Multiplexed Affinity-Based Protein Complex Purification - Analytical

Jonathan Ashby , Samantha Schachermeyer , Songqin Pan , and Wenwan Zhong. Analytical ... Meghan J. McFadden , Murray S. Junop , and John D. Brennan...
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Anal. Chem. 2008, 80, 7068–7074

Multiplexed Affinity-Based Protein Complex Purification Jishan Li, Jianping Ge, Yadong Yin, and Wenwan Zhong* Department of Chemistry, University of California, Riverside, California 92521 Here we proved the principle of a multiplexed affinitybased protein complex purification (MAPcP) technique that targets simultaneous extraction of multiple protein complexes with superior purity. Microspheres of various sizes and coupled with different affinity probes extract several protein complexes concurrently and specifically. After the coextraction, flow-field flow fractionation (FlFFF) rapidly washes the microspheres as well as separates them based on their sizes to recover the clean individual complex for downstream analysis. Demonstration of the parallel extraction of two immuno-complexes from the yeast whole cell lysate showed that MAPcP can enhance the sample purity significantly compared to the traditional centrifugation and magnetic pull-down methods used for small scale protein purification. Simultaneous isolation of multiple protein complexes can facilitate the elucidation of the functional relationship among protein complexes and improve our understanding of the biological network. Protein complexes formed by several interacting proteins are the actual functional machineries carrying out the cellular processes in the cell.1-3 These complexes are different in size, number of proteins, and stabilities. The study of protein complexes can help to identify as many binding partners for a single protein as possible within the shortest time, thus is greatly desirable in the construction of a high quality protein-protein interaction network and in the promotion of understanding protein functions and dynamic processes within the cell.1-3 Analyzing the entire protein complex can also help to identify proteins that do not directly interact with the target protein but are essential for the integrity and function of the protein machinery. Sucrose-gradient ultracentrifugation4 and blue native polyacrylamide gel electrophoresis (BN-PAGE)5,6 have been used to separate native protein complexes by density or size, but the resolving power is not high * Corresponding author. E-mail: [email protected]. Phone: 951-8274925. Fax: 951-827-4713. (1) Carmi, S.; Levanon, E. Y.; Havlin, S.; Eisenberg, E. Phys. Rev. E: Stat., Nonlinear, Soft Matter Phys. 2006, 73, 031900/031901–031909/031906. (2) Cusick, M. E.; Klitgord, N.; Vidal, M.; Hill, D. E. Hum. Mol. Genet. 2005, 14, R171–R181. (3) Stoevesandt, O.; Koehler, K.; Wolf, S.; Andre, T.; Hummel, W.; Brock, R. Mol. Cell. Proteomics 2007, 6, 503–513. (4) Righetti, P. G.; Castagna, A.; Antonioli, P.; Boschetti, E. Electrophoresis 2005, 26, 297–319. (5) Heinemeyer, J.; Lewejohann, D.; Braun, H.-P. Methods Mol. Biol. (Totowa, NJ, U.S.) 2007, 355, 343–352. (6) Randelj, O.; Rassow, J.; Motz, C. Methods Mol. Biol. (Clifton, NJ) 2007, 390, 417–428.

