Multiplexed Size Separation of Intact Proteins in Solution Phase for

Jul 2, 2009 - This enables simultaneous size separation of independent proteome samples, each into 16 well resolved liquid fractions, covering 10−15...
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Anal. Chem. 2009, 81, 6201–6209

Multiplexed Size Separation of Intact Proteins in Solution Phase for Mass Spectrometry John C. Tran† and Alan A. Doucette* Department of Chemistry, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4J3 Reliable size-based protein separation is an invaluable biological technique. Unfortunately, size separation in solution is underutilized, owing perhaps to the poor resolution of conventional techniques. Here, we report an enhanced multiplexed GELFrEE (gel-eluted liquid fraction entrapment electrophoresis) device which incorporates eight independent separation channels, operating with high repeatability. This enables simultaneous size separation of independent proteome samples, each into 16 well resolved liquid fractions, covering 10-150 kDa in 1.5 h. A novel strategy to increase sample loads while maintaining electrophoretic resolution is presented by distributing the sample among the eight channels with subsequent pooling of collected fractions. Liquid chromatographytandem mass spectrometry (LC-MS/MS) analysis of the S. cerevisiae proteome following GELFrEE separation and sodium dodecyl sulfate (SDS) removal demonstrates the resolution and high correlation achieved between molecular weight and fraction number for the identified proteins. This device is highly orthogonal to solution isoelectric focusing, enabling our disclosure of a fully multiplexed high-throughput two-dimensional liquid electrophoretic (2D LE) platform that separates analogously to 2D polyacrylamide gel electrophoresis (PAGE). With 2D LE, a total of 128 well-resolved liquid fractions are obtained from 1 mg of S. cerevisiae proteins covering ranges 3.8 < pI < 7.8 and 10 kDa < MW < 150 kDa in an unprecedented 3.25 h total separation time. While mass spectrometry (MS) occupies a central role in highthroughput proteomic analysis,1 sample preparation ahead of MS analysis is pivotal to successful protein characterization. With MS, protein identification can occur via the established bottom-up approach, affording impressive proteome coverage2 and also facilitating protein quantification3 from complex biological systems. Alternatively, a complementary top-down (intact protein analysis) MS approach to proteome analysis incorporates the potential for full sequence coverage, enabling unambiguous protein identifica* Corresponding author. Alan A. Doucette, Department of Chemistry, Dalhousie University, 6274 Coburg Road, Halifax, Nova Scotia, Canada, B3H 4J3. E-mail: [email protected]. Fax: (902) 494-1310. Phone: (902) 494-3714. † Present address: Department of Chemistry, University of Illinois, Urbana, Illinois 61801. (1) Aebersold, R.; Mann, M. Nature 2003, 422, 198–207. (2) Washburn, M. P.; Wolters, D.; Yates, J. R. Nat. Biotechnol. 2001, 19, 242– 247. (3) Gygi, S. P.; Rist, B.; Gerber, S. A.; Turecek, F.; Gelb, M. H.; Aebersold, R. Nat. Biotechnol. 1999, 17, 994–999. 10.1021/ac900729r CCC: $40.75  2009 American Chemical Society Published on Web 07/02/2009

