooled) and analyzed by LC-MS/MS. 2.6. LC-MS/MS Methodology. Samples were analyzed on a hybrid LTQ-Orbitrap mass spectrometer from Thermo Fisher Scientific coupled with a New Objectives PV-550 nanoelectrospray ion source and an Eksigent NanoLC-2D chromatography system. Peptides were analyzed by trapping on a 2.5 cm ProteoPrep II precolumn (New Objective) and analytical sepa-

ration on a 10 cm × 75 µm i.d. fused silica column packed in house with Magic C18 AQ, terminated with an integral fused silica emitter pulled in house. Peptides were eluted using a 5-40% ACN/0.1% formic acid gradient performed over 40 min at a flow rate of 300 nL/min. During each 1-s full-range FTMS scan (nominal resolution of 60 000 fwhm, 300-2000 m/z), the three most intense ions were analyzed via MS/MS in the linear ion trap. MS/MS settings used a trigger threshold of 1000 counts, monoisotopic precursor selection (MIPS), and rejection of parent ions that had unassigned charge states, were previously identified as contaminants on blank gradient runs, or were previously selected for MS/MS (data dependent acquisition using a dynamic exclusion for 150% of the observed chromatographic peak width). Column performance was monitored using trypsin autolysis fragments (m/z 421.76), and via blank injections between samples to assay for contamination. 2.7. Data Analysis. Centroided ion masses were extracted using the extract_msn.exe utility from Bioworks 3.3.1 and were used for database searching with Mascot v2.2.04 (Matrix Science) and X! Tandem v2007.01.01.1 (www.thegpm.org). Journal of Proteome Research • Vol. 8, No. 10, 2009 4595

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Jmeian and El Rassi of proteins in Tables 2-5, the matching probabilities were at least 80%. This comprehensive list was generated on the basis of peptide identifications at greater than 80% probability and protein identifications at greater than 90.0% probability and contained at least 1 identified peptide.

3. Results and Discussion As mentioned above in section 2.7, Table 1 contains high “quality” protein identification from pooled tryptic digest of each of the IMAC and RP fractions, whereas Tables 2-5 contain “tentative” identification of most of the protein spots that were visible on the 2-DE gels stained by Coomassie Blue. Tables 2-5 are needed to comprehensively describe the multi fractionation and assess its effect on the resolution in 2-DE of serum proteomics. However, and whenever a protein identity is evoked, a reference to Table 1 is also given to unequivocally support the protein identity.

Figure 2. Chromatograms of the elution of medium- and lowabundance proteins from the (A) IDA-Zn2+, (B) IDA-Ni2+, and (C) IDA-Cu2+ columns. Mobile phases: (0-5.0 min) 0.05 M Na2HPO4, 0.05 M NaCl, pH 7.0, followed 0.05 M Na2HPO4, 0.05 M NaCl, 100 mM imidazole, pH 7.0 (v/v) (5.1-10.0 min). Flow rate, 0.5 mL/ min; wavelength, 280 nm.

Searches were conducted using the following search parameters: a 10 ppm parent ion mass tolerance; a 0.6 Da fragment ion tolerance one missed tryptic cleavage; one or more of the following variable modifications: pyroglutamate cyclization of N-terminal Gln, oxidation of Met, acrylamide adducts of Cys, formylation or acetylation of the protein N-terminus and acylation by fixed modification. Scaffold (version Scaffold_2.02.01, Proteome Software Inc., Portland, OR) was used to validate MS/MS based peptide and protein identifications. For the high quality protein identification in Table 1, peptide identifications were accepted if they could be established at greater than 80.0% matching probability. Proteins identifications were accepted if they could be established at greater than 99.0% probability and contained at least 2 identified peptides. The matching of proteins generated by the Scaffold software with identification probabilities greater than 95% were reported in Table 1. For the comprehensive list 4596


