Synergistic Design of Electric Field and Membrane in Facilitating

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Synergistic Design of Electric Field and Membrane in Facilitating Continuous Adsorption for Cleanup and Enrichment of Proteins in Direct ESI-MS Analysis Yu Zhou,† Hailin Shen,‡ Tie Yi,§ Dawei Wen,† Nannan Pang,† Jie Liao,| and Huwei Liu*,† Beijing National Laboratory for Molecular Sciences, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, Institute of Analytical Chemistry, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China, Manchester Interdisciplinary Biocentre, the University of Manchester, Manchester M1 7DN, United Kingdom, College of Information Science and Engineering, Central South University, Changsha 410083, China, and Medical Experiment and Analysis Center, General Hospital of Chinese PLA, Beijing 100853, China We designed and fabricated a novel microdevice to facilitate continuous adsorption phenomena for biological sample preparation. Using the device, we also developed an online, highly integrated, multifunctional strategy, with a promise of accepting a large volume of crude tissue extracts with the end point generation of a reliable MS identification within 20 min. Under an external electric field, charged membranes can adsorb multiple layers of proteins, which exceed the capacity limit of common resins or membranes. It enlarges sample loading and trapping efficiency, thus bypasses the tradeoff between sample capacity and downstream detection sensitivity. This integrated approach, formed by synergistic utilization among electric field, membrane, and fluidic handling at the microscale, reduces the overall complexity of crude samples in one step for direct MS analysis. The sample preparation goals, including enrichment, desalting, removal of noncharged contaminants, and initial fractionation, can be rapidly performed in a single device. The strategy facilitates reproducible MS quantification by circumventing traditional laborious and time-consuming sample preparation steps. In addition, MEPD extended the ion trap linear dynamic range from 2 to at least 4 orders of magnitude by eliminating ion suppression effect, enriching target analyte(s), and decreasing sample loss during integrated sample preparation. Mass spectrometry (MS), despite its success in proteomics, still faces significant technical challenges due to the complex nature of biological samples and the ion suppression in MS ion source. Reducing sample complexity and obtaining sufficient quantities of target proteins from its biological source are critical

steps for proteomic research.1,2 However, it is hard to achieve required sample purity in a single step in the present, and a series of purification procedures is commonly used to convert a raw biological sample into a purified product. Thus, trade-offs need to be made between sample quality and throughput based on the type of analysis to be performed. The challenge is made more complex by the protein’s instability and modifications. For example, laborious and time-consuming sample handlings lead to the risk of sample losses due to proteolytic degradation and modification.3 Success in proteomics depends upon careful study design and high-quality biological samples.4 Advances in chromatographic and electrophoretic techniques,5-9 such as nano HPLC,10 UPLC,11 MudPIT,12 MDLC,13 monolithic columns,14,15 and miniaturization of total analysis systems on a chip,16 etc., have opened up exciting possibilities to get comprehensive fractionation. However, no technique is suitable to accept crude extracts as starting materials. Crude extracts from biological origins are often turbid and contain lipid droplets; hence, without sample preparation procedures including desalting, buffer exchange, matrix removal, and target enrichment, they are seldom (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12)

* To whom correspondence should be addressed. E-mail: [email protected]. Phone: (+) +86-10-62754976. Fax: (+) +86-10-62751708. † Peking University. ‡ University of Manchester. § Central South University. | General Hospital of Chinese PLA.

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(13) (14) (15) (16)

Domon, B.; Aebersold, R. Science 2006, 312, 212–217. Cravatt, B. F.; Simon, G. M.; Yates, J. R., III Nature 2007, 450, 991–1000. Gorg, A.; Weiss, W.; Dunn, M. J. Proteomics 2004, 4, 3665–3685. Boguski, M. S.; McIntosh, M. W. Nature 2003, 422, 233–237. Ye, M.; Jiang, X.; Feng, S.; Tian, R.; Zou, H. TrAC, Trends Anal. Chem 2007, 26, 80–84. Cooper, J. W.; Wang, Y.; Lee, C. S. Electrophoresis 2004, 25, 3913–3926. Righetti, P. G.; Castagna, A.; Antonioli, P.; Boschetti, E. Electrophoresis 2005, 26, 297–319. Wittmann-Liebold, B.; Graack, H.-R.; Pohl, T. Proteomics 2006, 6, 4688– 4703. Kraly, J.; Fazal, M. A.; Schoenherr, R. M.; Bonn, R.; Harwood, M. M.; Turner, E.; Jones, M.; Dovichi, N. J. Anal. Chem. 2006, 78, 4097–4110. Bonneil, E.; Tessier, S.; Carrier, A.; Thibault, P. Electrophoresis 2005, 26, 4575–4589. Motoyama, A.; Venable, J. D.; Ruse, C. I.; Yates, J. R., III Anal. Chem. 2006, 78, 5109–5118. Link, A. J.; Eng, J.; Schieltz, D. M.; Carmack, E.; Mize, G. J.; Morris, D. R.; Garvik, B. M.; Yates, J. R., III Nat. Biotechnol. 1999, 17, 676–682. Wagner, K.; Unger, K. K. Bioforum Int. 2001, 5, 116–119. Svec, F. Electrophoresis 2006, 27, 947–961. Josic, D.; Clifton, J. G. J. Chromatogr., A 2007, 1144, 2–13. Fortier, M.-H.; Bonneil, E.; Goodley, P.; Thibault, P. Anal. Chem. 2005, 77, 1631–1640. 10.1021/ac800816k CCC: $40.75  2008 American Chemical Society Published on Web 10/28/2008