tions as well as complete characterizations of post-translational modifications.4 While recent technological innovations in MS detection platforms have enabled high-throughput top-down analysis on chromatographic time scales5 and characterization for proteins as high as 200 kDa,6 the limitations imposed by frontend manipulation of the sample, particularly in the context of separating intact proteins, continue to present a major bottleneck in the top-down analytical workflow. Regardless of the MS strategy, effective protein separation is critical for decreasing sample complexity and increasing the dynamic range of detection. An ideal separation encompasses both high resolution and high sample recovery in a form compatible with downstream analysis. Well established chromatographic and electrophoretic techniques, applied at the level of peptides, are readily coupled online to MS, either individually or in multidimensional formats.7 A complementary strategy for providing greater reduction of sample complexity is to incorporate intact protein prefractionation, which further improve the dynamic range of MS detection.8,9 In contrast to peptide separation, however, the effective fractionation of proteins at the intact level continues to present a challenge in solution-based platforms and remains heavily reliant on gel-based technology.10 While the classical approach of 2D PAGE11 provides unrivalled resolution, the sample bias, irreproducibility, poor recovery for intact proteins, and poor throughput of this platform warrants exploration of liquid-based alternatives.12-14 Protein recovery is a greater concern in solution platforms than its peptide-based counterparts. For example, high molecular weight (MW) and hydrophobic compounds are more difficult to recover from reversed-phase liquid chromatography (4) Kelleher, N. L. Anal. Chem. 2004, 76, 196A–203A. (5) Parks, B. A.; Jiang, L.; Thomas, P. M.; Wenger, C. D.; Roth, M. J.; Boyne, M. T., II; Burke, P. V.; Kwast, K. E.; Kelleher, N. L. Anal. Chem. 2007, 80, 2857–2866. (6) Han, X. M.; Jin, M.; Breuker, K.; McLafferty, F. W. Science 2006, 314, 109–112. (7) Fournier, M. L.; Gilmore, J. M.; Martin-Brown, S. A.; Washburn, M. P. Chem. Rev. 2007, 107, 3654–3686. (8) Chen, E. I.; Hewel, J.; Felding-Habermann, B.; Yates, J. R. Mol. Cell. Proteomics 2006, 5, 53–56. (9) 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. (10) Laemmli, U. K. Nature 1970, 227, 680–685. (11) O’Farrell, P. H. J. Biol. Chem. 1975, 250, 4007–4021. (12) Nilsson, C. L.; Davidsson, P. Mass Spectrom. Rev. 2000, 19, 390–397. (13) Wang, H.; Hanash, S. Mass Spectrom. Rev. 2005, 24, 413–426. (14) Righetti, P. G.; Castagna, A.; Antonioli, P.; Boschetti, E. Electrophoresis 2005, 26, 297–319.

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(RPLC).15 Ion exchange chromatography has questionable recovery for certain components of a proteome,16 and capillary electrophoresis brings concerns of interaction between proteins and the silica surface. Effective solution-based separation, applied to intact proteins, could enhance the detection workflow in both bottom-up and top-down proteomics.9 The molecular weight is arguably the most accessible intrinsic property of a protein, thus size-based protein separation in solution is a highly valued asset in protein studies. Size-based proteome separation would certainly complement other promising solution platforms such as ion exchange chromatography, solution isoelectric focusing, and RPLC, by affording the potential for multidimensional separation of intact proteins. Existing strategies for size separation in solution (size exclusion chromatography and centrifugation) provide poor resolution and questionable yields. As an alternative, we recently developed an effective solution phase size separation platform for biological samples, termed gel-eluted liquid fraction entrapment electrophoresis (GELFrEE).17 The approach is a form of continuous elution tube gel electrophoresis,18-20 which repackages the familiar techniques of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) into a column format. In this strategy, proteins are electrophoretically migrated (eluted) from the end of the gel column through continuous application of the electric field. With GELFrEE, proteins are subsequently trapped in a collection chamber, resulting in a time-based partitioning of the proteome over a broad mass range. GELFrEE differs from other continuous elution tube gel electrophoresis in that it uses a smaller column, thus ensuring faster separation; the unique GELFrEE entrapment strategy affords sample enrichment as opposed to dilution, and through customization of the entrapment interval (longer trapping times at high MW proteins), the likelihood of trapping given proteins in single fractions is maximized. This technique is also extremely promising for proteome analysis since it can rapidly separate intact proteins in a predictable manner, while maintaining unbiased high recoveries. Here, we present an enhanced approach to GELFrEE separation in the form of an integrated eight column platform (multiplexed GELFrEE or mGELFrEE). The MS profiling of a complex proteome mixture demonstrates the exceptional correlation between protein MW and fraction number; intact protein analysis by electrospray ionization-mass spectrometry (ESI-MS) is also demonstrated following an SDS cleanup of the GELFrEE fractions. In addition to the improved throughput afforded by a multiplexed device, we present a unique strategy to enhance resolution at high sample loadings by pooling fractions collected following separation of a single sample dispersed through multiple columns. Finally, we illustrate the coupling of mGELFrEE to a custom built solution isoelectric focusing (sIEF) device.21 This semipreparative twodimensional liquid electrophoresis (2D LE) platform separates (15) Santoni, V.; Molloy, M.; Rabilloud, T. Electrophoresis 2000, 21, 1054–1070. (16) Tran, J. C.; Wall, M. J.; Doucette, A. A. J. Chromatogr., B 2009, 877, 807– 813. (17) Tran, J. C.; Doucette, A. A. Anal. Chem. 2008, 80, 1568–1573. (18) Lewis, U. J.; Clark, M. O. Anal. Biochem. 1963, 6, 303–315. (19) Jovin, T.; Naughton, M. A.; Chrambach, A. Anal. Biochem. 1964, 9, 351– 369. (20) Meng, F. Y.; Cargile, B. J.; Patrie, S. M.; Johnson, J. R.; McLoughlin, S. M.; Kelleher, N. L. Anal. Chem. 2002, 74, 2923–2929. (21) Tran, J. C.; Doucette, A. A. J. Proteome Res. 2008, 7, 1761–1766.

