Anal. Chem. 2008, 80, 1568-1573
Gel-Eluted Liquid Fraction Entrapment Electrophoresis: An Electrophoretic Method for Broad Molecular Weight Range Proteome Separation John C. Tran and Alan A. Doucette*
Department of Chemistry, Dalhousie University, 6274 Coburg Road, Halifax, Nova Scotia, Canada B3H 4J3
Although well-established as a technique for protein purification, the application of continuous elution tube gel electrophoresis to proteome fractionation remains problematic. Difficulties associated with sample collection, particularly at the high mass range or at low sample loadings, continue to plague the technique. Furthermore, an upper mass limit is imposed as slow-moving higher molecular weight proteins are progressively diluted during the collection phase. In short, with current technology, effective separation over a broad mass range has not been achieved. In this work, we present improved techniques for continuous elution tube gel electrophoresis to accommodate broad mass range separation of proteins. Our device enables rapid partitioning of a proteome into discrete mass range fractions in the solution phase. High recovery is achieved at submicrogram to milligram sample loadings. We demonstrate comprehensive, reproducible separations of protein mixtures, as well as separation of a proteome in as fast as 1 h, over mass ranges from below 10 to 250 kDa. Finally, we identified proteins from a prefractionated standard protein mixture using liquid chromatography tandem mass spectrometric (LC-MS/ MS) analysis. Separations are crucial for proteome analysis, with preferred systems offering high resolution, reproducibility, and recovery. In proteomics, well-established peptide separation techniques are available to reduce sample complexity prior to analysis.1 At the intact protein level, two-dimensional electrophoresis (2DE)2 continues to be widely employed, yet, apart from an unrivalled degree of resolution,3 2DE has many disadvantages.4 A recent report by Brunner et al. describing a “targeted” shotgun approach for comprehensive proteome analysis illustrates the importance * To whom correspondence should be addressed. E-mail: alan.doucette@ dal.ca. Fax: 902-494-1310. Phone: 902-494-3714. (1) Washburn, M. P.; Wolters, D.; Yates, J. R. Nat. Biotechnol. 2001, 19, 242247. (2) O’Farrell, P. H. J. Biol. Chem. 1975, 250, 4007-4021. (3) Gorg, A.; Weiss, W.; Dunn, M. J. Proteomics 2004, 4, 3665-3685. (4) 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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of intact protein prefractionation in the detection strategy.5 The rising popularity of top-down approaches for protein analysis6 also fuels the need for more effective protein separation strategies. Nonetheless, with noted exceptions,7-10 optimizing or developing new approaches for proteome separation at the intact level remains an underdeveloped area. Proteome separations are most beneficial when the elution order occurs in a predictable fashion. Such predictability can permit the isolation of a particular protein, or class of proteins (enrichment of isoforms or PTM proteins), and can also assist in the identification process.11 The molecular weight (MW) is an intrinsic property of a protein, and its measurement is usually unaffected by sample or solvent conditions. Being orthogonal to both charge and hydrophobicity, the MW of a protein presents a highly desirable mode of separation. Unfortunately, very few solution-based systems are established that separate proteins according to size. For example, membrane filtration and ultrafiltration strategies are inherently labor intensive and offer a poor degree of resolution and recovery. Although size exclusion chromatography has been coupled to other separation platforms,12-15 it has not seen very widespread use in proteomics, since it offers relatively low peak capacity. A size-based protein separation platform with a high degree of resolution, throughput, and sample recovery would present a more desirable and useful system. (5) Brunner, E.; Ahrens, C. H.; Mohanty, S.; Baetschmann, H.; Loevenich, S.; Potthast, F.; Deutsch, E. W.; Panse, C.; de Lichtenberg, U.; Rinner, O.; Lee, H.; Pedrioli, P. G. A.; Malmstrom, J.; Koehler, K.; Schrimpf, S.; Krijgsveld, J.; Kregenow, F.; Heck, A. J. R.; Hafen, E.; Schlapbach, R.; Aebersold, R. Nat. Biotechnol. 