suitable for direct loading onto chromatographic columns, a MS, or a microchip, due to the possibility of deteriorating separation efficiency and causing irreparable column damage and fouling.17 Current initial sample clarification must rely on traditional methods, including fractional precipitation, phase-partition, ultrafiltration, and dialysis.18 It is therefore difficult to achieve high throughput and automation because, in general, all procedures involve manual intervention and are labor-intensive and timeconsuming. Microfabricated devices promise to be the automated and integrated technology for bimolecular sample processing.19-21 Various preconcentration elements (including chromatographic,22 electrophoretic,23 membrane concentration24,25) and purification steps (separation,26 desalting,27 and detergent removal), as well as chemical reactions (digestion28 and labeling29), have been exploited and integrated into microfluidic platforms for biomolecule analysis. Several strategies cleverly utilize electrophoretic mobilities, and some with the aid of membranes, to construct electrokinetic trapping or sweeping for online electrophoretic concentration. Significant concentration performances are demonstrated as an increase of 1000-fold or even as high as 106-108.25,30-32 Despite those great advancements, the integration of current microfabricated devices with the mainstream MS platforms in proteomics poses additional challenges.33 First, the microfabricated devices suffer an intrinsic scaling problem: the tradeoff between limited sample capacity and downstream detector sensitivity due to limited amount of sample handling.34 Second, efficient electrokinetic sample processing, originally designed for CE with optical detector, often requires special buffer and reagent arrangements and can not tolerate complex sample condition. For example, high conductive samples result in obvious joule heating which may impair focus or concentration performance. Their online couplings with ESI-MS are difficult due to various operational constraints, such as buffer requirements, flow rate compatibilities, and scaling problems. In addition, methods using elec(17) Mukhopadhyay, R. Anal. Chem. 2005, 77, 429A–432A. (18) Visser, N. F. C.; Lingeman, H.; Irth, H. Anal. Bioanal. Chem. 2005, 382, 535–558. (19) Lazar, I. M.; Grym, J.; Foret, F. Mass Spectrom. Rev. 2006, 25, 573–594. (20) Freire, S. L. S.; Wheeler, A. R. Lab Chip 2006, 6, 1415–1423. (21) Lichtenberg, J.; de Rooij, N. F.; Verpoorte, E. Talanta 2002, 56, 233–266. (22) Yu, C.; Davey, M. H.; Svec, F.; Frechet, J. M. J. Anal. Chem. 2001, 73, 5088–5096. (23) Kim, S. M.; Burns, M. A.; Hasselbrink, E. F. Anal. Chem. 2006, 78, 4779– 4785. (24) Khandurina, J.; Jacobson, S. C.; Waters, L. C.; Foote, R. S.; Ramsey, J. M. Anal. Chem. 1999, 71, 1815–1819. (25) Liu, J.; Sun, X.; Farnsworth, P. B.; Lee, M. L. Anal. Chem. 2006, 78, 4654– 4662. (26) Rocklin, R. D.; Ramsey, R. S.; Ramsey, J. M. Anal. Chem. 2000, 72, 5244– 5249. (27) Xu, N.; Lin, Y.; Hofstadler, S. A.; Matson, D.; Call, C. J.; Smith, R. D. Anal. Chem. 1998, 70, 3553–3556. (28) Astorga-Wells, J.; Bergman, T.; Joernvall, H. Anal. Chem. 2004, 76, 2425– 2429. (29) Lee, C.-C.; Sui, G.; Elizarov, A.; Shu, C. J.; Shin, Y.-S.; Dooley, A. N.; Huang, J.; Daridon, A.; Wyatt, P.; Stout, D.; Kolb, H. C.; Witte, O. N.; Satyamurthy, N.; Heath, J. R.; Phelps, M. E.; Quake, S. R.; Tseng, H.-R. Science 2005, 310, 1793–1796. (30) Wang, Y.-C.; Stevens, A. L.; Han, J. Anal. Chem. 2005, 77, 4293–4299. (31) Zhou, K.; Kovarik, M. L.; Jacobson, S. C. J. Am. Chem. Soc. 2008, 130, 8614–8616. (32) Wu, X. Z.; Hosaka, A.; Hobo, T. Anal. Chem. 1998, 70, 2081–2084. (33) Koster, S.; Verpoorte, E. Lab Chip 2007, 7, 1394–1412. (34) Whitesides, G. M. Nature 2006, 442, 368–373.