3.1. On-Line Depletion of High-Abundance Proteins and Fractionation/Concentration of Low- and MediumAbundance Proteins. In this study, albumin depleted serum samples were further depleted of the next top-7 highabundance proteins by tandem affinity columns followed by on-line fractionation/concentration of the depleted sample by tandem IMAC and RP columns (see Figure 1). An aliquot of 0.5 mL of serum sample was used in this study representing ∼33 mg of total proteins. The depletion process described in this report removes ∼85%6,34,35 of the total protein mass, and thus, only ∼5 mg of total proteins remain after depletion, which was further fractionated on a series of three IMAC columns and an RP-column connected in tandem with the depletion columns. Figure 2 shows the chromatograms of the elution of the captured medium- and low-abundance proteins from the IDA-Zn2+, IDA-Ni2+ and IDA-Cu2+ columns using stepwise elution with 100 mM imidazole in 0.05 M Na2HPO4, 0.05 M NaCl, pH 7.0. A quick visual inspection of the peak heights in the three chromatograms in Figure 2 reveals that the amount of proteins bound to IDA-Cu2+ is the highest followed by IDAZn2+ and then IDA-Ni2+, an observation which is in agreement with Figure 3A-C. The IDA monolithic columns used in this study were similar to those reported by Luo et al.33 which were found to exhibit an adsorption capacity of ∼ 20 mg for bovine serum albumin on an IDA-Cu2+column (50 × 4 mm i.d.) at pH 7.4. The monomer solution, initiator, and polymerization temperature as well as the chromatographic conditions in the study in ref 33 and the present study are closely related. Also, earlier investigations reported the high adsorption capacity of IDA metal chelate columns. For instance, a Cu-loaded Sepharose 6B column exhibited an adsorption capacity of 101 mg/mL column for immunoglobulin and 164 mg/mL for transferrin.36 Although Porath et al.24 reported that the adsorption loading capacity is in the following order: IDA-Cu2+ > IDA- Zn2+ > IDANi2+, this adsorption loading capacity is more or less of the same order of magnitude. On the basis of these literature data, the loading capacities for the IDA-metal chelate columns used in the fractionation are much higher than the amount of proteins being fractionated which is ∼5 mg. Moreover, to ensure that the columns are not overloaded, the protein fractionation was performed in 2.5 cycles whereby each cycle consisted of 10 consecutive accumulated injections on the tandem columns. Thus, it is safe to assume that all the columns were underutilized in this study to avoid overloading.

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Table 1. The LC-MS/MS Results of the Proteins Identified in the Pooled Tryptic Digest of Each of the IMAC and RP Fractionsa Zn identified proteins

Afamin R-1-Acid glycoprotein 2 R-1-Antichymotrypsin R-1-Antitrypsin R-1B-Glycoprotein R-2-HS-Glycoprotein Angiotensinogen Antithrombin-III Apolipoprotein A-I Apolipoprotein A-II Apolipoprotein A-IV β-2-Glycoprotein 1 C-reactive protein Ceruloplasmin Complement C3 Complement C4-A Complement component C6 Complement component C7 Complement component C8 R chain Complement component C8 β chain Complement component C9 Complement factor B Complement factor H Complement factor I Corticosteroid-binding globulin Fibronectin Ficolin-3 Gelsolin Hemopexin Heparin cofactor 2 Hyaluronan-binding protein 2 Ig R-1 chain C region Ig γ-1 chain C region Ig κ chain C region Ig κ chain V-III region B6 Ig λ chain C regions Ig µ chain C region Immunoglobulin J chain Inter-R-trypsin inhibitor heavy chain H1 Inter-R-trypsin inhibitor heavy chain H2 Inter-R-trypsin inhibitor heavy chain H4 Keratin, type I cytoskeletal 10 Keratin, type I cytoskeletal 9 Keratin, type II cytoskeletal 1 Kininogen-1 Leucine-rich R-2-glycoprotein Lumican Pigment epithelium-derived factor Plasma protease C1 inhibitor Prothrombin Retinol-binding protein 4 Serotransferrin Serum amyloid P-component Thyroxine-binding globulin Transthyretin1 Vitamin D-binding protein Vitronectin Zinc-R-2-glycoprotein

Ni

Cu

RP

accession molecular unique spectra unique spectra unique spectra unique spectra number (_HUMAN) weight (kDa) peptide countb peptide countb peptide countb peptide countb

AFAM A1AG2 AACT A1AT A1BG FETUA ANGT ANT3 APOA1 APOA2 APOA4 APOH CRP CERU CO3 CO4A CO6 CO7 CO8A CO8B CO9 CFAB CFAH CFAI CBG FINC FCN3 GELS HEMO HEP2 HABP2 IGHA1 IGHG1 IGKC KV301 LAC IGHM IGJ ITIH1 ITIH2 ITIH4 K1C10 K1C9 K2C1 KNG1 A2GL LUM PEDF IC1 THRB RET4 TRFE SAMP THBG TTHY VTDB VTNC ZA2G

69 24 48 47 54 39 53 53 31 11 45 38 25 122 187 193 105 94 65 67 63 86 139 66 45 263 33 86 52 57 63 38 36 12 12 11 49 16 101 106 103 60 62 66 72 38 38 46 55 70 23 77 25 46 16 53 54 34

9

2

25

3

50 43

127 108

5

7

23 4

67 8

17

4

5 0 8

7

5

56

5

9 4 11

15

7

11 13 5 10 3

20 27 10 18 6

3

10

16 52 24 8 2 5

37 128 50 13 4 7

21

48

8

19

2 2

4 7

10 7 2 4 2

21 12 2 9 3

6

9

6 3 6 8 5 7

12 3 10 12 8 11

10

25

3 7

4

4 25

4

7

16 18 7 4 6 16 19

42 44 14 6 10 34 37

7

10

8 68 6

16 197 12

4

8

3

5

2

3

3

5

7 3 3 2 4 2 3

23 5 15 2 14 4 7

3 15 6

5 26 8

6 12 6 4 5

9 27 11 6 6

4

6

2

3

3 4 27

5 17 67

10

25

4

21

6 5 5

13 13 11

5

10

3

5

7 2 7

11 5 11

2

6

6

a

The table was generated on the basis of peptide identification at greater than 80% probability and protein identifications at greater than 99% probability and contained at least 2 identified peptides. b Spectra count: the number of spectra that contributed to the protein identification.