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analogously to 2D PAGE and yields 128 well resolved fractions in an unprecedented 3.25 h. Both the mGELFrEE and 2D LE platforms make important contributions to top-down and bottomup proteomics as front-end separation tools for mass spectrometry analysis. EXPERIMENTAL PROCEDURES Materials. Milli-Q grade water was purified to 18.2 MΩ cm-1. All reagents for gel electrophoresis were obtained from BioRad Canada (Mississauga, Canada). The 3.5 kDa MWCO dialysis membranes were purchased from Fisher Scientific (Ottawa, Canada). Lyophilized S. cerevisiae, B. subtilis, trypsin (TPCK treated), protein standards (bovine ubiquitin, chicken lysozyme, equine myoglobin, bovine carbonic anhydrase), and other chemicals were purchased from Sigma (Oakville, Canada). Sample Preparation. Lyophilized cells of S. cerevisiae were suspended in SDS PAGE running buffer,10 whereas cells of B. subtilis were suspended in pure water and independently lysed by two passes in a French press at 8 000 psi, and then centrifuged at 13 000g, and the supernatants were stored at -20 °C until use. The concentrations were determined using a Bradford assay. A 50 mL urine sample, obtained from a healthy donor, was subject to protein precipitation with three volumes of cold acetone (-20 °C, overnight). The resulting pellet was reconstituted in running buffer (concentration 5 g/L). Between 100 and 200 µL of sample was loaded in a mGELFrEE column after combining the sample with 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. mGELFrEE Device. Design. With reference to Figure 1a, the device consists of four main components: (1) a cathode chamber, (2) the gel columns, (3) the collection chamber, and (4) an anode chamber. The 250 mL electrolyte chambers and collection chamber are constructed of polyoxymethylene (Delrin) and Teflon, respectively. Eight parallel glass columns extend from the cathode chamber to the collection chamber, using O-rings and/ or rubber gaskets around the glass tubes to prevent leakage. A 1.9 cm spacing between the columns matches the dimensions of a multichannel pipettor. The collection chamber consists of eight channels bore into the Teflon plate of thickness 1.0 cm. Access holes are drilled from the top of the plate into these channels to permit multiple extraction of sample from the chamber. A 3.5 kDa MWCO regenerated cellulose acetate membrane is sandwiched between the collection and anode chamber. Once the gel tubes are positioned in the opposing side of the collection chamber, a void volume of approximately 140 µL per chamber is achieved. Gel Column. The 4 mm i.d. polyacrylamide gel columns were cast into 6 mm o.d. × 6.0 cm glass tubes. Unless otherwise noted, the gel column contained a 1 cm long resolving gel, cast to 15% T, 2.67% C, as well as a 2.4 cm long stacking gel (4% T, 2.67% C), prepared using the classical Laemmli protocol for SDS PAGE.10 Samples were loaded into the void volume of the glass tube (cathode end) above the stacking gel. GELFrEE Operation. The GELFrEE device was operated at 360 V in a stop and go cycle, collecting fractions from the eight gel columns at defined time points by extracting the solution contained in the collection chambers. Following extraction, the collection chamber was replenished with 100 µL of Laemmli running buffer per channel. During operation, the cathode end