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enough and protein complexes may decompose in the separation matrix. Affinity capture of specific protein complexes by immunoprecipitation (IP) using magnetic or resin beads coupled with antibodies against the bait protein can provide high recognition specificity, and the abundant nonspecific proteins can be removed by stringent wash cycles to facilitate recognition of the genuine interacting proteins.7-12 The bait protein can also be tagged with multiple high affinity probes, such as the IgG binding domain of protein A, hemagglutinin (HA) epitope, and polyhistidine (6xHis), so that two consecutive purification steps and one enzymemediated release of complex from the stationary phase can be employed to further improve sample purity. Such a process is called tandem affinity purification (TAP).7-12 However, proteins may degrade and the complex may decompose during the long TAP process, and the purity is still not satisfied in some situations. Often a large percentage of the proteins identified in the extracted complexes are classified as nonspecifically bound.10 It has been known that one way for the cell to regulate cellular processes is through variation in protein abundance and proteinprotein interaction.2,3,13 Such regulations could be related to normal cellular activities, disease development, or response to external stimuli.2,3,13 Often changes are made to a group of proteins to orchestrate the final effects. Therefore, studies targeting multiple protein complexes functioning downstream or upstream of each other or those involved in parallel pathways carrying out similar tasks are important in systems biology. They can help to elucidate the functional relationship among these complexes, reveal how the cellular processes are regulated, and promote understanding in the complex intercellular web of interactions that contribute to the structure and function of a living cell.2,3,13-15 Moreover, certain proteins, such as the RNA-dependent protein kinase (PKR), carry out multiple functions in the cell by binding to different regulators or effectors at various protein (7) Das, P. M.; Ramachandran, K.; van Wert, J.; Singal, R. BioTechniques 2004, 37, 961–969. (8) Elion, E. A.; Wang, Y. Methods Mol. Biol. (Totowa, NJ, U.S.) 2004, 284, 1–14. (9) Herrmann, J. M.; Westermann, B.; Neupert, W. Methods Cell Biol. 2001, 65, 217–230. (10) Markham, K.; Bai, Y.; Schmitt-Ulms, G. Anal. Bioanal. Chem. 2007, 389, 461–473. (11) Portelius, E.; Tran, A. J.; Andreasson, U.; Persson, R.; Brinkmalm, G.; Zetterberg, H.; Blennow, K.; Westman-Brinkmalm, A. J. Proteome Res. 2007, 6, 4433–4439. (12) Selbach, M.; Mann, M. Nat. Methods 2006, 3, 981–983. (13) Arnold, A.; Sielaff, I.; Johnsson, N.; Johnsson, K. Chem. Biol. 2007, 1, 458– 479. (14) Guerrero, C.; Tagwerker, C.; Kaiser, P.; Huang, L. Mol. Cell. Proteomics 2006, 5, 366–378. (15) Zhou, M.; Veenstra, T. D. Proteomics 2007, 7, 2688–2697. 10.1021/ac801251y CCC: $40.75  2008 American Chemical Society Published on Web 08/21/2008

domains. Simultaneous isolation of the protein complexes formed on different domains and comparison of the changes of these complexes upon external stimuli will lead us to useful information about how the protein and the related cellular pathway are regulated. However, the current affinity-based fractionation techniques target only one protein complex in each isolation process. Isolation of different protein complexes for concurrent comparison can only be conducted with either parallel extraction processes using split sample aliquots, which requires large sample amounts, or tandem extractions from the same sample, which elongates the incubation duration and thus reduces the stabilities of the complexes. Variations in the sample amount used for each extraction and complex situation could also be introduced and could prevent reliable comparison. Different from the traditional separation technologies like electrophoresis, gel filtration chromatography, and liquid chromatography, field flow fraction (FFF) utilizes no separation matrix or stationary phase, which could be a big advantage in analyzing large but fragile protein complexes, because no small pores of the polymer network or tortuous channels of the stationary phase can trap the complex and destroy its structure.16-18 In FFF, an external field is applied in the orthogonal direction to the channel flow and drives analytes to the accumulation wall.19-21 The diffusion force moves the analytes back to the center of the channel, and at equilibrium analytes distribute at different height levels within the parabolic flow profile inside the ribbon-shaped channel and exit the channel at different times.19-21 The elution time is determined by the nature of the external force and the physical property of the analyte. For example, flow-field flow fraction (Fl-FFF) employs a cross liquid flow as the external field and separates analytes by their hydration size. Attempts to apply conventional FFF techniques to separate large protein complexes, organelles, and cells have achieved some success16-18 but suffered from the low resolving power that occurred with BN-PAGE, sucrose density gradient, and gel filtration chromatography. Since Fl-FFF is well-known for its superior capability of separating microbeads within the micrometer size range, we can combine microsphere-based immuno-precipitation with efficient Fl-FFF separation of the microparticles to achieve specific recognition of protein complexes and ultrahigh sample purity, forming a multiplexed affinity-based protein complex purification (MAPcP) technique. In this proof-of-principle study, we demonstrated that two immuno-complexes could be extracted concurrently from the yeast whole cell lysate with significantly improved sample purity in comparison to that from the traditional centrifugation and magnetic pull-down methods. Magnetic particles were also compatible with MAPcP to facilitate sample handling and enhance sample recovery. Simultaneous extraction of multiple complexes with the remarkably simple process of MAPcP can facilitate the elucidation of the functional relationship among protein complexes and enhance our understanding of the biological network. (16) Chmelik, J. Proteomics 2007, 7, 2719–2728. (17) Williams, S. K. R.; Lee, D. J. Sep. Sci. 2006, 29, 1720–1732. (18) Reschiglian, P.; Zattoni, A.; Roda, B.; Michelini, E.; Roda, A. Trends Biotechnol. 2005, 23, 475–483. (19) Kowalkowski, T.; Buszewski, B.; Cantado, C.; Dondi, F. Crit. Rev. Anal. Chem. 2006, 36, 129–135. (20) Myers, M. N. J. Microcolumn Sep. 1997, 9, 151–162. (21) Giddings, J. C. Science 1993, 260, 1456–1465.