As described in the Introduction and based on the selectivity of the IDA-metal chelate columns, the columns were arranged in the order of decreasing selectivity (i.e., IDA-Zn2+, IDA-Ni2+,

IDA-Cu2+ and RP columns). The 2-DE electropherograms for each fraction collected from the 4 columns are shown in Figure 3. When the gels were stained with fluorescent SYPRO, ∼1450 Journal of Proteome Research • Vol. 8, No. 10, 2009 4597

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Table 2. Proteins Identified on the IDA-Zn spot number

1

2-6 7 8, 41 9, 42 10-25 26-32 33

34-36 37

38

39

40

43 44 45 46 47

Column detection method

protein identity

Complement factor H Isocitrate dehydrogenase [NAD] subunit γ, mitochondrial Complement factor B 5-Hydroxytryptamine receptor 7 Complement C4A Complement component C4B Hemopexin R-2-HS-glycoprotein R-2-HS-glycoprotein 5-hydroxytryptamine receptor 7 Complement C3 precursor Complement C3 Arginine deiminase Clusterin Sad1/unc-84 domain-containing protein 1 Isocitrate dehydrogenase [NAD] subunit γ, mitochondrial Complement factor Metabotropic glutamate receptor 1 Complement C3 Complement C4-A Ceruloplasmin Acidic repeat-containing protein Clusterin Isocitrate dehydrogenase [NAD] subunit γ, mitochondrial Complement factor B Complement factor H Complement factor I Cardiotrophin-like cytokine factor 1 RNA-binding motif protein Complement C3 Complement C3 Complement C4-A Arginine deiminase Acidic repeat-containing protein Cardiotrophin-like cytokine factor 1 Isocitrate dehydrogenase [NAD] subunit γ, mitochondrial C4B3 Fragment Complement C4-A Complement C4-B SERPINB8 protein SCAD-SRL. Serum amyloid p component

LC-MS/MS

MALDI-TOF LC-MS/MS

spot Number

1-7, 29-33, 44, 45, 50 8-10 12, 13 13-15 16 17

MALDI-TOF MALDI-TOF MALDI-TOF MALDI-TOF LC-MS/MS

MALDI-TOF LC-MS/MS

18-20 21-26, 34 27, 28 35, 36 38-43 46

47

48, 52 49 LC-MS/MS

LC-MS/MS 50

53 LC-MS/MS 54, 55 56

58-61 62 MALDI-TOF LC-MS/MS MALDI-TOF MALDI-TOF MALDI-TOF

spots were detected on the four gels with 464 spots in the IDAZn2+ column fraction, 457 spots in the IDA-Ni2+ column 4598

Table 3. Proteins Identified on the IDA-Ni2+ Column


protein identity

detection method

Ceruloplasmin

MALDI-TOF

Complement factor B Serine/cysteine protease inhibitor complement C1 inhibitor Complement C3 R-1-antichymotrypsin Vitronectin Acidic repeat-containing protein Arginine deiminase R-1-B-glycoprotein Hemopexin R-1-antitrypsin Apolipoprotein H R-2-HS-glycoprotein Complement factor I 5-hydroxytryptamine receptor 7 CD5 antigen-like Ceruloplasmin Complement C3 Ig R-1 chain C region Metal transporter CNNM4 Protein Shroom3 Complement C3 Complement C3 Hemopexin Complement C4-A Isocitrate dehydrogenase [NAD] subunit γ, mitochondrial Ig R-1 chain C region Arginine deiminase Coiled-coil domain-containing protein 87 Ceruloplasmin Complement C3 60S ribosomal protein L14 Complement factor I Complement C3 Acidic repeat-containing protein TUWD12.- Homo sapiens Complement C4-A Acidic repeat-containing protein Serum amyloid P component Complement C4-A Serum amyloid P-component Coiled-coil domain-containing protein

MALDI-TOF LC-MS/MS LC-MS/MS MALDI-TOF LC-MS/MS

MALDI-TOF MALDI-TOF MALDI-TOF MALDI-TOF MALDI-TOF LC-MS/MS

LC-MS/MS

MALDI-TOF LC-MS/MS

LC-MS/MS

LC-MS/MS

MALDI-TOF LC-MS/MS

MALDI-TOF LC-MS/MS

fraction, 380 spots in the IDA-Cu2+ column fraction and 143 spots in the RP column fraction. From the total 1450 spots detected by fluorescent SYPRO, only 384 spots were subsequently detected by Coomassie Blue. In these 384 spots, the number of identified proteins by mass spectrometry (MALDI