2007, 25, 576-583. (6) Kelleher, N. L.; Lin, H. Y.; Valaskovic, G. A.; Aaserud, D. J.; Fridriksson, E. K.; McLafferty, F. W. J. Am. Chem. Soc. 1999, 121, 806-812. (7) Nilsson, C. L.; Davidsson, P. Mass Spectrom. Rev. 2000, 19, 390-397. (8) Shi, Y.; Xiang, R.; Horvath, C.; Wilkins, J. A. J. Chromatogr., A 2004, 1053, 27-36. (9) Wang, H.; Hanash, S. Mass Spectrom. Rev. 2005, 24, 413-426. (10) Lubman, D. M.; Kachman, M. T.; Wang, H. X.; Gong, S. Y.; Yan, F.; Hamler, R. L.; O’Neil, K. A.; Zhu, K.; Buchanan, N. S.; Barder, T. J. J. Chromatogr., B 2002, 782, 183-196. (11) Pal, M.; Moffa, A.; Sreekumar, A.; Ethier, S. P.; Barder, T. J.; Chinnaiyan, A.; Lubman, D. M. Anal. Chem. 2006, 78, 702-710. (12) Bushey, M. M.; Jorgenson, J. W. Anal. Chem. 1990, 62, 161-167. (13) Opiteck, G. J.; Jorgenson, J. W.; Anderegg, R. J. Anal. Chem. 1997, 69, 2283-2291. (14) Lecchi, P.; Gupte, A. R.; Perez, R. E.; Stockert, L. V.; Abramson, F. P. J. Biochem. Biophys. Methods 2003, 56, 141-152. (15) Hou, W. M.; Ethier, M.; Smith, J. C.; Sheng, Y. L.; Figeys, D. Anal. Chem. 2007, 79, 39-44. 10.1021/ac702197w CCC: $40.75
© 2008 American Chemical Society Published on Web 01/30/2008
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is arguably the best method for size-based protein separation. Interestingly, the most serious limitation of SDSPAGE relates to the recovery of protein from the gel.16 As an interfering compound, the presence of SDS may provide an impending limitation toward mass spectrometric (MS) analysis. However the benefits of SDS in realizing predictable size-based separations, as well as assisting protein solubilization, arguably outweigh this disadvantage. In light of this, it would be most beneficial to take advantage of the high resolving power of SDSPAGE for molecular weight separation, while avoiding the laborious tasks of spot excision, in-gel digestion, and peptide extraction. As an alternative to solvent extraction, proteins have been electrophoretically eluted from SDS-PAGE. The Whole Gel Eluter from Bio-Rad applies the strategy of electroelution across an entire gel. Davidsson and Nilsson have used this system for fractionation and analysis of proteins from human cerebrospinal fluid.17 Although the device achieves broad size-based separation, the restricted number of fractions available with this device limits the flexibility to optimize resolution. Furthermore, sample loading capacity is limited by the dimensions of the slab gel. Finally, the Whole Gel Eluter would be difficult to multiplex and therefore would not readily integrate into a multidimensional solution-based platform in a high-throughput format. As a different strategy to preparative gel electrophoresis, proteins can be eluted from the end of a gel column by continuous application of the electric field, wherein proteins are trapped by a molecular weight cut off (MWCO) membrane and subsequently collected.18-21 This technique is generally referred to as continuous elution tube gel electrophoresis. Although the ability to purify a target protein with extremely high resolution has been wellestablished,22,23 broad fractionation of an entire proteome with such methodology has been problematic.24-26 In general, systems based on this approach are biased toward lower MW protein. Other significant limitations include long separation times and an unacceptably large dilution of sample during separation, particularly at high masses. These difficulties need to be overcome before continuous elution electrophoretic techniques can be generally adopted for comprehensive, broad mass range proteome separation. Our group is interested in size-based protein separations that provide useful intrinsic information for targeted or comprehensive protein analysis. To this end, we introduce an improved means of protein fractionation using a customized platform for continuous (16) Rabilloud, T. Proteomics 2002, 2, 3-10. (17) Davidsson, P.; Nilsson, C. L. Biochim. Biophys. Acta 1999, 1473, 391-399. (18) Lewis, U. J.; Clark, M. O. Anal. Biochem. 1963, 6, 303-315. (19) Racusen, D.; Calvanic, N. Anal. Biochem. 1964, 7, 62-66. (20) Jovin, T.; Chrambach, A.; Naughton, M. A. Anal. Biochem. 1964, 9, 351369. (21) Shain, D. H.; Yoo, J. Y.; Slaughter, R. G.; Hayes, S. E.; Ji, T. H. Anal. Biochem. 1992, 200, 47-51. (22) Masuoka, J.; Glee, P. M.; Hazen, K. C. Electrophoresis 1998, 19, 675-678. (23) Davidsson, P.; Westman, A.; Puchades, M.; Nilsson, C. L.; Blennow, K. Anal. Chem. 1999, 71, 642-647. (24) Meng, F. Y.; Cargile, B. J.; Patrie, S. M.; Johnson, J. R.; McLoughlin, S. M.; Kelleher, N. L. Anal. Chem. 2002, 74, 2923-2929. (25) Du, Y.; Meng, F.; Patrie, S. M.; Miller, L. M.; Kelleher, N. L. J. Proteome Res. 2004, 3, 801-806. (26) Zerefos, P. G.; Vougas, K.; Dimitraki, P.; Kossida, S.; Petrolekas, A.; Stravodimos, K.; Giannopoulos, A.; Fountoulakis, M.; Vlahou, A. Proteomics 2006, 6, 4346-4355.