Figure 1. (A) Schematic of the multiplexed gel-eluted liquid fraction entrapment electrophoresis (mGELFrEE) device. The main parts include I, cathode chamber; II, eight parallel gel columns (only six are shown); III, collection chamber; and IV, anode chamber. A photograph of the device is shown in part B, with a close-up of the eight gel columns provided in part C. These parallel glass tubes contain a discontinuous polyacrylamide gel, cast according to the method of Laemmli.10 To enhance visualization, a 4 cm long “resolving” polyacrylamide gel (15% T) is used (1 cm gels are employed during typical operation). A separated, prestained molecular weight protein ladder is visible (larger proteins at top). During operation, separated proteins will migrate through the gel column until they elute from the gel, where they are entrapped by a 3.5 kDa MWCO membrane at the base of the collection chamber. Access ports allow fractions to be successively removed from the collection chamber as the run progresses. With each collection, larger proteins are removed, resulting in a broad mass separation of the proteome in approximately 1.5 h.

of the device was raised to a 45° angle, ensuring sample contact with the stacking gel phase. sIEF Separation. A total of 1 mg of S. cerevisiae was suspended in sIEF cocktail buffer (4 M urea, 2 M thiourea, 50 mm diothiothreitol (DTT)) and separated on a custom designed eight-channel sIEF system21 using 1% w/v Biolyte 3/10 carrier ampholytes (Bio-Rad). Following separation, the resulting fractions were removed to separate vials, and the eight chambers of the sIEF device were each extracted with 100 µL of Laemmli running buffer. This wash was combined with the original sample, and a portion was subject to SDS PAGE as described below. The remaining sample was precipitated with three volumes of cold acetone (-20 °C, overnight). The pellet was reconstituted in 100 µL of gel loading buffer and subsequently separated with mGELFrEE. RPLC Separation. A total of 400 µg of S. cerevisiae, suspended in solvent A (H2O/0.1% formic acid) was injected onto a 1 mm × 10 cm polymeric reversed-phase column (259 VHP from Vydac, Hesperia, CA) and separated using a gradient from 2-30% B (ACN/0.1% formic acid) over 2 min, 30-40% B over 5 min, 40-50% over 10 min, 50-60% B over 2 min, followed by a ramp to 100% B over 3 min at a flow rate of 50 µL/min. Analytical SDS PAGE. mGELFrEE fractions were analyzed by discontinuous SDS PAGE10 using 15% T resolving slab gels. For this, 15-20 µL of the fractions was combined with 5 µL of gel loading buffer, of which 15-20 µL were loaded onto individual

lanes of the slab gel along with the appropriate standards. Gels were silver stained22 and scanned using a flatbed scanner. Sample Digestion and Bottom-Up LC-MS/MS. A total of 100 µg of S. cerevisiae protein extract was loaded onto a mGELFrEE column and partitioned into 16 fractions. SDS was removed from the resulting fractions based on the protocol described by Wessel and Flugge.23 A Bio-Rad DC Protein Assay was used to assess protein recovery from 5 and 25 µg protein extracts of S. cerevisiae, prepared in SDS PAGE running buffer. The extracts underwent chloroform/methanol/water (CMW) precipitation23 followed by resuspension in water with 1% SDS to ensure complete protein resolubilization. The efficiency of SDS removal following CMW precipitation was determined using a methylene blue colorimetric assay, as described by Arand et al.24 After protein precipitation, the resulting pellets were redissolved in 50 µL of 100 mM NH4HCO3 and digested overnight at 37 °C through addition of 250 ng of trypsin. Digests were stopped by acidification with formic acid and then evaporated to a final volume of 15 µL per sample. A 3 µL portion of this was injected onto a 150 mm × 180 µm Biobasic C18 column (ThermoFisher Scientific, San Jose, CA) and separated using a gradient from 2 to 40% B (ACN/0.1% formic acid) over 85 min, followed by a ramp to 80% B over 5 min at a flow rate of 2 µL/min. A nanospray source was used to interface to the LTQ linear ion trap mass spectrometer (ThermoFisher) operated in data dependent mode (1 MS scan followed by MS/MS of top five ions). Data were searched using Sequest, part of BioWorks 3.2, against the S. cerevisiae subset of the Uniprot database. Positive identification was based in part on criteria defined by Washburn et al.2 Peptides with charge +1, +2, and +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. Furthermore, two or more unique peptides, being assigned to the same protein, were required for positive identification. In cases where a unique peptide sequence could be assigned to multiple proteins, the peptide was assigned to the protein of highest MW. This criteria provided a peptide false positive rate of 0.44% (1% for proteins), determined through reverse database searching. Intact Protein Analysis by MS Following mGELFrEE. Six protein standards, which include ubiquitin, cytochrome c, lysozyme, myoglobin, R-lactalbumin, and carbonic anhydrase were separated by mGELFrEE (10 µg per protein) and precipitated to remove SDS.23 A final washing step was incorporated into the precipitation protocol, whereby 400 µL of methanol was added to the resulting pellet, then centrifuged and discarded. Pellets were solubilized with 5 µL of 88% formic acid and subsequently diluted to 100 µL with water. Half of the resulting fraction was injected onto a homepacked 0.5 mm × 5 cm C4 column (5 µm, 300 Å pores) and separated at 20 µL/min using a gradient from 5 to 30% B over 3 min, then to 80% B over 20 min. The column flow was interfaced via an ESI source to a MicroTOF LC system from Bruker Daltonics (Billerica, MA). Spectra were processed with the protein deconvolution tools in DataAnalysis V3.3 software, available from Bruker. (22) Shevchenko, A.; Wilm, M.; Vorm, O.; Mann, M. Anal. Chem. 1996, 68, 850–858. (23) Wessel, D.; Flugge, U. I. Anal. Biochem. 1984, 138, 141–143. (24) Arand, M.; Friedberg, T.; Oesch, F. Anal. Biochem. 1992, 207, 73–75.