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Table 4. Proteins Identified on the IDA-Cu spot number

1, 2 3

Column

protein identity

Ceruloplasmin Complement C3 Inter-R-trypsin inhibitor heavy chain H2 Apolipoprotein A-I Ceruloplasmin Ig R-1 chain C region

detection method

MALDI-TOF LC-MS/MS

4

Apolipoprotein A-II Afamin Transferrin

MALDI-TOF

5-8 9-11

Complement C3 R-1B-glycoprotein

MALDI-TOF MALDI-TOF

12, 13

R-1-antichymotrypsin

MALDI-TOF

14-17 18, 19 20

Kininogen R-1-antitrypsin Serine/threonine Kinase TAO1 Proteasome 26 S subunit MSS1 Zinc finger protein SLB protein Leucine rich R-2-glycoprotein Zinc-R-2-glycoprotein Vitamin D-binding protein Complement C3 R-2-HS-glycoprotein R-1-Antitrypsin Complement component C9 Apolipoprotein A-IV precursor Plasma protease C1

MALDI-TOF MALDI-TOF MALDI-TOF

21 22 23 25 26, 35, 37 27-30 31-33 34

36 38

spot number

protein identity

detection method

41 42 43

Apolipoprotein J Lysophospholipase-like 1. 1c3d


44

Complement C3 Arginine deiminase Soluble guanylate cyclase gcy-37 Prothrombin Immunoglobulin J chain Kinase suppressor of Ras 2 Coagulation factor X Inter-R-trypsin inhibitor heavy chain H4 Dihydrodipicolinate synthase Protein Shroom3 Apolipoprotein A-I Ceruloplasmin

LC-MS/MS

45

46

47-51 52

MALDI-TOF

Kininogen-1


Apolipoprotein A-II Apolipoprotein A-I Protein PUF6

53

MALDI-TOF MALDI-TOF MALDI-TOF LC-MS/MS

40

Ig R-1 chain C region β-2-Glycoprotein 1 Bifunctional protein glum LETMD1 protein

LC-MS/MS

MALDI-TOF LC-MS/MS

LC-MS/MS

Glycerol kinase 1 Kininogen-1

54

Complement C3 Haptoglobin Arginine deiminase Ig R-1 chain C region,

LC-MS/MS

MALDI-TOF LC-MS/MS

Periostin Serine-protein kinase

39

LC-MS/MS

LC-MS/MS

LC-MS/MS

Table 5. Proteins Identified on the RP-Column spot number

protein identity

detection method

2-4 1, 5, 6 7

Antithrombin R-1-acid glycoprotein-2 Retinol-binding protein precursor

MALDI-TOF LC-MS/MS MALDI-TOF

and LC-MS/MS) was 250 proteins and they are listed in Tables 2-5. The relatively high number of detected protein spots on the three IDA-metal chelate columns is a result of the sequential depletion and fractionation which removed high-abundance proteins and distributed medium-abundance proteins on the various fractionation columns, thus, enhancing protein resolution by 2-DE, see Figure 3. For example, mediumabundance protein such as hemopexin (Figure 3A, spots nos. 10-25), which was captured by the IDA-Zn2+ column, has

56-58, 61 59

5-hydroxytryptamine receptor 7 Ig λ-chain C regions Isocitrate dehydrogenase [NAD] subunit γ, mitochondrial Transthyretin Transthyretin Protein PUF6 Cardiotrophin-like cytokine factor 1

MALDI-TOF LC-MS/MS

allowed the easy detection and identification of other proteins in the IDA-Ni2+ fraction, and kininogen (Figure 3C, spots nos. 14-17), which was not captured by IDA-Zn2+ and IDA-Ni2+, has allowed the detection of other proteins on these two columns. Kininogen was retained on the IDA-Cu2+ column, thus, reducing the number of proteins that would otherwise crowd the area on the 2D gels of either the IDA-Zn2+ or the IDA-Ni2+ fractions, which in turn would decrease the resolution and thus the number of detected spots. 3.2. The Contribution of the Depletion/Fractionation Strategy to the Depth of the Proteomic Analysis. As mentioned in the preceding section, the depletion and fractionation strategy described here has increased the number of proteins detected from 345 to 1450 and identified on the 2-DE gels. In fact, the number of detected spots was ∼1450 using SYPRO Journal of Proteome Research • Vol. 8, No. 10, 2009 4599

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Figure 3. 2-DE electropherograms performed on 0.5 mL of depleted serum and fractionated/concentrated on (A) IDA-Zn2+, (B) IDA-Ni2+, (C) IDA-Cu2+ and (D) the RP-column. 2-DE experiments were performed on a 17 cm IPG strip (pH 4-7) and subsequently on a 10-20% Tris-HCl 18.3 cm × 19.3 cm gel stained with Bio-Safe Coomassie. Conditions for the first dimension: 200 V for 2 h, 500 V for 2 h, 1000 V for 3 h and 3500 V for 21 h. Conditions for the second dimension: 30 min at 16 mA/gel, then 24 mA/gel for 5 h.