trokinetic injections suffer from injection biases, which are, to some extent, incompatible with a quantitative method. Those issues preclude them to be practical clarification tools and limit their applications in direct coupling to current MS-based strategies. In fact, the challenge of sample preparation exists throughout the whole proteomics pipeline, not only prior to but also between and after the multidimensional separation. If multiple dimensions are to be coupled online, the compatibility requirement becomes more stringent. Sample preparation, especially rapid desalting and buffer exchange, is highly desirable for true high-throughput proteomics.35 Ideally, all aspects of the preparation goals, from enrichment, removal of contaminants, and desalting to fractionation, can be rapidly performed on a single device, and the purified samples can be smoothly transferred to downstream MS analysis. To solve those problems, we tried to explore a new concept, continuous adsorption phenomena,36 to design a microelectro purification device (MEPD) for an online, automated, highly integrated startto-finish strategy for biological sample preparation with quantitative sample recovery and smooth interface with online ESI-MS analysis. Unlike online electrophoretic concentration methods mainly exploiting electrophoretic mobility, we utilized electric field and conductive membranes in a different way. With a flow-through format and the aid of rapid three layer fluidic handling controlled by a commercially available HPLC liquid delivery system and workstation, the synergy among electric field, membrane, and multiple-layer fluidic handling facilitates continuous adsorption phenomenon and an all-in-one integrated sample preparation strategy at the microscale. Enrichment, desalting, removing noncharged contaminants, buffer exchange, and prefractionation can be preformed on demand simultaneously or sequentially in one device. With charged membranes as templates, the electric field suppresses protein-protein electrostatic repulsion to facilitate and stabilize multiple layer adsorptions. This phenomenon has the potential to enlarge sample capacity, thus bypasses the tradeoff between limiting sample loading capacity and downstream detector sensitivity. This technique has the potential to speed up and automate current time-consuming and laborious initial sample preparation for MS analysis. These features, together with large sample loadability, low-cost fabrication procedures, tolerance to sample turbidity, simple device regeneration, and ability for direct integration into commercially available instruments, suggest a promise to improve throughput and data quality in proteomics fractionation, especially in initial sample clarification and target protein enrichment. EXPERIMENTAL SECTION Chemicals and Reagents. HPLC-grade solvents including acetonitrile and methanol were obtained from Fischer Scientific (Fair Lawn, NJ), and cytochrome c (horse heart) and myoglobin (horse heart) were obtained from Sigma-Aldrich (St. Louis, MO). Bradford assay (Coomassie blue protein assay) was from Applygen Technologies Inc. (Beijing, China). All other chemicals and (35) Huck, C. W.; Bakry, R.; Bonn, G. K. Curr. Proteomics 2005, 2, 269–285. (36) Ngankam, A. P.; Van Tassel, P. R. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 1140–1145.


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Figure 1. Protein analysis using a microelectric purification device. (a) MEPD expanded view, showing MEPD layers; (b) instrument configuration of MEPD; (c) illustration of a highly integrated sample preparation strategy using MEPD device in a continuous flow through format. (Stage 1: concentration, desalting, and noncharged contaminants removal; here the membrane surface with grafted functional groups adsorbs multiple layers of proteins under an electric field. Stage 2: protein release and fractionation by organic solvent gradient; Stage 3: device regeneration with the mobile phase containing a high content of organic solvent while reversing the potential direction). (d) A typical total ion chromatogram (TIC) spectra of a crude porcine heart tissue extract (extracted by 150 mM TCA solution without further desalting) processed by the MEPD as shown in part c. (e) ESI mass spectra at elution time indicated in part d. (Left panel, charge states for 12 kDa protein; right panel, charge states for 14 kDa protein).

solvents were of analytical grade. All the reagents were used without further purification. MEPD Unit Fabrication. As shown in Figure 1a, The MEPD unit was in the form of a set of thin, flat sheet channels. Differential etch depths were achieved on 30 mm × 30 mm × 0.6 mm polycarbonate chips using a Legend 36 EXT excimer laser directwrite micromachining system (EPILOG).37 The channel (50 µm wide × 600 µm deep) was produced by laser cutting around the 8922


channel perimeter followed by stripping the remaining polymer from the center. Polymer slides with microchannels were used for fluidic channels and seals. With the device held in place using stainless steel knurled-head screws, two ion exchange membranes, two electrodes, and three polymer slides with microchannels were (37) Pugmire, D. L.; Waddell, E. A.; Haasch, R.; Tarlov, M. J.; Locascio, L. E. Anal. Chem. 2002, 74, 871–878.

mounted as in Figure 1a. Two ion exchange membranes were sandwiched between three polymer layers with flow channels (50 µm wide, 40 mm long, 600 µm deep), confining the shapes of sample stream and buffer streams. The pressure from the sample stream would enlarge the membrane functional area. The membrane supports were polystyrene cross-linked with divinylbenzene, in which sulfonic and quaternary ammonium functional groups form strong ion exchanger. Membranes were about 0.2 mm thick and reinforced with a screen to provide mechanical stability. The membranes, which were made in flat sheets, contained 30-50% water and had a network of pores too small to permit water transport. Sample fluid access was provided by mechanically drilling through the chips of headers at either end of the microchannels. Fused silica capillary tubing was directly mounted on the access by using thermal bonding and using finger tight adapter to construct the micro-to-macro interface. System Setup. The system to interface with MS has three main components: the MEPD unit, voltage controller, and liquid delivery system (Figure S-1 in the Supporting Information). The MEPD is the core of the system (Figure 1a), which confines the shapes of the sample stream (middle channel in Figure 1a) and buffer streams (upper and lower channels in Figure 1a). Buffer streams are circulated through the MEPD unit via peristaltic pumps from a buffer tank (10 mM NH4HCO3) outside MEPD. A homemade power supply was used to create a variable electric field perpendicular across the microchannels of the device, to adjust the force that acted on the charged particles or dipoles inside the microchannel. Positively charged ions moved toward the cation-selective membranes while negatively charged ones migrated toward the anion-selective membrane. The convective flow-through design enables online coupling the MEPD system to many analysis processes performed in a continuous-flow format. With capillary tubing as the micro-to-macro interface, there are many possible configurations (Figure 1b) for connecting the MEPD with sample introduction modules and detectors. Owing to its modular design, we facilely integrated the MEPD unit into a commercial LC-MS system, which featured fast and reproducible sample loading using an autosampler to improve sample throughput. The sample stream and solvent gradient were fully controllable by workstation software and an HPLC pump and could be optimized for different types of samples to be processed. The counterflow buffer streams helped to remove salt contaminant retained on membranes and electrodes. In a typical experiment, the complex sample mixture was injected into a stream of media flow (0.1% formic acid) and loaded onto the MEPD by the autosampler, and then concentration and desalting were accomplished rapidly and simultaneously. After the purification stage, by switching off or changing the MEPD voltage potential, target proteins were fractioned and eluted out off the MEPD into the mass spectrometer using a linear gradient from 0%-30% solvent B over 10 min at 0.3 mL min-1. Mobile-phase buffers were for solvent A, 0.1% aqueous formic acid; for solvent B, 0.1% formic acid in acetonitrile/water (80:20). An LC column can be coupled with the MEPD to extend the separation dimension. To remove the contaminating proteins and other molecules from the MEPD, a solution of high organic solvent content (50%