EXPERIMENTAL SECTION Bioreagents and Chemicals. Sodium phosphate monobasic, sodium hydroxide, FL-70 Detergent, sodium dodecyl sulfate (SDS), phosphate buffered saline (PBS) (pH 7.4), sodium azide (NaN3), Tween 20, and sodium chloride were purchased from Fisher Scientific (Fairlawn, NJ). Human immunoglobulin (IgG), recombinant protein A, mouse anti-T7 monoclonal IgG, rabbit antigreen fluorescence protein (GFP) IgG, recombinant GFP, Alexa Fluor 488 Protein Labeling Kit, BenchMark Protein Ladder, and SilverQuest Silver Staining Kit were obtained from Invitrogen (Carlsbad, CA). Components for making the sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) (SDS, acrylamide, N,N′methylenebisacrylamide, ammonium persulfate, N,N,N′,N′-tetramethylethylenediamine) were from Sigma (St. Louis, MO). All solutions were prepared in deionized water (18 MΩ) from a Milli-Q water purification system (Millipore, Billerica, MA). Conjugation of Protein to Microsphere. SPHERO carboxyl polystyrene particles (Lake Forest, IL) of different sizes, 6.7, 5.28, 4.51, and 3.17 µm, were employed in the present study. Coupling human IgG and protein G to microspheres was performed as follows. First, 1 mg of protein was added to 4 mL of the 0.1 M phosphate buffer (pH 7.3) containing around 108 microspheres. Mixed with 160 mg of 1-ethyl-3-(3-(dimethylaminopropyl)carbodiimide hydrochloride (EDC) (Sigma), the reaction mixture was stirred at room temperature for 4 h. Then the microspheres were centrifuged down at 5 900g for 1 min. Following removal of the supernatant, the microspheres were washed twice, each with 4 mL of PBS-0.05% Tween 20. The conjugated beads were then resuspended in 2 mL of the PBS-1% BSA-0.05% NaN3 solution and stored in a refrigerator (4 °C). Preparation of Yeast Total Protein Extract. The fission yeast strain was kindly provided by Dr. Jian-Kang Zhu in the Department of Botany and Plant Science of University of California Riverside (UCR). The yeast culture medium was prepared by adding 5 g of Bacto yeast extract (BD Biosciences, San Jose, CA), 30 g of glucose (Fisher Scientific), and 250 mg of adenine hemisulfate (Sigma) to 1 L of water and then autoclaved. One liter of the medium was inoculated with 10 mL of a saturated yeast solution harvested overnight in an incubator shaker (New Brunswick Scientific Co, Inc., New Jersey) at 30 °C. Cells were then grown to an OD600 (optical density at 600 nm) of 3 and spun down. The harvested cells were then ground to very fine powder in liquid nitrogen and stored at - 80 °C until use. Right before use, the broken cells were dissolved in the lysis buffer at a 2:1 buffer to cell volume ratio. The lysis buffer was composed of 50 mM HEPES-KOH at pH 7.6, 300 mM potassium acetate, 10% glycerol, 1 mM EGTA, 1 mM EDTA, 0.1% NP-40, 0.2 mM dithiothreitol (DTT), 5 mM magnesium acetate, 1 mM sodium fluoride, 20 mM sodium β-glycerophosphate, 1 mM phenylmethanesulfonyl fluoride (PMSF), and 1 mM benzamidine (All of the above chemicals were from Fisher Scientific.). The total lysate was then used for immuno-precipitation. Preparation of Microspheres Modified by Superparamagnetic Particles. The water-soluble magnetite colloidal nanocrystals were prepared as described in ref 32. The nanocrystals used in the present study to modify the 5.28-µm polystyrene microspheres had an average diameter of 10 nm. First, 100 µL of the nanocrystals (about 1016/mL) were suspended in 4 mL of 0.1 M Analytical Chemistry, Vol. 80, No. 18, September 15, 2008