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Safety Considerations. Appropriate precautions must be taken for the handling of acrylamide, a neurotoxin, as well as for the operation of the mGELFrEE and sIEF devices, which require high voltages. RESULTS Summary of mGELFrEE Technology. GELFrEE17 is a semipreparative electrophoretic platform which achieves molecular weight partitioning of proteome mixtures in the solution phase. A key feature of GELFrEE is the use of unusually short (1 cm) gel columns, which translates into rapid (∼1.5 h) separation times for proteome mixtures over a very broad mass range, easily exceeding 100 kDa. Here, a multiplexed platform (mGELFrEE) has been constructed (Figure 1), taking advantage of the compact nature of the original design. The main components of the mGELFrEE system, namely, cathode, gel columns, collection chambers, and anode as well as operation of the mGELFrEE device, follow our previously reported single channel GELFrEE device.17 The collection chambers of the mGELFrEE device (see Figure 1C) are spaced to work with an eight-channel micropipettor and provides a simple, yet efficient means of trapping proteins with the assistance of a 3.5 kDa dialysis membrane at the anode terminus of the chambers. The benefits of SDS PAGE are exploited in this mGELFrEE device, including the generation of predictable size separation for intact proteins, while maintaining high solubility during separation and sample collection. Moreover, collection of proteins in the solution phase is preferred for downstream analysis of protein (digested, or intact) through MS. Increased Throughput. Figure 1C depicts the separation of prestained protein markers in the eight mGELFrEE columns. Distinct protein bands are well resolved within each of these columns, and the similar migration of the bands suggests high repeatability between the columns. Gel images, depicting typical separations of various proteomes which include B. subtilis, S. cerevisiae, and human urine, under standard mGELFrEE operating conditions, are shown in Figure 2. The simultaneous separation of the three independent mixtures is demonstrated over a mass range between 10 kDa to over 120 kDa in a rapid time scale of 1.5 h. Size-based partitioning of these complex mixtures is shown in this figure, with the majority of proteins appearing in one or two gel lanes. Comparison of band intensities of the individual fractions to the original (unfractionated) control mixture confirms the high recoveries, which are typical of a GELFrEE experiment.17 This figure also demonstrates the enhanced throughput of the mGELFrEE device, where simultaneous separations operate entirely independently in any given channel. In other words, the type of sample loaded onto a given column does not significantly alter the separation profile obtained on other columns. Many parameters have influence on the repeatability of the experiment, including the composition of the running buffer and dimensions of the gel column. It is seen from Figure 2 that the mass range collected at any given time interval was similar for each column, demonstrating how predictable MW separation is achieved during mGELFrEE separation. To properly illustrate the repeatability of the mGELFrEE separation, identical samples of S. cerevisiae were fractionated through individual gel columns. Proteins fractions collected from four independent columns were analyzed by SDS PAGE and are shown in Figure 3A. A blank was also introduced into an mGELFrEE gel column, and the absence 6204