acetonitrile in water, v/v) was applied to the MEPD for regeneration after each processing. Crude Tissue Extract Preparation. To isolate the soluble fraction of the heart muscle, 100 g of heart tissue was homogenized in 100 mL of 150 mM trichloroacetic acid (TCA). The homogenized sample was centrifuged at 4000g for 20 min at 4 °C. The supernatant fraction, containing soluble protein, was then collected as starting materials for online MEPD-ESI-MS analysis to get direct protein identification. The centrifugation step separated the soluble protein from the membrane fraction and insoluble cell debris. The cytochrome c in crude extracts was purified38 and measured using a Bradford assay (Coomassie blue protein assay) according to the manufacturer’s protocol. C18 Reversed-Phase Column Desalting. To compare with the MEPD, the samples were desalted on a C18 reversed-phase (RP) LC column, 4.6 mm × 150 mm column packed with 5 µm C18 sorbent (BonChrom C18 column), which was employed in tandem with ESI-MS.39 The HPLC system was connected in-line to an orthogonal ESI mass spectrometer via a six-port, two-position switching valve, allowing desalting followed by MS analysis of intact proteins. The protein solution was injected onto a RP-LC column. An aqueous buffer was used to elute the salts while the proteins were concentrated at the top of the column. After elution of the salts, the proteins were eluted with H2O-acetonitrile mobile phase according to a typical desalting protocol.40 Elution condition was 100% eluent A for 6 min, then 100% eluent B for 6 min at flow rate of 1 mL min-1. Eluent A was 0.1% aqueous formic acid. Eluent B was 0.1% formic acid in acetonitrile. MS and Data Analysis. All experiments were performed using an Agilent XCT ion trap mass spectrometer with an HP1100 solvent delivery system (Agilent, Palo Alto, CA). Crude samples were loaded onto the MEPD by an autosampler, then purified and analyzed by online MEPD-ESI-MS. For all measurements, the mass spectrometer was operated in ultrascan-mode with a typical resolving power of at least 1000. All analyses were performed using the positive mode, and the m/z range covered a full mass window (500-1200 Da). MS data was processed using DataAnalysis 3.3 (Bruker Daltonik GmbH) for an Agilent ion trap MS, and peak lists were generated using the software’s default parameters. The model protein’s spectra were deconvoluted, and protein molecular masses were determined from the series of multiply charged ions by using the component analysis function of DataAnalysis 3.3. The quantitative result can be measured by spectral count, peak area intensity (or intensity ratio) of the two most intense peaks (or peak pairs) in the protein multiply charged series. The mass error tolerance values were typically under 300 ppm. At 300 ppm mass accuracy, there is sufficient mass specificity to resolve different model proteins with known molecular weight. With this level of mass accuracy, confident identifications can be made in the instances of coeluting proteins. RESULTS AND DISCUSSION MEPD Principle and All-in-One Strategy. As a proof of allin-one integrated sample preparation strategy (Figure 1c), the MEPD was applied to the direct identification of the cytochrome c present in a crude extract in TCA solution (150 mM) from Analytical Chemistry, Vol. 80, No. 23, December 1, 2008