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phosphate buffer at pH 7.3, followed by the addition of 400 µg of human IgG and 40 mg of EDC. The mixture was incubated for 4 h at room temperature (RT), and then the pH value of the solution was adjusted to 4 by the addition of phosphoric acid to precipitate the nanocrystals. The HIgG conjugated nanocrystals were then distributed in 0.1 M phosphate buffer. Aggregates were removed with the help of a magnet until a clear nanocrystal suspension was obtained. About 108 of the 5.28-µm carboxylated polystyrene microspheres were activated for 20 min in 0.1 M sodium acetate buffer (NaAc, pH 5.0) by 140 mg of EDC and 20 mg of NHS. Then the microspheres were centrifuged and washed once by the NaAc buffer before being mixed with the HIgG conjugated nanocrystals. After 3.5-h incubation at RT, the nanocrystal decorated microspheres were washed and stored in the PBS solution containing 1% BSA and 0.02% NaN3 at 4 °C. Immuno-Precipitation (IP) Procedure. All IPs took place at 37 °C in 500 µL of either the incubation buffer (IB, PBS buffer containing 1% BSA and 0.2% Tween 20) or the whole cell lysate from yeast. During the extraction, one tablet of the protease inhibitor cocktail from Roche Applied Science (San Jose, CA) was added to the whole cell lysate to prevent protein degradation. For the extraction of the GFP complex, ten million protein G-coupled 6.7-µm microspheres were first reacted with 1 µg/mL rabbit-antiGFP IgG (RIgG) in IB, then incubated with 1 µg/mL GFP in the yeast total extract for another hour, capturing the RIgG/GFP complex by the protein G molecules on the microsphere surface. Similarly, extraction of the protein A complex by ten million of the HIgG conjugated 4.5-µm microspheres took place first in IB with the presence of 10 µg/mL recombinant protein A (ProA). After one-hour incubation at RT, the microspheres were isolated and incubated with 1 µg/mL anti-T7 mouse IgG (MIgG) in the yeast whole cell lysate for another hour, forming the ProA/MIgG complex on the microspheres. After extraction, the beads were either stringently washed with the wash buffer (PBS with 0.05% Tween 20) five times and isolated by centrifugation or redistributed in the Fl-FFF running buffer (10 mM pH 7.5 PBS, 0.05% Fl70, 50 mM NaCl) at a concentration of 1011/L and subjected to Fl-FFF separation. The process for protein A complex extraction using the modified, magnetic microspheres was the same as above, except that a magnet was used to bring down the beads instead of centrifugation. Flow-Field Flow Fractionation of Microspheres after Extraction and Subsequent Gel Analysis of Eluted Proteins. The F-1000 Fractionator from Postnova Analytics Inc. (Salt Lake City, UT) was used for size-based separation of the microspheres. The separation channel has a void volume (Vo) of 1.41 mL, with a thickness of 0.0254 cm, a breadth of 2.0 cm, and a length of 27.7 cm. The accumulation wall was made from a regenerated cellulose membrane with a molecular-weight-cutoff value of 100 kD. The column eluent flowed into an HPLC ultraviolet-visible (UV-vis) detector (SPD-20A, Postnova Analytics Inc.) operated at 214 nm for absorption detection. All separations were performed in the open mode with an exiting cross-flow. The outlet channel flow rate was maintained the same as the inlet channel flow rate by a needle valve. Beads loaded with protein complexes were separated in the F-1000 system at a channel flow rate of 1 mL/ min and a cross-flow rate of 2 mL/min for 2 min, and then the channel flow rate was increased to 3 mL/min to complete the 7070

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Figure 1. Schematic of the MAPcP process.

separation. Microspheres were collected after the Fl-FFF separation. The captured proteins were eluted off the beads by 20 µL of 0.1 M glycine-HCl at pH 2.5 with 0.05% Tween 20 and subjected to gel electrophoresis (Mini-PROTEAN Tetra Electrophoresis System, Bio-Rad Life Science, Hercules, CA) using SDS-PAGE (12% running gel with 5% stacking gel) without further concentration. The protein bands in the 1D SDS-PAGE gel were visualized with the SilverQuest silver staining kit (Invitrogen). RESULTS AND DISCUSSION Multiplexed Affinity-Based Protein Complex Purification. The whole process of MAPcP is illustrated in Figure 1. Several protein complexes are immunoprecipitated simultaneously by groups of microspheres in one pot. These microspheres are of different sizes. When coupled with different affinity probes, they offer highly specific extractions of multiple complexes. Fl-FFF separates the microspheres based on their sizes after the coextraction to recover the individual complex for downstream analysis. The advantage of using microspheres as the solid support for affinity capture relies on their high surface area-to-volume ratio, which can improve the binding efficiency. Experiments done in our laboratory demonstrated that each 5-µm particle can be coupled with 105 IgG (data not shown), meaning that up to 1012 target molecules (30 ng of protein with Mw 100 kDa) can be extracted if ten million beads are used under ideal conditions. Because the overall volume of beads is about 0.6 µL with a total surface area of 6.35 cm2, a small sample volume with condensed protein concentrations can then be used to enhance extraction efficiency. Additionally, utilization of microspheres simplifies FlFFF separation by selectively choosing their sizes. Employing Fl-FFF in MAPcP offers several advantages. First, Fl-FFF can separate particles based on their size, therefore multiple complexes can be extracted simultaneously using par-