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Figure 2. Silver stained gel images of fractions collected following mGELFrEE separation demonstrate the high-resolution MW separation attainable for various proteome samples. Here, 100 µg of extracts of (A) S. cerevisiae, (B) human urine, and (C) B. subtilis were loaded onto individual columns of mGELFrEE and simultaneously separated. The collection times indicated represent the accumulated duration of the applied voltage. By customizing the collection entrapment interval, a linear size distribution is apparent on the SDS PAGE gels, though it is noted that such gels separate in a logarithmic fashion. The control lanes represent equivalent loadings of the unfractionated sample assuming 100% recovery in mGELFrEE. The high-throughput nature of mGELFrEE is realized as independent samples are simultaneously separated by the device.

of detectable proteins from this channel illustrates that the mGELFrEE device prevents sample carryover across the collection chamber. Minor differences in the mass range of the collected proteins are noticeable for the lower MW (earlier) fraction, as is seen with channel 6 in the figure. With these fractions, a short collection time is employed (1 min in this case), and thus a minor difference in the length of the gel column translate into observable differences in the collected mass range, on the order of ∼5 kDa. Such differences are less obvious in the higher MW fractions, both as a consequence of the slower rate of protein migration (thus longer trapping intervals) as well as the mass resolution at this MW on an SDS PAGE gel. Run-to-run repeatability was also assessed, and the results are shown in Supplementary Figure S1 in the Supporting Information. High repeatability was achieved with particular attention in maintaining constant gel and running buffer compositions, since changes resulting from inconsistent buffer preparation would translate into variations in the mass ranges collected at a given trapping time interval. Increased Loading Capacity. In chromatography, accommodating a larger quantity of sample can be achieved simply by moving to a larger diameter column. Unfortunately, as with all electrophoretic separations, increased cross-sectional area will deteriorate the resolution as a consequence of Joule heating. In Chrambach’s preparative gel electrophoresis device,19 Joule heating was minimized in a large diameter column by incorporating a cooling device. However, cooling devices add to the complexity and expense of the platform and also impede the rate of protein mobility, leading to longer separation times.

Figure 3. Silver stained gel images of fractions collected from the mGELFrEE device reveal the high reproducibility of mGELFrEE columns and also illustrate how sample loading capacity can be increased when employing the pooled fraction protocol. (A) Silver-stained gel images show the reproducibility of fractions from the separation of 100 µg of S. cerevisiae among parallel columns of the mGELFrEE device. The fractions collected from channels 2, 4, 6, and 8 are shown, with channel 5 representing the loading of a reagent blank, and illustrate no detectable cross talk between the collection chambers. Thus, the mass range of any collected fraction is consistent for identically cast columns in a single mGELFrEE run. (B) A total of 800 µg of S. cerevisiae protein extract was loaded onto a single 4 mm i.d. mGELFrEE column. The effect of overloading is apparent as protein components are observed in multiple fractions following separation. (C) A total of 800 µg of S. cerevisiae protein extract was distributed across eight mGELFrEE columns (100 µg/column). Fractions collected across the eight columns at each time point were pooled. The effectiveness of this protocol as a means of achieving high resolution at higher sample loading is seen when comparing this gel image to that of the overloaded gel column.

The column dimensions employed in the mGELFrEE device can easily accommodate 100 µg of protein mixtures per column, as depicted in Figure 2. However, at higher sample loadings, band broadening becomes significant and proteins are no longer retained in individual fractions. Figure 3B shows gel images of fractions collected from a separation of 800 µg of S. cerevisiae in a single mGELFrEE column. This overloaded column displays poor resolution compared to an equivalent separation of 100 µg of this sample (Figure 2A). What is presented is an innovative pooled fraction protocol that maintains high resolution and rapid separation, at increased sample loading. The pooled fraction protocol works by distributing the sample over the eight parallel columns of the mGELFrEE system. Since loading capacity is dependent on the cross-sectional area of the gel column, a theoretical 8-fold increase in sample loading is generated. Following separation, the collected fractions are pooled from each entrapment period. Of course, for such a strategy to be effective, the repeatability of the eight columns must be high (as demonstrated in Figure 3A) in order to maintain high resolution in the pooled fractions. Figure 3C illustrates the effectiveness of the pooled fraction protocol. Here, an 800 µg sample of S. cerevisiae was distributed among the eight gel columns (100 µg per column). After separation, corresponding fractions were combined and analyzed by analytical gels. The resolution depicted in Figure 3C for the 800 µg sample is comparable to the separation profile obtained from an unpooled 100 µg sample (Figure 2A). It is also far superior to that of the overloaded 800 µg sample shown in Figure 3B. The mGELFrEE device therefore maintains resolution at higher sample loadings, equivalent to the number of gel columns available in the mGELFrEE device. In theory, such a protocol could also be applied to