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porcine heart tissue. The preparation of starting material from tissue has only two steps, including disrupting tissue in a suitable buffer (150 mM trichloroacetic acid) and centrifuging the crude extract (4000g, 20 min). The crude extract, at extreme pH value, contained a high content of salts, cell debris, and lipids, which were incompatible to common chromatographic methods or MS analysis without further clarification and desalting. Here we could use it as the starting material for direct online MEPD-ESI-MS analysis. The integrated sample preparation strategy can be divided into three sequential stages prior to MS analysis. In stage 1, concentration, desalting, and depletion of noncharged contaminants are carried out rapidly and simultaneously. By controlling electric field (under a constant potential on the electrode of 3 V in this example), the membranes work as a selective separation barrier and absorber simultaneously, which retains proteins while it allows the transport of small molecules. Buffer (salt) exchange is carried away by the counter-flow buffer stream. Neutral species, such as glucose, cell debris, and lipids, do not respond to the electric field, and as a result, they are delivered to waste. An electric field can accelerate mass transport, provide additional driving force needed in adsorption and release, and suppress protein-protein electrostatic repulsion to facilitate multiple layer adsorptions. The minimization design also leads to fast processing. At this stage, the sample stream flows through MEPD to waste to avoid potential instrument contamination. In stage 2, the adsorbed proteins are released and fractionated using a linear organic gradient from 0%-30% solvent B (0.1% formic acid in 80% acetonitrile and 20% H2O) over 10 min at 0.3 mL min-1. The solvent A is 0.1% formic acid in H2O. Elutes are compatible to ESI and switched from waste to the ESI source for direct MS analysis. In addition, we expect that solvent gradients, electric potential, solution ionic strength, and pH are other variables that can be used to further tune the conditions, enabling a rational design of reversible surfaces with a very wide range of adsorption and desorption capabilities. Stage 3 is for regenerating the MEPD. Membrane fouling poses a big problem for every micro and membrane device.17 To solve this problem, we flush the device online with the mobile phase containing a high content of organic solvent while reversing the potential direction after every run, with the elutes delivered to waste. To extend the lifetime and performance, the device should be washed offline with high ionic strength buffer (2 M NaCl) after every analytical batch. We evaluated the MEPD’s clarification performance by using online ESI-MS analysis, because ESI is sensitive to sample purity. Sample matrix, coeluting compounds, and cross-talk can contribute to ion suppression and lead to deleterious effects or even a failed MS analysis. At intact-protein level measurements, achieving high quality charge-state distribution is a prerequisite for assuring confidence in all other downstream parameters, including confidence either

in identification or in quantification.41 Figure 1d shows the typical elution profile of the crude extract processed by online MEPDESI-MS. The corresponding spectra are shown in Figure 1e. Parts d and e of Figure 1 indicated that high-quality spectra were obtained within 15 min. With little background noise, the spectra were easily deconvoluted and protein molecular masses were determined from the series of multiply charged ions. In contrast, with no treatment or with ZipTip sample pretreatment (according to the manufacturer’s protocol), we got failed MS analysis (data not shown). The results show that the MEPD can reduce overall sample complexity in one step and provide samples with enough purity and amount compatible to direct ESI-MS analysis. However the crude extract contains thousands of proteins, and only two proteins can get a prominent MS charge-state distribution. The major reason should be the signals of low abundance proteins are seriously suppressed due to coeluting with high concentration proteins. For complete separation of thousands of proteins after treatment by MEPD, one or two-dimensional chromatographic techniques have to be used. In fact, for every separation technique, there are compromises among speed, resolution, recovery, and capacity. At the initial clarification stage, enrichment and cleanup are best accomplished using a rapid procedure with high capacity and some degree of resolution, but the resolution is not a necessity at this stage. Rapid sample handling avoids the risk of sample losses due to proteolytic degradation and modification. High sample loading and trapping efficiency both facilitate the enrichment of low abundance proteins from a large volume of starting materials and provide enough amounts for downstream comprehensive separation. As a result of the convective flow of solutes through porous membranes, the MEPD exhibits low back pressure, short residence times, and high volumetric throughputs when compared to conventional chromatographic packed beds. In a trapping mode, this structure facilitates large sample loadability with no limit in theory. For example, we can facilely load microliter level volumes by the autosampler, milliliter by the syringe pump, and volumes larger than the milliliter by liquid pump. By loading a larger volume of sample, we could improve the signal-to-noise ratio significantly (Figure 2). The result indicates that further sensitivity improvement is achievable using a large volume of starting material, which will facilitate processing low abundance proteins. Without an amplification technique in analogy to PCR, low-abundance proteins can be analyzed only if a large amount of starting material, such as a cell or tissue extract, is used.42 The trapping capacities could be estimated by continuously applying sample on the device to the point where the control sample starts to leak. Sample leakages were monitored by an increase of MS response in flow-through fractions. According to the manufacturer’s specification following the ion-exchange mechanism, we estimated the theoretical maximum binding capacity of the ion-exchange membrane to be 6 µg mm-2. In contrast, in a case study, under an applied electric potential (10 V), 100 µL of control sample (cytochrome c, 1 mg mL-1 in 20 mM NH4HCO3) was injected into a stream of media flow (0.1% formic acid, 0.2

(38) Keilin, D.; Hartree, E. F. Proc. R. Soc. London, Ser. B 1937, 122, 298–308. (39) Pohl, T.; Kamp, R. M. Anal. Biochem. 1987, 160, 388–391. (40) Simpson, R. J. Proteins and Proteomics: A Laboratory Manual; Cold Spring Harbor Laboratory Press: New York, 2003; pp 265-268.

(41) Hayter, J. R.; Robertson, D. H. L.; Gaskell, S. J.; Beynon, R. J. Mol. Cell. Proteomics 2003, 2, 85–95. (42) Gygi, S. P.; Corthals, G. L.; Zhang, Y.; Rochon, Y.; Aebersold, R. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 9390–9395.