Figure 1. (A) Photo of the GELFrEE device: 1 ) anode, 2 ) column, 3 ) collection chamber, 4 ) cathode. Part B shows a closeup of the gel showing separation of a prestained MW protein ladder: 5 ) stacking gel, 6 ) resolving gel. The resolving gel column was 3 cm long, cast to 15% T, and operated under standard conditions. Although not shown here, during operation proteins that eluted from the gel column were subsequently trapped and recovered in discrete fractions from the collection chamber.
elution tube gel electrophoresis. We demonstrate optimized conditions that enable a broad mass range proteome separation in a fast, effective, reproducible, and high-yield format. Although the device employs a gel column for separation, the proteins are ultimately eluted from the column and collected in the solution phase (i.e., free of the gel). Therefore, we have termed this separation technique GELFrEE (Gel-Eluted Liquid Fraction Entrapment Electrophoresis). MATERIALS AND METHODS Materials. Milli-Q grade water was purified to 18.2 MΩ cm-1. All reagents for gel electrophoresis were obtained from Bio-Rad (Mississauga, ON). The 3.5 kDa MWCO dialysis membranes were purchased from Fisher Scientific (Ottawa, ON). All proteins, including trypsin (TPCK treated, cat. T8802), lyophilized Bacillus subtilis, solvents, and other chemicals were purchased from Sigma (Oakville, ON). Sample Preparation. Lyophilized cells of B. subtilis were suspended in pure water and lysed in a French press at 8000 psi. The lysed bacteria were centrifuged at 13 000g, and the supernatant was collected and stored at -20 °C until ready to use. Standard protein solutions were prepared by weight to the appropriate concentration. For consistency, 200 µL of sample was loaded in the gel column, combining 180 µL of the sample with 20 µL of 5× gel loading buffer (0.25 M Tris-HCl pH 6.8, 10% w/v SDS, 50% glycerol, 0.5% w/v bromophenol blue). Samples were heated at 95 °C for 5 min prior to loading. GELFrEE Device. Design. Apart from the column, the device is constructed of Teflon and is shown in Figure 1A. Referring to the labels in this figure, the device consists of four main components: a cathode chamber (1), the gel column (2), a collection chamber (3), and an anode chamber (4). Analytical Chemistry, Vol. 80, No. 5, March 1, 2008
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Gel Column. The 0.6 cm diameter polyacrylamide gel column was cast into a 0.8 cm o.d. × 6.0 cm glass tube. Unless otherwise noted, the gel column contained a 1 cm long resolving gel (determined through the volume of unpolymerized gel added to the gel), cast to 15% T, 2.67% C, along with a 1.5 cm stacking gel of 4% T, 2.67% C. Gels were prepared using the SDS-PAGE Laemmli buffer system.27 Samples were loaded into the void volume of the glass tube above the stacking gel. Collection Chamber. During separation, eluted samples were trapped and recovered in the collection chamber. This consists of a round channel bored into a 1.0 cm thick Teflon disk with inner diameter to match the outer diameter of the glass tube containing the gel column. A 3.5 kDa MWCO membrane was sandwiched between the collection chamber and the anode chamber, sealed by pressure once the chambers were clamped together (see Figure 1A). An access port was drilled into the top of the chamber, allowing multiple fractions to be removed without disassembling the device. The void volume of the collection chamber can be adjusted by controlling the depth of the gel column inserted into the chamber. For consistency, the void volume of the collection chamber was kept constant at approximately 140 µL. GELFrEE Operating Conditions. Operation of the device can be described in three distinct stages: (1) sample loading, (2) separation, and (3) collection. The electrolyte chambers of the device, as well as the void volume above the gel column were completely filled with electrode running buffer (0.192 M glycine, 0.025 M Tris, 0.1% SDS).27 A volume of 100 µL of the running buffer was also introduced into the collection chamber. To assist with sample loading, the cathode (loading) end of the device was raised at a 45° angle, which enabled the sample to reach the start of the stacking gel through force of gravity. Separation occurred with a constant application of 240 V. After the sample plug migrated into the gel (∼10 min), the device was laid horizontal for the remainder of the separation. After the dye front had visibly entered the collection chamber, the first fraction was collected. During collection, the power supply was paused and the content in the collection chamber was manually transferred (pipetted) to a clean vial. A fresh 100 µL portion of running