fluorescent stain. For MALDI-TOF and LC-MS/MS analysis, the 2-DE gels were subsequently stained with Coomassie Blue. In general, Coomassie Blue visible spots were excised and submitted to MALDI-TOF, and they totaled 384 spots. The Coomassie Blue visible spots were first analyzed by MALDI-TOF, and the spots that revealed the presence of sufficient amount of proteins (they totaled 40 spots) but could not be identified by MALDI-TOF were subsequently submitted to LC-MS/MS analysis. The 384 selected spots resulted in the tentative identification of 250 protein spots (see Tables 2-5). The number of protein spots identified by MALDI-TOF alone was 136 and those identified by LC-MS/MS alone were 114 proteins. In MALDI-TOF, 47 protein spots were tentatively identified in the fraction collected from the IDA-Ni2+ column, 46 protein spots in the fraction obtained from the IDA-Cu2+ column, 39 protein spots from the IDA-Zn2+ column and 4 protein spots in the RP-column fraction. In LC-MS/MS, and more precisely in the 40 selected spots submitted to LC-MS/MS, 32 proteins were tentatively identified in the IDA-Ni2+ fraction, 35 proteins in the IDA-Zn2+ fraction, 44 proteins in the IDA-Cu2+ fraction and 3 proteins in the RP fraction. An additional 45 proteins were identified in the pooled tryptic digests form the IMAC and RP columns fractions. Thus, the combined total number of pro4600


teins identified by MALDI-TOF and LC-MS/MS is 295 proteins and protein spots. This is an impressive number demonstrating the ability and usefulness of the platform described here that involved simultaneous depletion and fractionation of serum proteins. In comparison to a recent study by us,8 which involved the depletion by tandem affinity columns and the subsequent capturing and concentration of medium- and lowabundance proteins on an RP column only, the subsequent fractionation of proteins on four tandem columns (3 IMAC columns and 1 RP column) into multiple fractions increased the number of detected proteins by ∼4 times (1450 spots vs 357 spots) and the number of identified proteins by ∼5.8 times (295 spots vs 51 spots) with respect to our recent study.8 The increase in the number of identified proteins could be attributed to the reduction of proteins present in a single spot. It should be mentioned that, even after fractionation, some spots contained multiple proteins as indicated in Tables 2-5. Also, by comparing the four 2-DE gels shown in Figure 3, it can be seen that many proteins migrate at the same location on the different gels (i.e., the pI and molecular weight were similar). For example, hemopexin (Figure 3A, spots nos. 10-25), serine/cysteine protease inhibitor (Figure 3B, spots nos. 12, 13), SLB protein (Figure 3C, spot no. 23) and antithrombin (Figure


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Figure 4. Protein map of human serum proteins of the four overlaid fractions (i.e., IDA-Zn2+, IDA-Ni2+, IDA-Cu2+ and RP column fractions) collected from the depletion/fractionation strategy shown in Figure 1. Only proteins that were detected by CBB are illustrated in this figure. The proteins from the IDA-Zn2+ column fraction are represented by circles, those from IDA-Ni2+ column fraction are represented by rectangles, those from the IDA-Cu2+ fraction are represented by triangles and those from RP column fraction are represented by diamonds.

3D, spots nos. 2-4) were detected at similar pI and molecular weights on the four electropherograms. Also, the albumin spots would have covered all of these proteins, if albumin was not depleted in the first place. For at glance demonstration of the usefulness of the multi fractionation subsequent to the depletion of high-abundance proteins, the various spots detected and identified in the four different fractions (i.e., 3 IMAC fractions and 1 RP fraction) were overlaid as shown in Figure 4. It is clear from this figure that fractionating the sample prevented many proteins to comigrate in 2-DE, thus, emphasizing the importance of fractionation in reducing the complexity of the serum proteome. When the platform described in this manuscript is used, the detection of low- and medium-abundance proteins that may serve as protein biomarker could be achieved. One particular example is ficolin-3 (FCN3), which was detected in the IDANi2+ subproteome. In a recent study37 aimed at investigating the differential expression of ficolin-3 in patients with systemic lupus erythematosus (SLE) or its clinical subsets, the authors reported that the elevation of this protein and its association with specific manifestations in SLE may indicate a pathogenetic role of ficolin-3 in SLE. 3.3. Retention of Proteins on the Different Fractionation Columns. The contribution of each column to fractionating serum proteins can be seen in Figure 3. For example, the IDAZn2+ column selectively captured proteins such as hemopexin and complement factor H as shown in Figure 3A, Tables 1 and 2. Hemopexin is a protein that has high affinity to heme that reaches plasma as a result of hemolysis or tissue damage.38,39 Hemopexin contains 19 His residues and is best retained by IDA chelated with Ni2+ and Cu2+ followed by Co2+ and Zn2+ and has a pI value ranging from 5.46 to 6.36.39 It was found that the binding of heme to hemopexin diminishes the retention of the protein to IDA-metal chelate columns (e.g., Ni2+, Cu2+ and Zn2+).39 These findings are in agreement with the results in this present study. As can be seen in Figure 3A, the