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Figure 2. Online MEPD-ESI-MS analysis using a small volume injection and large volume infusion, demonstrating the enrichment effect that can be obtained by large volume sample loading using an LC pump or syringe pump. (a) Standard protein mixture (20 µL, 100 pg µL-1 cytochrome c mixture in 100 mM NH4HCO3) is loaded onto the MEPD by the autosampler. S/N ) 6 at m/z 688. (b) The standard protein mixture is continuously loaded by pumping sample at a flow rate of 0.1 mL min-1 for 10 min (100 pg µL-1 cytochrome c mixture in 100 mM NH4HCO3). S/N ) 68 at m/z 688.

mL min-1) and loaded onto the MEPD without leakage. The trapping capacity can be estimated, from the ratio of sample load to area of ion exchange membrane (∼2 mm2), to be 50 µg mm-2. The result exceeded the maximum limit of the ion-exchange mechanism, indicating formation of multiple layer adsorptions. The assumption is consistent with the result from the optical waveguide lightmode spectroscopy.36 We tried various conditions, and the adsorptions varied with several parameters including electric field strength, medium flow rate, nature of the samples, and pH environment. The general trends are that the increase in potential strength and the decrease in medium flow rate result in better adsorptions. However the exact adsorption mechanism needs more investigation. To assess the extent of the technical variability in this system, a standard protein (cytochrome c, 18 ng µL-1) spiked in NH4HCO3 (100 mM) was profiled continuously over a period of 24 h. Measuring spectral count of the most intense peaks in protein multiply charged series, the RSD of 25 replicates was 7.6%, demonstrating that the MEPD is reusable and reproducible. A flow-through MEPD can be reused for extended periods (>300 h) without membrane exchange. We suggest that cross-contamination in a continuous-flow format may be much less problematic than in a packed-bed mode or a conventional membrane format. MEPD Validation in Different Matrixes and Comparison with C18 RP LC Column Desalting. Rapid desalting and solvent exchange adapter are highly desirable. To demonstrate the versatility of the MEPD’s purification ability in different buffer

systems, we chose cytochrome c as a model protein. Five common buffers including TCA (150 mM, pH 1.0), NH4HCO3 (100 mM, pH 8.5), (NH4)2SO4 (100 mM, pH 5.1 and 500 mM, pH 4.8), phosphate buffered saline (PBS, pH 7.4), and urea (8 M, pH 8.5) were used as model matrixes. These buffers are often used for the extraction, dissolution, storage, and separation of biological samples. ESI has low salt tolerance. Typically, samples are desalted prior to MS analysis using an RP-LC column, either “online” or “offline”. The effectiveness of the homemade MEPD for desalting of samples prior to MS was compared with online C18 LC column desalting. The results are summarized in Figure 3. For comparison, the same amount (26 µL) of protein at the same concentration (10 ng µL-1 spiked in different matrixes) was desalted and concentrated. In the untreated sample spectra, chemical noises and salt adduct peaks obscure relevant analyte peaks. Parts m-q in Figure 3 shows the electrospray mass spectra of cytochrome c with MEPD treatment in different complex matrixes. A prominent charge-state distribution ranging from +22 (m/z 563) to +15 (m/z 825) is clearly evident, where each peak has a high signal-to-noise ratio. Compared to the C18 column, the MEPD has similar signal intensity but less interfering noise. At an extreme pH value, the spectral signal of the online C18 column desalting (Figure 3i) is almost indistinguishable from the background noise, but the MEPD (Figure 3o) has good desalting performance. For a lower concentration of cytochrome c (100 pg µL-1, in 100 mM NH4HCO3) (Figure 2a), the MEPD cleanup still worked but the C18 column desalting could not enrich and desalt efficiently and just got chemical noise (data not shown). Similar results were obtained for myoglobin (data not shown). Note that an analogous increase in concentration and signal could be achieved for online MEPD-ESI-MS by loading a larger volume of sample (Figure 2b). It could improve the signal-to-noise ratio significantly, indicating that further improvement in sensitivity is achievable using MEPD. It should be pointed out that the MEPD can be used for other charged macromolecules. For example, thymosin R1 (Mr 3108) is hard to be retained due to its small diameter and hydrophilic property.43 It was used to test the feasibility of MEPD in this application. The peptide (10 pg µL-1, 20 µL) has not been detected in the online C18 column desalting ESI mass spectrum (Figure 3l). By contrast, it generates an intense peak at +4 (m/z 778) in the MEPD-ESI-MS spectrum (Figure 3r), indicating that MEPD can be applied to a wide range of proteins and peptides. The results demonstrate that with a very simple device and virtually no optimization, MEPD can be performed in continuous flow, yielding product quality and quantity comparable to or even better than standard C18 column desalting methods. Compared to MEPD cleanup, C18 column desalting has some drawbacks, such as (1) the incompatibility of these stationary phases to extreme pH, (2) the relatively low capacity and retention for hydrophilic compounds, and (3) the relatively high elution volume necessary for quantitative recovery of analyte. Different types of salts may show a different influence on the proteins adsorption onto the MEPD. Any kind of salt could affect protein adsorption, not just competing for the binding sites on the charged surface. From our experiments, sulfate ions at high (43) Goldstein, A. L.; Badamchian, M. Expert Opin. Biol. Ther. 2004, 4, 559– 573.