ticles of various sizes. The orthogonal field in Fl-FFF is a crossflow that exits the channel through a wall made of porous membrane. Two elution modes exist in Fl-FFF. When the size of the analyte is smaller than 500 nm, a low to moderate channel flow rate is used to achieve sufficient separation and the elution time increases with size because of the size dependence on the diffusion coefficients, which is called the normal mode.20,21,24 In this mode, because the force generated by the cross-flow is independent of the size and density of the particles but the diffusion force is reversely proportional to the particle size, elution times of the microspheres have well defined relationship with their sizes.20-22 If the analyte size exceeds 2 µm, the diffusion force becomes negligible and a fast channel flow rate is needed to generate a lift force to move the large analyte away from the accumulation wall. In such a steric/hyperlayer mode, larger analytes exit the column earlier than smaller analytes because they experience larger lift force and their mass centers locate at higher levels within the parabolic flow.20,22 In addition, microspheres undergo continuous wash by the two flow streams inside the separation channel, and the proteins bound nonspecifically could be washed off the particles and escape through the porous membrane. More importantly, free protein molecules can be separated from the microspheres very well to prevent the cosedimentation of nonspecific proteins with the particles, a phenomenon that occurs to centrifugation and magnetic pull-down due to the rapid aggregation of particles under the external forces and contributes to the low purity level of IP. Higher purity of the protein complex sample could then be achieved. Further more, Fl-FFF is highly biocompatible.17,18 Impacts of carrier flow composition on size-based separation are not prominent.23 Effective particle separation could be achieved within the pH range of 7-9 and the salt range of 0-100 mM. Buffer compositions also seemed to have negligible impact on the separation.23 Thus, physiological buffers can be used as the carrier flow along with the open separation channel without packing material, well preserving the protein structure and complex integrity. Dual-Glow Condition for Separation of Free Proteins from Particle Populations. Protein molecules are much smaller than 500 nm with large diffusion coefficients. Using one constant flow rate and one elution mode may not be adequate to separate the free proteins from the particle population. The coeluted proteins would then interfere with reliable identification of the interacting proteins in each complex. In order to achieve superior sample purity, we employed a dual-flow separation condition in MAPcP.25-28 The Fl-FFF program starts with a low flow rate to separate the free protein molecules from the microsphere population in a normal elution mode, and then it switches to a higher channel flow rate to resolve the microspheres by size in the steric/ (22) Giddings, J. C. Am. Lab. 1992, 24, 20D, 20F-20M. (23) Li, J.; Zhong, W. J. Chromatogr., A 2008, 1183, 143–149. (24) Contado, C.; Dalpiaz, A.; Leo, E.; Zborowski, M.; Williams, P. S. J. Chromatogr., A 2007, 1157, 321–335. (25) Lee, H.; Kim, H.; Moon, M. H. J. Chromatogr., A 2005, 1089, 203–210. (26) Hee Moon, M.; Williams, P. S.; Kang, D.; Hwang, I. J. Chromatogr., A 2002, 955, 263–272. (27) Williams, P. S.; Giddings, M. C.; Giddings, J. C. Anal. Chem. 2001, 73, 4202–4211. (28) Botana, A. M.; Ratanathanawongs, S. K.; Giddings, J. C. J. Microcolumn Sep. 1995, 7, 395–402.

Figure 2. Demonstration of the separation effect of using the dualflow condition. (a) Separation of HSA using a channel flow of 1 mL/ min and a cross-flow of 2 mL/min; (b) separation of the 3.17- and 5.28-µm particles with the same flow condition as shown in part a; (c) separation of the 3.17- and 5.28-µm particles with the dual-flow condition in which the channel flow rate was switched from 1 to 3 mL/min after the first 2 min while the channel flow rate remained as 2 mL/min; (d) separation of HAS from the particles using the same dual-flow condition as indicated in part c.