buffer was then introduced into the collection chamber, and the power supply was resumed to continue separation. This process was repeated over the course of separation, where an additional fraction was collected between each stop-and-go cycle. The period between each collection cycle was varied according to the time of the eluting proteins, whereby successive fractions were collected following longer electrophoretic separation. The reported elution times refer to the accumulated period of voltage application to the system. Analytical SDS-PAGE. GELFrEE fractions were analyzed by discontinuous SDS-PAGE27 using 15% T resolving slab gels. For this, 20 µL of the fractions were combined with 5 µL of 5× gel loading buffer, of which 20 µL was loaded onto individual lanes of the slab gel along with the appropriate standards. Gels were either silver-28 or coomassie-stained, as indicated, and scanned using a flatbed scanner. Sample Digestion and Liquid Chromatography-Mass Spectrometry (LC-MS). An amount of 40 µg each of a 12-protein (27) Laemmli, U. K. Nature 1970, 227, 680-685. (28) Shevchenko, A.; Wilm, M.; Vorm, O.; Mann, M. Anal. Chem. 1996, 68, 850-858.
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mixture was loaded onto the GELFrEE device (1 cm, 15% T resolving gel column) and partitioned into 17 fractions. These fractions were precipitated using the protocol described by Wessel and Flugge.29 After precipitation, the pellet was redissolved in 100 µL of 100 mM NH4HCO3 and digested with trypsin according to standard protocols. Following overnight digestion, samples were acidified with formic acid and a 1 µL portion of this was injected onto a 150 mm × 180 µm Biobasic C18 column (Thermo Scientific, San Jose, CA) and separated using a standard gradient from water to acetonitrile (2%/min). A nanospray source was used to interface to the LTQ linear ion trap mass spectrometer (Thermo Scientific), which was operated in data-dependent mode to acquire MS/MS spectra for peptide sequencing. Data were searched using Sequest, part of Bioworks 3.5, against the Uniprot database. Positive identification was based in part on criteria defined by Washburn et al.1 Peptides with charge +1, +2, +3 were accepted with Xcorr scores greater than 1.9, 2.2, and 3.75, respectively. The peptides were further filtered with ∆Cn g 0.1, and Rsp e 4. Safety Considerations. Appropriate precautions must be taken for the handling of acrylamide, a neurotoxin, as well as for the operation of the GELFrEE device, which requires high voltages. RESULTS AND DISCUSSION Overview of the GELFrEE Design. Denatured-PAGE is a powerful technique for mass separation, providing very predictable runs and excellent resolution. The prestained MW protein ladder was visible within the gel column shown in Figure 1. A clear separation was observed, being comparable to that of conventional analytical slab gels. As with all preparative gel electrophoresis devices, the composition and dimensions of the gel column will establish the attainable resolution, mass range, and separation speed. Our device employs short resolving gels to afford rapid, broad mass range proteome fractionation (details provided below). Following separation, as proteins are ultimately recovered in solution, the objective of a continuous elution gel electrophoresis system is to maintain the resolution afforded by the gel column. The collection chamber of the GELFrEE device achieves this goal, recovering protein fractions in high yield and with minimal dilution. Collection Chamber. With other continuous elution systems, proteins that elute from the gel column are recovered from a trapping membrane filter using a counterflow of buffered solution. The limitation of this approach is that large MW species are progressively diluted during sample collection. As others have also shown, Figure 2A illustrates how higher MW proteins migrate slower than smaller species, giving rise to the logarithmic mass elution profile. It has been reported that band spreading in gel electrophoresis follows a linear dependence with time (t), as opposed to t1/2 as would be predicted by a diffusion model.30,31 A consequence of this is that larger (slower moving) species elute over progressively longer time windows in a continuous elution system. To provide an example, from Figure 2A, a 10 kDa protein will elute in 7 min on a 14% T gel, whereas a 100 kDa protein elutes in 50 min. Assuming that the 10 kDa protein band can be (29) Wessel, D.; Flugge, U. I. Anal. Biochem. 1984, 138, 141-143. (30) Yarmola, E.; Calabrese, P. P.; Chrambach, A.; Weiss, G. H. J. Phys. Chem. B 1997, 101, 2381-2387. (31) Yarmola, E.; Chrambach, A. J. Phys. Chem. B 1998, 102, 4813-4818.