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Figure 3. Direct ESI-MS analysis of online desalted samples after C18 LC column and MEPD processing. Comparison of ESI mass spectra of 10 ng µL-1 cytochrome c (parts a-e, g-k, m-q, Mr 12 KD) and 10 pg µL-1 peptide thymosin R1 (part f, l, r, Mr 3108) in various matrixes without treatment (parts a-f), processed by C18 LC column (parts g-l), and MEPD (parts m-r).

concentration (500 mM (NH4)2SO4) are known to bind to the protein charge surface, to form salt adducts, and to alter their multiply charged distribution (data not shown). How different salts will affect the adsorption remains unclear at this stage, and the precise mechanism of MEPD adsorption requires further investigation. Quantification by MEPD-MS. The MS ability to rapid determine the absolute concentration of a protein (or proteins) present within a complex protein mixture is highly desirable for identifying novel disease-specific protein biomarkers, gaining better understandings of disease processes, and discovering novel protein targets for therapeutic interventions and drug developments.44 However, mass spectrometric signals may not always correlate precisely, accurately, or directly with the amount of analyte in a complex matrix. This deviation is mainly due to matrix inference and ion suppression regardless of the ionization technique or instrument employed.45 Critical challenges limiting current quan(44) Hanash, S. Nature 2003, 422, 226–232. (45) Hamdan, M.; Righetti, P. G. Mass Spectrom. Rev. 2002, 21, 287–302.

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titative confidence are the efficiency and reproducibility of sample preparations involved. The MEPD sample processing is a highly qualified candidate for reliable quantification, because it can reduce sample complexity in one-step automated fashion and obtain sufficient quantities of target proteins from various complicated mixtures. It circumvents a series of purification procedures commonly used and has no compromise among purification depth, processing throughput, and consistence. To confirm the quantitative nature of MEPD processing, a protein marker mixture (cytochrome c and myoglobin) was spiked into two simulated complex matrixes where ion suppression effects and interferences are common. One was the neat salt solution, and the other contained albumin (1 mg mL-1) and IgG (1 mg mL-1) as high abundance interfering proteins, NH4HCO3 (100 mM) as the salt and urea (2 M) as the chaotrope. A sample volume of 20 µL was processed by online MEPD-ESI ion trap MS, and a high quality charge-state distribution of intact protein was obtained. The mass spectra of intact proteins have enough specificity both to significantly distinguish the signal of a sample from that

Figure 4. Comparison of the ability of MEPD quantitative recovery of control protein mixtures from two simulated complex matrixes. Matrix A contains albumin (1 mg mL-1) and IgG (1 mg mL-1) as high abundance interfering proteins, NH4HCO3 (100 mM) as the salt and urea (2 M) as the chaotrope. Matrix B is a neat salt solution, NH4HCO3 (100 mM). A protein marker mixture (cytochrome c and myoglobin) were added at different concentrations, from 10-4 to 10 mg mL-1, representing different protein abundance levels. The two most intense peaks of cytochrome c (m/z 688, m/z 728) and myoglobin (m/z 679, m/z 707) in multiply charged distributions were selected. Plot of their spectral count against spiked standard protein concentrations demonstrate a large linear range. Peak area intensity agreed well with spectral count measurements for different target peaks. (data not shown).

of the blank or chemical noise and to obtain a quantitative result with sufficient precision. In Figure 4, standard curves of the spiked protein marker mixture were generated with concentrations ranging from 10-4 to 10 mg mL-1 using both neat salt solutions (100 mM NH4HCO3) (dashed line) and the simulated complex matrix (solid line). We used the average of three replicates of MS spectral count to fit the linear model. Figure 4 reveals three significant results. First, the curves described a linear correlation between the measured intensity of the MS signals and the spiked target proteins over 4 orders of magnitude with a correlation coefficient above 0.99 both at normal scale (data not show) and at logarithmic scale, suggesting the ability of quantitative sample recovery from complex mixtures, which could facilitate a simple and straightforward quantification strategy. Second, the linear dynamic range of over 4 orders of magnitude was obtained. Traditionally, the linear dynamic range of the ion trap measurement is limited,46 typically less than 2 orders of magnitude due to ion suppression effect, analyzer and detector saturation, and the complex nature of biological samples. We assume the MEPD extended the ion trap linear dynamic range from two extreme: (1) to make lower abundance protein detectable by decreasing the matrix effect and (2) to quantitatively measure high-concentration proteins by eliminating ion-ion interaction and other interfering signals to circumvent MS analyzer and detector saturation. In addition, the MEPD enhanced dynamic range by enriching target analyte(s) (46) Liu, H.; Sadygov, R. G.; Yates, J. R., III Anal. Chem. 2004, 76, 4193–4201.

and decreasing sample loss during integrated sample preparation. Third, there was little difference between the presence and the absence of high-abundance proteins, indicating that highabundance proteins have no significant interference with the analysis of low-level proteins. Using the standard curve, we estimated the detection limit and the quantitation limit using the S/N ratio according to the revised version of ISO/WD 13530. The LOD (for cytochrome c, m/z 688) is 56 pg µL-1 for which the S/N equals 3. And the LOQ, 3 times the LOD, is about 168 pg µL-1. On the basis of quantitative recovery of the MEPD processing, we designed a strategy for absolute protein measurements from complex mixtures (Figure 5A) and used a porcine heart muscle extract containing cytochrome c (extracted by 150 mM TCA without further clarification) to demonstrate the quantitative validity. Like other absolute quantitative proteomics strategies, we used an appropriate internal standard (IS) to compensate for sample losses during sample workup. Theoretically, the optimal IS would be an isotopically labeled analogue of the analyzed protein; however, obtaining such an IS is a tedious process. An alternative simplified approach uses a species variant of the analyzed protein (i.e., the same protein from another species) as the IS, if it has a high sequence homology with the target endogenous analyte (EA).47 Employing this approach, horse heart cytochrome c was (47) Czerwenka, C.; Maier, I.; Potocnik, N.; Pittner, F.; Lindner, W. Anal. Chem. 2007, 79, 5165–5172.