hyperlayer mode.The effect is demonstrated in Figure 2. With a channel flow of 1 mL/min and a cross-flow of 2 mL/min (the normal elution mode), human serum albumin (HSA) was eluted within the first 3 min (trace a). Under the same constant flow condition, the 3.17- and 5.28-µm particles were eluted at around 6 min with the smaller particles coming out first (trace b), indicating a normal elution mode even though the particle sizes were larger than 2 µm.22 The deviation of elution order from theoretical prediction may be attributed to the electrostatic repulsive interaction between the cellulose membrane and the particles at pH 7.4. On the contrary, if a dual-flow condition was employed by switching the channel flow rate from 1 to 3 mL/min after the first 2 min, the particles were well resolved after 4 min with the 5.28µm beads eluted first (trace c), indicating the occurrence of the steric/hyperlayer separation mode. The HSA was eluted before the switch of flow rates and well separated from the particles in such a dual-flow condition (trace d). The residue nonspecific proteins could be further cleaned up by the flows and washed away through the porous membrane composing the separation channel wall. We chose a membrane with a molecular weight cutoff size of 100 kDa to allow most of the small protein molecules to escape the channel. Extraction of Two Immuno-Complexes by MAPcP. Once a good separation condition was determined, extraction of two immuno-complexes was performed to demonstrate the effectiveness of MAPcP. One of the complexes was formed between recombinant protein A and mouse monoclonal anti-T7 immunoglobin (MIgG), representing a strong affinity interaction and was extracted by the 4.51-µm microspheres conjugated with human IgG (HIgG). The second complex formed by rabbit antigreen fluorescence protein (GFP) IgG (RIgG) and GFP was isolated by the 6.7-µm microspheres coupled to protein G, an example of the typical antigen-antibody interaction. Protein A and GFP are also common affinity tags used in immunoprecipitation.8,13,29-31 Incu(29) Waugh, D. S. Trends Biotechnol. 2005, 23, 316–320. (30) Stahl, S.; Hober, S.; Nilsson, J.; Uhlen, M.; Nygren, P.-A. Biotechnol. Bioprocess. 2003, 27, 95–129. (31) Chang, I.-F. Proteomics 2006, 6, 6158–6166.

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Figure 3. Extraction of two immuno-complexes from crude yeast cell lysate. (A) Fractogram of microsphere separation by Fl-FFF after complex extraction. Particles were collected from each peak to retrieve the individual complex. (B) SDS-PAGE of each complex recovered from particles separated by Fl-FFF or collected by centrifugation. GFP complex from MAPcP (lane 1) or from centrifugation (lane 2); Protein A complex from MAPcP (lane 3) or from centrifugation (lane 4); lane L, protein molecular weight marker.

bation of microspheres with their corresponding target proteins was done in the whole cell lysate prepared from 500 mL of yeast cell suspension (OD600 ∼ 3). Because of the high affinity between the protein G- and HIgG-coupled microspheres, in this proof-ofprinciple study the particles were incubated separately and then mixed before Fl-FFF separation. Figure 3A is the fractogram for the two particles after complex extraction, showing that effective particle separation was still obtained even though the particle surface was loaded with large protein complexes. Each particle fraction was collected individually, and the proteins were then eluted for SDS-PAGE analysis (Figure 3B). The cleanup effect of MAPcP was compared to that from five stringent washes with centrifugation. Figure 3B clearly illustrates that the recovered protein complexes remained intact during Fl-FFF. Proteins involved in each complex (recombinant protein A ∼ 42 kDa, IgG heavy chain ∼ 50 kDa and light chain ∼ 25 kDa, GFP ∼ 27 kDa) showed up clearly in both fractions prepared by either MAPcP or centrifugation with comparable amounts. Since we used a 7072