a greater number of even smaller diameter columns, which could further enhance resolution while maintaining sample loading. Mass Spectrometry Analysis of mGELFrEE Fractions. The separation profiles of mGELFrEE are demonstrated by conventional SDS PAGE analysis of resulting fractions. Puchades and co-workers25 have demonstrated that the classical Wessel and Flugge23 precipitation protocol can sufficiently remove SDS from protein fractions collected in a preparative gel electrophoresis separation enabling MALDI MS analysis. We have also shown that this SDS depletion protocol can be applied to GELFrEE fractions enabling LC-ESI MS/MS analysis of simple standard protein mixtures.17 On the basis of a methylene blue colorimetric assay,24 the amount of SDS remaining from our chloroform/ methanol/water precipitations was reduced to below the detection limit of 2 mg/L (i.e., >500-fold reduction of SDS), being consistent with those values reported by Purchades et al.25 Furthermore, protein recovery in our precipitations ranged from slightly over a 50% yield, with 12% RSD, when 5 µg of a yeast proteome extract was used to just under 70% recovered, with 10% RSD, when 25 µg of yeast protein was precipitated. A 5 µg sample is a reasonable estimate of the amount of protein present in a mGELFrEE fraction, based on a 100 µg protein load on the column. We also note that the precipitation protocol does not appear to be biased to protein size over the MW range examined here. This was determined from side-by-side comparisons of precipitated and unprecipitated mGELFrEE fractions in SDS PAGE gels (results not shown). To fully evaluate the separation profiles of complex proteome mixtures attained through GELFrEE as a prefractionation tool ahead of MS, we sought to analyze the resulting 16 fractions from (25) Puchades, M.; Westman, A.; Blennow, K.; Davidsson, P. Rapid Commun. Mass Spectrom. 1999, 13, 344–349.

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Figure 4. Plots summarize results from LC-MS/MS analysis of mGELFrEE fractions collected from yeast extracts (data corresponds to the yeast sample shown in Figure 2A). (A) The plot shows the number of proteins identified by mass spectrometry in each of the fraction. The customized collection entrapment interval enables a relatively even distribution of proteins across the collected fractions. (B) The plot shows the size distribution of proteins identified following separation by mGELFrEE. These identified proteins are compared to the total size distribution of yeast proteins as predicted from the genome. The similarity of these distributions suggests that the proteins detected in mGELFrEE reflect the molecular weight distribution of the predicted proteome.

the separation of the S. cerevisiae proteome (shown in Figure 2A). Proteins from these fractions were precipitated to remove SDS, digested with trypsin, and subjected to LC-MS/MS analysis. The number of proteins identified in each fraction is summarized in Figure 4A. MS analysis resulted in the identification of 1120 proteins, of which 428 were unique (1% protein false positive rate). The separation of mGELFrEE is shown to be relatively unbiased across the MW range covered. This is demonstrated in Figure 4B where the profile of identified proteins from mGELFrEE fractions overlaps nicely with the profile distribution as predicted by the yeast genome. It is noted that a slightly greater proportion of proteins were identified in the low mass range 10-30 kDa, though a small number of large proteins identified is consistent with the predicted yeast proteome profile. An ideal proteome fractionation tool would evenly partition the mixture across the attained fraction. Considering the MW distribution profile of proteins in yeast, it is evident from Figure 4A that by collecting the fractions over customized time intervals, a more even partitioning of the sample mixture can be obtained across the multiple fractions. As we show, the greater sampling of fractions in the low to mid mass range (