Figure 2. Elution profile of standard MW protein markers for GELFrEE separations using resolving gel columns cast to 10% T (2), 12% T (9), 14% T (b), and 15% T (1). The graph in (A) plots MW vs the elution times, as measured from the estimated time that the tracking dye first entered the resolving gel until the time when the standard protein was first detected in a collected fraction. The graph in (B) represents data from these same runs, plotting MW against the fraction number in which the proteins were recovered from the device.
collected in a 40 s window, it would take approximately 5 min to collect the 100 kDa protein. With conventional continuous elution systems that use constant elution flow rate, higher MW proteins are often observed in multiple fractions and are consequently diluted by the larger collection volume. The current strategy employed to collect samples while maintaining resolution differs significantly from that of traditional devices. Here, proteins are eluted from the gel and subsequently confined in the collection chamber (Figure 1) for a defined time interval. Over the course of a run, as the migration rate decreases for larger proteins, the time interval for collection of subsequent fractions is simply increased to match the bandwidth of the larger MW proteins. As seen from Figure 2B, protein fractions were therefore collected in an approximate “linear” MW profile (noting the log scale for MW). Another ramification was that proteins remained focused during collection, ideally being recovered in single fractions over the entire mass range, and at consistently high yield. With the use of this device, an approximate 2-fold increase in concentration (relative to initial sample loading) can be maintained during sample collection. The number of fractions collected with the GELFrEE device was therefore an approximate representation of the peak capacity attainable with this system.
Composition of the Resolving Gel Column. The composition of the resolving gel (i.e., % T) is an important parameter in optimizing the desired degree of fractionation over a given mass range. Gels cast to lower % T generally favor collection of high-mass species, whereas a higher %T favors the low mass range. From Figure 2, a 250 kDa protein begins to elute in 1 h on a 10% T gel, whereas a 75 kDa protein took the equivalent time to elute from a 15% T gel. The lower mass limit for a 10%, 12%, and 15% T gel were 20, 15, and 6 kDa, respectively. Proteins with masses below this limit will coelute and therefore cannot be separated, although it is noted that these proteins will still be recovered (in the first fraction). As seen below, we have found that a 15% T gel generally afforded effective mass resolution over the mass range of ∼10 to ∼150 kDa. Length of the Resolving Gel Column. The rapid separations described in Figure 2 were achieved by employing high electric fields on short gel columns. It is desirable to use the shortest gel column possible while maintaining resolution. Figure 3A depicts the fractions collected from the separation of a proteome extract of B. subtilis using a 1 cm long (resolving) gel column, cast at 15% T. As shown, the last protein fraction, collected 90 min following initial voltage application, contains protein at estimated MW range between 120 and 150 kDa. Proteins above 100 kDa were easily recovered 1 h into the run. In this same run on the short gel column, proteins with MW extending below 10 kDa were resolved in the collected fractions. This figure therefore demonstrates the rapid, broad mass range separation of a proteome under a single set of operating conditions. The use of extremely short resolving gel columns in preparative electrophoresis is somewhat unconventional. However, due to the nature of protein dispersion which contributes to band broadening in gel electrophoresis, longer gels may not necessarily afford higher overall resolution, particularly over the entire mass range of the sample.31 Also, assuming a constant electric field, longer gels require proportionally longer separation times. Figure 3B shows an equivalent separation profile for B. subtilis conducted on a 3 cm gel column. As the figure suggests, the gain in resolution with this longer gel was not significantly improved, particularly in the mass range >30 kDa. In addition, the latter separation required over 4 h to span a similar mass range. For reasons of sample recovery from the collection chamber (details below), a run on the 3 cm gel generated