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membrane with a molecular weight cutoff size of 100 kDa, small proteins like GFP should have been washed off through the membrane and not retained in the channel if it was free. The recovery of GFP after Fl-FFF strongly indicated that GFP remained attached onto the particle surface during the fractionation, and the Fl-FFF process did not break down the interactions between proteins. Furthermore, the extracted protein complexes were remarkably clean. Most of the nonspecific protein bands (marked by /) disappeared in the samples prepared from Fl-FFF. The protein G coupling might render a surface with less nonspecific binding than the HIgG conjugation, and fewer noninteracting proteins were observed in the fraction extracted by the protein G beads. Even though the particles were incubated separately, the particles carrying two protein complexes were well resolved in Fl-FFF while the complex structure remained intact. This example clearly demonstrated that MAPcP could be applied to extract two protein complexes simultaneously, which makes it possible to contemporarily compare the compositions in complexes formed by different bait proteins or various protein domains. In situations of purification of endogenous protein complexes, particles will be coupled to antibodies targeting different bait proteins and the cross-reactivity between protein G and IgG will not exist, allowing coincubation of particles within the same sample. No tandem purification step was needed to achieve high sample purity, which dramatically simplified the extraction process and lowered down the sample consumption. Even though six rounds of separation and collection were needed here to fractionate the 10 million microspheres used to obtain enough proteins for downstream analysis, each round took less than 10 min and the whole process was finished within 1 h after the affinity binding, which is much shorter than TAP and should effectively avoid sample decomposition and protein degradation. We also evaluated the reproducibility of our method by conducting three repeated extraction of protein A-IgG complex from crude yeast cell lysate. High sample purity was consistently obtained with MAPcP. Additionally, we measured the intensities of the protein bands on the gel images using Adobe Photoshop CS3 (Microsoft) and calculated the average percentages of proteins recovered from MAPcP using 100% for the recovery from centrifugation. They were 101% ± 14% for protein A and 93% ± 7% for IgG, respectively. The minimal protein loss was determined to be originated from the particle loss probably inside the Fl-FFF channel or during the collection procedure. The particle loss was estimated by weighing the particles after protein elution. The particles were dried in the oven overnight at 37 °C before weighing and about 91% ± 6% (n ) 4) particles were recovered from MAPcP compared to that from centrifugation (100%). These results indicate that MAPcP leads to comparable sample recovery to the centrifugation method but yields much higher sample purity after the extraction, which will be beneficial to the identification of genuine protein interactions in protein complex analysis. Compatibility of MAPcP with Magnetic Pull-Down. In the above study, MAPcP was compared to the centrifugation-based wash and showed higher effectiveness in sample cleanup. However, magnetic particles are universally used in immunoprecipitation due to the simplicity in handling.11,32-35 Therefore, we (32) Berna, M. J.; Zhen, Y.; Watson, D. E.; Hale, J. E.; Ackermann, B. L. Anal. Chem. 2007, 79, 4199–4205.

compared the performance of MAPcP with that of the magnetic pull-down. The commercially available magnetic microparticles have a mass density of 1.50 g/mL and cannot be suspended freely in aqueous solutions, which prevents them from being well separated by simple Fl-FFF methods. Instead, we modified the polystyrene particle surface with the 10-nm polyacrylate-capped magnetite (Fe3O4) nanocrystals.36 The nanocrystals were first conjugated to HIgG through the carboxyl groups of the polyacrylate coating. We estimated the amount of HIgG conjugated to the nanocrystal using the fluorescent labeled rabbit antihuman IgG (data not shown). Measurement of the residue fluorescent Ab in the solution after immuno-reaction with the HIgG-conjugated nanocrystal and removal of the nanocrystal indicated that one nanocrystal was linked to 1.5 HIgG molecules, which could act as the linker between the nanocrystal and the microsphere. Therefore, another carbodiimide-mediated conjugation reaction was carried out between the HIgG coupled nanocrystals and the carboxylated microspheres. The TEM image (Figure 4A) clearly shows that a thin layer of the nanocrystals was laid on top of the microsphere after our treatment. Even though lumps of nanocrystals were observed occasionally along the microsphere surface probably due to the aggregation of nanocrystals during conjugation with HIgG, the modified particles readily responded to the magnetic field imposed by a NdFeB magnet and could easily be separated in Fl-FFF (Figure 4B). We then applied them to extract the protein A complex from the crude yeast cell lysate. Comparison was made among magnetic pull-down of the regular 5-µm magnetic particles or the modified particles and MAPcP of the modified particles. From the SDSPAGE results of the eluted proteins (Figure 4C), we can see both the magnetic pull-down procedures gave rise to a very high protein background, probably due to the high affinity of magnetic particles to proteins.37,38 Nevertheless, none of the interfering bands showed up in the fraction prepared by MAPcP, and the proteins involved in the complex, protein A and MIgG, were recognized more easily. This result demonstrates that MAPcP could result in much purer protein complex sample than the magnetic pulldown method because of the dual-flow method and the continuous wash condition in Fl-FFF, which should improve the accuracy in identification of the interacting proteins. In addition, particles modified with magnetic nanocrystals could be used in MAPcP to facilitate sample handling. CONCLUSIONS Our proof-of-principle study demonstrates that MAPcP can isolate multiple protein complexes simultaneously. High sample purity is also obtained to facilitate reliable identification of interacting proteins compared to the conventional particle-based (33) Ye, X.; Poustovoitov, M.; Santos, H.; Nelson, D. M.; Adams, P. D. Methods Mol. Biol. (Totowa, NJ, U.S.) 2004, 281, 261–270. (34) Ebert, M. P. A.; Niemeyer, D.; Deininger, S. O.; Wex, T.; Knippig, C.; Hoffmann, J.; Sauer, J.; Albrecht, W.; Malfertheiner, P.; Roecken, C. J. Proteome Res. 2006, 5, 2152–2158. (35) Yaneva, M.; Tempst, P. Methods Mol. Biol. (Totowa, NJ, U.S.) 2006, 338, 291–303. (36) Ge, J.; Hu, Y.; Biasini, M.; Dong, C.; Guo, J.; Beyermann, W. P.; Yin, Y. Chem.sEur. J. 2007, 13, 7153–7161. (37) Li, Y.; Leng, T.; Lin, H.; Deng, C.; Xu, X.; Yao, N.; Yang, P.; Zhang, X. J. Proteome Res. 2007, 6, 4498–4510. (38) Chen, C.-T.; Chen, Y.-C. Anal. Chem. 2005, 77, 5912–5919.