majority of serum hemopexin is retained on the IDA-Zn2+ column (Figure 3A, spots nos. 1-25). The little amount of the hemopexin that escaped the IDA-Zn2+ column was captured on the IDA-Ni2+ column (Figure 3B, spots nos. 21-26 and 34). This finding is also confirmed by the LC-MS/MS results shown in Table 1. As can be seen in Table 1, the number of hemopexin (Hemo_HUMAN) unique peptides and the spectra count (i.e., the number of spectra that contributed to the protein identification) detected in the IDA-Zn2+ fraction are 17 (26% sequence coverage) and 56, respectively. On the other hand, the numbers of hemopexin unique peptides and spectra detected in the IDA-Ni2+ fraction are 10 (20% sequence coverage) and 21, respectively. Despite the fact that hemopexin binds best to Ni2+ and Cu2+,39 it was preferentially captured by the IDA-Zn2+ because it is the first column in the series, see Figure 1. This explain why hemopexin was not detected on the IDA-Cu2+ or the RP column as can be seen in Figure 3C,D and Tables 1, 4, and 5. Instead, proteins such as R-1B-glycoprotein and antithrombin were detected in similar locations on the 2-DE of the IDA-Cu2+ fraction and the RP fraction. The escape of some hemopexin molecules from IDA-Zn2+ column to the IDA-Ni2+ column could be due to the heme-hemopexin association that weakens the binding of hemopexin to the IDA-Zn2+ column. In addition, protein folding, post-translational modification or protein-protein interaction obscuring the metal binding motif may also contribute in detecting hemopexin in the IDA-Ni2+ column fraction. Figure 3B and Tables 1 and 3 show proteins captured selectively on the IDA-Ni2+ column. As can be seen in Figure 3B, removing the majority of hemopexin by the first column (i.e., IDA-Zn2+ column) facilitated the detection of R-1Bglycoprotein (Figure 3B, spots nos. 18-20). Also, as shown in Figure 3B, proteins such as serum amyloid p (spots nos. 58-61), complement factor I (spot no. 46) and plasma protease C1 inhibitor (spot no. 11) were captured selectively on the IDANi2+ column. These results are also in agreement with the LCJournal of Proteome Research • Vol. 8, No. 10, 2009 4601

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MS/MS results presented in Table 1. The number of unique peptides and the spectra count for complement factor I (CFAI_HUMAN) are 8 (10% sequence coverage) and 19, respectively, whereas for plasma protease C1 inhibitor (ICI_HUMAN), they are 10 (20% sequence coverage) and 25, see Table 1. It should be mentioned that a trace amount of serum amyloid p was detected in the IDA-Zn2+ fraction (Figure 3A, spot 47). Serum amyloid p (SAMP_HUMAN) contains only 4 His residues, and each His pair is separated by at least 14 different amino acid residues. According to Sulkowski and his collaborators’s pattern,28,30 one may conclude that at least 3 His moieties are available on the protein surface so that the serum amyloid p can bind to IDA-Zn2+ as shown in Figure 3A, spot 47. Again, these findings are in agreement with the LCMS/MS results presented in Table 1. Although the same 7 unique peptides (30% sequence coverage) were detected on both the IDA-Zn2+ and IDA-Ni2+, the spectra count for the IDANi2+ is 25 compared to 15 in the IDA-Zn2+ fraction. An elevated total spectra count is a reflection of a stronger binding of the protein of interest to IDA-Ni2+. As shown in Table 1, complement component C8 alpha was detected in the IDA-Ni2+ fraction, while complement component C8 beta was detected in the IDA-Zn2+ fraction. By examining both sequences, it is found that complement component C8 beta contains 14 His, while complement component C8 alpha contains only 9 His groups. The presence of more His groups on the complement component C8 beta surface available for interaction with the chelated metal could be the reason for its presence in the IDA-Zn2+ fraction.