approximately 3 times as many fractions over the course of the separation. Moreover, the increased work load was compounded by an increase in sample dilution during collection of high-mass proteins as they begin to elute across multiple fractions. The goal of using short columns is to achieve rapid broad mass range fractionation, as opposed to the targeted purification of a single protein. A 1 cm gel column provided very effective partitioning (peak capacity ∼15) of a proteome sample, over an extremely broad mass range. Maximal throughput was also realized by minimizing the total separation time. To the best of our knowledge, the results revealed in Figures 2 and 3 represent the largest mass range partitioned and recovered using a semipreparative continuous elution gel electrophoretic device under such rapid separation times. Recovery from the Collection Chamber. An important feature of the GELFrEE collection chamber is the trapping Analytical Chemistry, Vol. 80, No. 5, March 1, 2008
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Figure 3. (A) Silver-stained SDS-PAGE images show the analysis of GELFrEE fractions from the separation of B. subtilis (200 µg loading) at 240 V over the broad mass range from ∼10 to ∼150 kDa. A 1 cm long resolving gel was employed, with all collected fractions represented in the gel image. Collection times are indicated above each fraction lane. The SDS-PAGE image in (B) shows separation of an equivalent sample using a 3 cm long resolving gel. Every third fraction is displayed in this image. To account for the increased gain in heat with the longer gel, the running voltage was reduced to 200 V.
efficiency of the 3.5 kDa MWCO membrane. Cellulose acetate, having an isoelectric point of 4.2,32 would be negatively charged at an operating pH 8 and should therefore repel SDS-bound proteins.33 Indeed, when a 50 ng/µL solution of BSA was loaded into the collection chamber (bypassing the column), the coomassie gel profiles used to assay the sample suggest negligible sample loss at up to 15 min of trapping time. However, at longer trapping times (30 min), a noticeable decrease in BSA recovery was observed. To prevent this in a GELFrEE experiment, the trapping time interval for a given fraction was maintained below 15 min. This limitation points to the need for rapid separations to maintain high recovery during MW fractionation, which was provided by the short gel columns. To illustrate protein recovery in the submicrogram range, a sample containing 500 ng each of BSA, cytochrome c, and ubiquitin was loaded and recovered from the MW separation device and yielded the gel images shown in Figure 4 (only 1/12 of the collected fraction volume was loaded in the gel). The resulting silver-stained bands demonstrate that a significant portion of the sample was effectively recovered from the GELFrEE device. We have found that as little as 100 ng of various proteins can be loaded on the column and subsequently be detected in a silver-stained gel in a similar experiment with comparable yields (results not shown). It is noted that loadings of 8 ng of a given protein in a gel lane were approaching our detection limit. The relative intensities of the protein bands displayed in Figure 5 provide a semiquantitative measure of the high recovery of five standard proteins following fractionation at loadings between 20 and 100 µg per protein. As with Figure 4, the standard protein lane provided in Figure 5 would represent equivalent loadings in these gels assuming 100% recovery from GELFrEE. Maintaining high concentration for recovered proteins was realized by minimizing the number of fractions containing the protein of interest (often a single fraction). In terms of upper loading capacity, a 0.6 cm (32) Pontie, M.; Chasseray, X.; Lemordant, D.; Laine, J. M. J. Membr. Sci. 1997, 129, 125-133. (33) Pincet, F.; Perez, E.; Belfort, G. Langmuir 1995, 11, 1229-1235.
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Figure 4. Control lanes consisting of 40 ng each of (A) BSA, (B) cytochrome c, and (C) ubiquitin are shown in the left column of the silver-stained SDS-PAGE images. The corresponding fractions recovered from GELFrEE, shown on the right half, were obtained in the same SDS-PAGE gel and would also represent 40 ng loadings assuming 100% recovery from the device. The collection time intervals for GELFrEE fractionation were 5 min (cytochrome c or ubiquitin) and 10 min (BSA), with a sample loading of 500 ng per protein onto the GELFrEE device.