Figure 4. (A) TEM image of the polystyrene microsphere modified by paramagnetic nanoparticles. (B) Size-based separation of the nanocrystal modified microspheres. The dual-flow condition was employed here. (C) SDS-PAGE of proteins extracted through three different procedures. Lane 1, proteins extracted by regular magnetic particle with the magnetic pull-down procedure; lane 2, proteins extracted by the modified polystyrene microspheres with the magnetic pull-down procedure; lane 3, proteins extracted by the modified polystyrene microspheres with the MAPcP procedure; lane L, protein molecular weight marker.

IP with centrifugation or magnetic pull-down. By isolating multiple protein complexes in one preparation, we eliminate variations brought in by different processes and could achieve more accurate comparison of protein complexes in their compositions. MAPcP may also facilitate study of the dynamic changes upon external stimuli on the cells. Such information will be extremely valuable for better understanding of the function of protein complexes in the cell. Moreover, MAPcP is compatible with magnetic particles which makes sample handling much easier. Even though the magnetic nanocrystal-modified polystyrene particles were emAnalytical Chemistry, Vol. 80, No. 18, September 15, 2008

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ployed in our demonstration, with an improved Fl-FFF method, regular magnetic particles could be separated.39 Successful separation of 10 polystyrene microspheres within the size range of 1-40 µm was achieved by Fl-FFF;40 using the current system and running conditions, we have achieved baseline separation of three types of protein-conjugated microspheres (6.17-, 5.28- or 4.51, and 3.17-µm), both pointing out the possibility of using MAPcP to target a number of protein complexes (up to three with current conditions) if necessary. Each separation takes only 10 min, and with 6-5 rounds of collection, the entire fractionation-based “wash” takes about 1 h. Employment of filter microplates and vacuum assemblies can automate the process, further shortening the processing time and reducing the sample handling. Even though MAPcP showed negligible effects on the integrity of the immuno-complexes used in our demonstration, interacting proteins could be lost due to their low affinity to the bait protein, as also observed in the presently available immunoprecipitation methods. In such cases, in vivo protein cross-linking can be employed with cell-membrane-permeable cross-linkers and (39) Jiang, Y.; Miller, M. E.; Hansen, M. E.; Myers, M. N.; Williams, P. S. J. Magnet. Magnet. Mater. 1999, 194, 53–61. (40) Ratanathanawongs, S. K.; Giddings, J. C. Anal. Chem. 1992, 64, 6–15.

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careful optimization of the cross-linking conditions.41-43 Moreover, MAPcP can be combined with subcellular fractionation techniques like the differential gradient centrifugation to enrich the target complexes before extraction and assist the detection of low abundant interacting proteins within the complexes.

ACKNOWLEDGMENT W.Z. thanks the University of California, Riverside, for startup funds, and Dr. Mingtang Xie and Dr. Yong-Jik Lee in the Department of Botany and Plant Sciences for help with yeast cell preparation and gel electrophoresis.

Received for review June 18, 2008. Accepted July 25, 2008. AC801251Y (41) Tagwerker, C.; Flick, K.; Cui, M.; Guerrero, C.; Dou, Y.; Auer, B.; Baldi, P.; Huang, L.; Kaiser, P. Mol. Cell. Proteomics 2006, 5, 737–748. (42) Agou, F.; Ye, F.; Veron, M. Methods Mol. Biol. (Totowa, NJ, U.S.) 2004, 261, 427–442. (43) Fancy, D. A. Curr. Opin. Chem. Biol. 2000, 4, 28–33.