As mentioned in the Introduction, it should be indicated again that the binding of proteins and peptides to IMAC columns is not solely based on His. The characterization of Cubinding peptides has shown that metal binding ligands involve methionine and cysteine in addition to histidine.41 It is also mentioned in the same study41 that proteins that do not usually bind to the immobilized metal may be detected with the metalbinding proteins because of protein-protein interaction. Finally, we can see that only 5 different proteins were identified on the RP-column. These proteins include antithrombin-III, R-1-acid glycoprotein-2, retinol-binding protein 4, Apolipoprotein A-I and Apolipoprotein A-II. As shown in Table 1, Apolipoprotein (A-II APOA2_HUMAN) was only detected on the RP fraction. This small protein has no His groups on its surface and thus was not retained by any of the IMAC columns. Retinol-binding protein 4 (RET4_Human) and R-1acid glycoprotein-2 (A1AG2_human) contain only two His groups (1.0%) and three His groups (1.50%), respectively. Since none of these proteins were retained on the IMAC columns, one may conclude that there is no accessible His groups on the surfaces of the proteins under investigation. Both antithrombin-III and Apolipoprotein A-I were detected also in the IDA-Cu2+ fraction as shown in Table 1. In fact, the number of unique peptides and the spectra count for these two proteins are much higher in the IDA-Cu2+ fraction. Again, this could be attributed to protein-protein interactions and post-translational modifications at the metal binding site.

Proteins retained on IDA-Cu2+ column are shown in Figure 3C and Tables 1 and 4. Examples of proteins captured by the IDA-Cu2+ column include kininogen (Figure 3C, spots nos. 14-17), vitamin D binding protein (Figure 3C, spots nos. 27-30), Zn-R-2-glycoprotein (Figure 3C, spots nos. 26, 35, 37), apolipoprotein AI (Figure 3C, spots nos. 47-51) and transthyretin (Figure 3C, spots nos. 56-58 and 61). The LC-MS/MS results in Table 1 also confirm these findings. All these proteins are only detected in the IDA-Cu2+ fraction with the number of unique peptides ranging from 4 to 27. Other proteins such as R-1-antichymotrypsin (Figure 3C, spots nos. 12-13) and R-1antitrypsin (Figure 3C, spot nos. 18, 19) were detected in the IDA-Ni2+ fraction as well as the IDA-Cu2+ fraction. However, the number of unique peptides and spectra in Table 1 as well as the 2-DE indicate that these two proteins are mainly present in the IDA-Cu2+ fraction. Kininogen, for example, possesses only 5 His of the 427 amino acid residues (i.e., only 1.17% His). Another example is Vitamin D binding protein in which the His residues represent 1.48% of the total amino acids. On the other hand, 4.11% of hemopexin is His. In general, the number of surface His is what determines the retention behavior of the protein to the immobilized metal and not its percentage. However, as the number of histidine increases, the likelihood to have more histidine on the surface increases. Complement C3 shows small affinity to the IDA-Zn2+ column (Figure 3A, spots nos. 34-36) and IDA-Ni2+ column (Figure 3B, spots nos. 48, 52) and much higher affinity to IDA-Cu2+ column (Figure 3C, spots nos. 3, 31-33 and 44). Also, trace amounts of complement C3 are present at many other locations (see Figure 3A-C and Tables 2-4). Complement C3 is a very large protein (187 KDa) that contains 1641 residues.40 Enzyme inhibitors were not used in this study and the proteolytic activity may have resulted in the formation of the smaller fragments detected in multiple locations.

In this investigation, we have shown that the column-based fluidic platform for the on-line depletion of high-abundance proteins and subsequent concentration/fractionation of medium- and low-abundance proteins is very suitable for reducing the complexity of serum samples. In addition, the depletion/ fractionation platform described here facilitated the in-depth proteomic analysis and the detection of low-abundance proteins. Abbreviations: AIBN, 2,2′-azobisisobutyronitrile; EDMA, ethylene glycol dimethacrylate; GMA, glycidyl methacrylate; HSA, human serum albumin; Igs, immunoglobulins; TFA, trifluoroacetic acid; IMAC, immobilized metal ion affinity chromatography; IDA, iminodiacetic acid.

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4. Conclusions

Acknowledgment. We would like to acknowledge the financial support in part by the National Science Foundation MRI award No. 0722494 for the purchase of LTQ-Orbitrap mass spectrometer for the Protein Core Facility, Department of Biochemistry and Molecular Biology, Oklahoma State University, and in part by the Coca Cola Co. Also, the author would like to thank Dr. Steven Hartson for his assistance in the LC-MS/MS analysis at the Protein Core Facility. References (1) Faca, V.; Pitteri Sharon, J.; Newcomb, L.; Glukhova, V.; Phanstiel, D.; Krasnoselsky, A.; Zhang, Q.; Struthers, J.; Wang, H.; Eng, J.; Fitzgibbon, M.; McIntosh, M.; Hanash, S. Contribution of protein fractionation to depth of analysis of the serum and plasma proteomes. J. Proteome Res. 2007, 6, 3558–3565. (2) Plavina, T.; Wakshull, E.; Hancock, W. S.; Hincapie, M. Combination of abundant protein depletion and multi-lectin affinity chromatography (M-LAC) for plasma protein biomarker discovery. J. Proteome Res. 2007, 6, 662–671. (3) Jmeian, Y.; El Rassi, Z. Liquid-phase-based separation systems for depletion, prefractionation and enrichment of proteins in biological fluids for in-depth proteomics analysis. Electrophoresis 2009, 30, 249–261.