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Figure 5. Absolute quantification of target protein from crude tissue extracts by online MEPD-ESI-MS: (A) illustration of absolute quantitative analysis of target intact proteins using online MEPD-ESI-MS and (B) MS spectra of spiked crude extracts. The spiked samples contain an internal standard at different concentrations, which cover the expected range of target protein. Peak pairs (m/z 645 and m/z 652) with high signal-to-noise ratio are chosen for the determination of concentration. The concentrations of internal standard in parts a-e are 40, 120, 160, 320, and 400 ng µL-1, respectively. (C) The measured average ratio of IS to EA versus the spiked IS concentration showing the linearity and precision of the method.

used as the IS for the MS quantitation of porcine heart cytochrome c. Pairs of IS and EA are discriminated based on the mass differential of the multiply charged distributions. In MS full scan mode, the signal intensity ratios of these ion pairs provide a direct measure of the absolute abundance of the corresponding protein present in the original samples after MEPD processing. The most intense mass peak with high signal-to-noise ratio was chosen for the determination of concentration. The absolute quantification is determined by comparing the abundance of the known IS with the EA, calculated as concentration IS/concentration EA, a ratio of IS to EA measured from the spectral count or peak area intensity in the mass spectra. We spiked a series of known, exact quantities of IS to the unknown EA sample to get a final IS concentration to be 40, 120, 160, 320, and 400 ng µL-1, respectively, with the same EA concentration and then carried out MEPD-ESI-MS analysis and recorded the mass spectra of intact proteins (parts a-e in Figure 5B). We can estimate the absolute EA concentration from each IS concentration level (from parts a-e in Figure 5B as 216, 221, 193, 203, and 224 µg mL-1, respectively). To evaluate the method validation, the average of the EA concentration from each level was calculated as 212 µg mL-1 in the crude extract (RSD ) 6.15%, n ) 5) and 8928


about 212 mg kg-1 in the heart tissue, which is consistent with results of the Bradford assay. With the peak intensity ratio (IS/EA) (from parts a-e in Figure 5B) plotted against the concentration of the spiked IS, a linear relationship is established (Figure 5C), suggesting the ability of this strategy to detect small changes between protein expressions on MS profile with confidence. The results illustrate that the MEPD method could benefit quantitative analysis. The advantages over traditional techniques include (1) enlarged sample load, versatile processing ability, and enhanced linear dynamic range help to recover and quantify lowlevel intact protein in nearly physiological salt conditions (for example, in the presence of high-abundance proteins, salts, and urea). The ability opens new possibilities for the direct analysis of intact proteins by MS at ever increasing sensitivity and in ever more complex samples, which reduce data complexity and keep the correlation between proteins and functions.48-51 (2) Automation and integration join the various stages of sample preparation (48) Bogdanov, B.; Smith, R. D. Mass Spectrom. Rev. 2005, 24, 168–200. (49) Forbes, A. J.; Patrie, S. M.; Taylor, G. K.; Kim, Y.-B.; Jiang, L.; Kelleher, N. L. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 2678–2683. (50) Han, X.; Jin, M.; Breuker, K.; McLafferty, F. W. Science 2006, 314, 109– 112.

into a single seamless procedure, which not only reduces ion suppression effects and sample loss but also increases sensitivity, throughput, and processing consistence and thus allows for better data quality. CONCLUSIONS We have developed a MEPD and an integrated sample preparation strategy for crude proteins extracts analyzed directly by ESI-MS. MEPD can be considered as a mixed-mode separation, encompassing elements of continuous adsorption media, electrodialysis, and electric-field assisted membrane chromatograph. The MEPD streamlines and speeds up the overall proteinprocessing workflow, from improving initial sample clarification through to a smoother transition to MS. It is possible to process a crude biological extract in an integrated format, which enables the entire protocol to be completed in less than 20 min with direct identification or quantification. Such strategies are best implemented on target intact protein purification, and it can be a good upfront sample clarification method for comprehensive proteins fractionation. To our knowledge, this is the first synergistic utilization among electric field, membrane techniques, and fluidic handling at the microscale. The synergy produces new functions and enhances system performance. Key advantages of the assay are its large sample loading capacity, tolerance to sample turbidity, high (51) Heck, A. J. R.; van den Heuvel, R. H. H. Mass Spectrom. Rev. 2004, 23, 368–389.

processing speed, facility to handling intact proteins, flexible macro-to-micro interface, no manual intervention, and amenable to automation. However, at the current stage, the MEPD cannot remove detergents such as SDS, Tween 20, etc. The possible solution should be membrane functionization to get specific enrichment. Although MEPD is coupled to ESI-MS in this study, it will be equally useful for MALDI-MS and even other types of proteomics approaches, including protein microarrays and structural proteomics. In addition, it has potential to be scaled down and used as a novel building block to create effective, complex, and highly integrated microfluidics networks. In combination with powerful multidimensional separation techniques and top-down strategies, considerable depth in proteome coverage may be achieved. ACKNOWLEDGMENT This work is supported by NSFC, Grants 20775090, 20627004, and 20675004. Mr. Guang Chen is acknowledged for his present of the membrane used in this study. SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

Received for review April 23, 2008. Accepted September 29, 2008. AC800816K


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Synergistic Design of Electric Field and Membrane in Facilitating

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