i.d. tube containing a 1.5 cm stacking gel easily accommodated over 200 µL of sample, or up to 1 mg for proteome mixtures. These results illustrate how the method of GELFrEE sample fractionation and collection provided very high protein recoveries over a wide range of sample amount loadings. Reproducibility. The reproducibility of the MW separation is dependent on the consistency of the buffers as well as the casting of gel columns. Parts A and B of Figure 5 display the gel profiles of a five-protein mixture, separated in independent runs under identical conditions and with identical collection times. The images reveal that the bulk of the collected proteins appeared in the same collected fractions, or in other words, these proteins eluted from the gel column in the same time period. Figure 5C represents an equivalent separation of the five-protein mixture, except that the fraction collection time was shifted to 1 min later than that of the previous images. Proteins are therefore expected to be observed in a lower fraction number. This illustrates how small changes in collection time (1 min in a typical 90 min run) will strongly influence the elution profile of the sample. Nonetheless, under a controlled set of operating conditions, the GELFrEE
Table 1. LC-MS/MS Analysis of Fractionated Standard Protein Mixture protein
MW (kDa)
fraction no.a
no. peptide
sequence coverage
Uniprot accession no.
β-gal amyl BSA oval carb R-cas β-lac myo lys cyt c ubiq
116 68 69 43 29 26 20 17 16 12 9
14 13 12 10 7 7 4 4 4 5 3
45 6 35 14 14 4 4 5 3 4 2
58 15 60 52 60 15 45 50 22 16 24
P00722 P69327 P02769 P01012 P00921 P02663 P02754 P068083 P00698 P0004 P62990
a Only the fraction containing the highest number of identified peptides for a given protein is listed.
tion. As shown in Table 1, all 11 proteins were easily detected with high sequence coverage. The elution profile for the detected proteins was highly predictable and proves that GELFrEE provided valuable intrinsic information.
Figure 5. Coomassie-stained SDS-PAGE images show the reproducibility of the GELFrEE separation in three independent separations of ovalbumin (I), carbonic anhydrase (II), β-lactalbumin (III), cytochrome c (IV), and ubiquitin (V) at (A) 200, (B) 100, and (C) 20 µg loading per protein. The fraction collection times for (A) and (B) were identical. Fractions from (C) began 1 min later, revealing the shift in eluted proteins to an earlier fraction number (i.e., collection times intervals were equivalent). The standard lane represents protein loads assuming 100% recovery from GELFrEE and also assuming a 1.8-fold increase in concentration which was theoretically experienced from volume reduction during sample loading to collection in GELFrEE.
device provided highly reproducible separations. High reproducibility ultimately enables mass range prediction of eluting proteins, based directly on the run time under a given set of conditions. This becomes particularly useful for targeted collection of a protein(s) of known MW. Furthermore, it provides intrinsic MW information to assist with comprehensive proteome analysis and higher degree of identification confidence. Application to Mass Spectrometric Analysis. As others have shown, proteins eluting off continuous elution tube gel electrophoresis can be further analyzed through MS.23-26 We would like to demonstrate that it is possible to analyze high-mass fractions using LC-MS/MS. With the use of a 1 cm, 15% T resolving gel, an 11-protein standard mixture over the mass range of 8-116 kDa was partitioned into 17 fractions. The fractions were subject to MS analysis following protein precipitation and enzymatic diges-
CONCLUSIONS The GELFrEE technique enables rapid, broad mass range proteome separations in the low-microgram to milligram range. This is attributed in part to the unique method of sample trapping and collection in the solution phase following electrophoretic elution from gel columns. This separation approach provides a highly desirable alternative and complementary tool for proteome fractionation at the intact level. Such separations afford valuable intrinsic MW information owing to the highly predictable and reproducible nature of the separation. Unlike other preparative devices, the compact design and simple construction of the device is readily compatible with multiplexing. We have most recently constructed a multiplexed (eight column) GELFrEE device, which is presently being integrated with other separation platforms to provide high-throughput multidimensional proteome separations. Results on this work will be presented in the near future. ACKNOWLEDGMENT This work was financially supported by the National Sciences and Engineering Research Council of Canada (NSERC), the Canada Foundation for Innovation, the Nova Scotia Research Trust, along with the Department of Chemistry and the Faculty of Science at Dalhousie University. Thanks to staff machinists Mike Boutilier and Rick Conrad (Department of Chemistry) for constructing the device. Received for review November 30, 2007.
October
24,
2007.
Accepted
AC702197W
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