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(22) Jmeian, Y.; El Rassi, Z. Tandem affinity monolithic microcolumns with immobilized protein A, protein G’, and antibodies for depletion of high abundance proteins from serum samples: integrated microcolumn-based fluidic system for simultaneous depletion and tryptic digestion. J. Proteome Res. 2007, 6, 947–954. (23) Okanda, F.; El Rassi, Z. Affinity monolithic capillary columns for glycomics/proteomics 1. Polymethacrylate monoliths with immobilized lectins for glycoprotein separation by affinity capillary electrochromatography and affinity nano-liquid chromatography in either a single column or columns coupled in series. Electrophoresis 2006, 27, 1020–1030. (24) Porath, J.; Carlsson, J.; Olsson, I.; Belfrage, G. Metal chelate affinity chromatography, a new approach to protein fractionation. Nature 1975, 258, 598–599. (25) Gooding, K. M.; El Rassi, Z.; Horvath, C. Metal interaction chromatography. In HPLC of Biological Macromolecules, 2nd ed.; Gooding, K. M., Regnier, F. E. , Eds.; Marcel Dekker: New York, 2002; Vol. 87, pp 247-280. (26) Porath, J.; Olin, B. Immobilized metal ion affinity adsorption and immobilized metal ion affinity chromatography of biomaterials. Serum protein affinities for gel-immobilized iron and nickel ions. Biochemistry 1983, 22, 1621–1630. (27) Porath, J. IMAC--Immobilized metal ion affinity based chromatography. Trends Anal. Chem. 1988, 7, 254–259. (28) Sulkowski, E. The saga of IMAC and MIT. BioEssays 1989, 10, 170– 175. (29) Sulkowski, E. Purification of proteins by IMAC. Trends Biotechnol. 1985, 3, 1–7. (30) Hemdan, E. S.; Zhao, Y. J.; Sulkowski, E.; Porath, J. Surface topography of histidine residues: a facile probe by immobilized metal ion affinity chromatography. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 1811–1815. (31) Scopes, R. K. Separation by adsorption II: ion exchangers and nonspecific adsorption. In Protein Purification: Principles and Practice, 3rd ed.; Springer-Verlag: New York, 1994; pp 180-183. (32) Corradini, D.; el Rassi, Z.; Horvath, C.; Guerra, G.; Horne, W. Combined lectin-affinity and metal-interaction chromatography for the separation of glycophorins by high-performance liquid chromatography. J. Chromatogr. 1988, 458, 1–11. (33) Luo, Q.; Zou, H.; Xiao, X.; Guo, Z.; Kong, L.; Mao, X. Chromatographic separation of proteins on metal immobilized iminodiacetic acid-bound molded monolithic rods of macroporous poly(glycidyl methacrylate-co-ethylene dimethacrylate). J. Chromatogr., A 2001, 926, 255–264. (34) Chromy, B. A.; Gonzales, A. D.; Perkins, J.; Choi, M. W.; Corzett, M. H.; Chang, B. C.; Corzett, C. H.; McCutchen-Maloney, S. L. Proteomic analysis of human serum by two-dimensional differential gel electrophoresis after depletion of high-abundant proteins. J. Proteome Res. 2004, 3, 1120–1127. (35) 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, 3304–3313. (36) Al-Mashikhi, S. A.; Nakai, S. Separation of immunoglobulin and transferrin from blood serum and plasma by metal chelate interaction chromatography. J. Dairy Sci. 1988, 71, 1756–1763. (37) Andersen, T.; Munthe-Fog, L.; Garred, P.; Jacobsen, S. Serum levels of ficolin-3 (Hakata antigen) in patients with systemic lupus erythematosus. J. Rheumatol. 2009, 36, 757–759. (38) Wong, J. C.; Holland, J.; Parsons, T.; Smith, A.; Williams, P. Identification and characterization of an iron-regulated hemopexin receptor in Haemophilus influenzae type b. Infect. Immun. 1994, 62, 48–59. (39) Mauk, M. R.; Rosell, F. I.; Lelj-Garolla, B.; Moore, G. R.; Mauk, A. G. Metal ion binding to human hemopexin. Biochemistry 2005, 44, 1864–1871. (40) Janssen, B. J.; Huizinga, E. G.; Raaijmakers, H. C.; Roos, A.; Daha, M. R.; Nilsson-Ekdahl, K.; Nilsson, B.; Gros, P. Structures of complement component C3 provide insights into the function and evolution of immunity. Nature 2005, 437, 505–511. (41) She, Y. M.; Narindrasorasak, S.; Yang, S.; Spitale, N.; Roberts, E. A.; Sarkar, B. Identification of metal-binding proteins in human hepatoma lines by immobilized metal affinity chromatography and mass spectrometry. Mol. Cell. Proteomics 2003, 2, 1306–1318.

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Journal of Proteome Research • Vol. 8, No. 10, 2009 4603

Multicolumn Separation Platform for Simultaneous Depletion and

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