Bifunctional Glass Membrane Designed to Interface SDS-PAGE

Feb 24, 2015 - We describe the construction and characterization of a novel membrane designed to allow proteins separated by gel electrophoresis (SDS-...
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Bifunctional Glass Membrane Designed to Interface SDS-PAGE Separations of Proteins with the Detection of Peptides by Mass Spectrometry Stephen J. Hattan,*,† Jie Du,‡ and Kenneth C. Parker† †

SimulTOF Systems, 60 Union Avenue, Sudbury, Massachusetts 01776, United States Toxikon Corporation, 15 Wiggins Avenue, Bedford, Massachusetts 01730, United States

‡

S Supporting Information *

ABSTRACT: We describe the construction and characterization of a novel membrane designed to allow proteins separated by gel electrophoresis (SDSPAGE) to be detected as peptides by mass spectrometry in an efficient and comprehensive manner. The key attribute of the membrane is a bifunctional design that allows for the digestion of protein(s) and retention of the resulting peptides with minimal lateral diffusion. Silane chemistries are used to differentially treat the opposing surfaces of a glass filter paper to enable this unique capability.

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To keep up with advances in mass spectrometry, efficient sample preparation schemes offering higher resolution are needed.13,14 Typically, proteomic experimental schemes (workflows) are based on either the “top-down” or “bottom-up” design.15−18 The “bottom-up” approach incorporates enzymatic digestion early in the workflow, and consequently, sample manipulation, separation, and eventual detection are all performed at the level of peptides. Generally, for mass spectrometers, the performance specifications19,20 of detection sensitivity, mass accuracy, and signal resolution are better in the mass range of peptides (10000 Da). Also, peptide-sized molecules yield more information-rich spectra when analyzed by MS/MS.21 Chromatographic separations of complex mixtures of peptides are more efficient and are, therefore, more widely applied than protein-based separations. The more complex, dynamic structure of proteins leads to greater variability in their interaction with chromatographic media,22 leading to lower predictability, reproducibility, and efficiency for LC separations. For all these reasons, “bottom-up” schemes are more commonly used in proteomic studies. The primary drawbacks to “bottom-up” approaches are that they increase sample complexity and confound the ability to distinguish protein polymorphs and modifications. The incorporation of an up-front digestion step into the workflow converts a complex mixture of proteins into an even more complex mixture of

he objective of this manuscript is to outline the construction of a novel membrane designed to enzymatically digest proteins and to retain the peptides generated close to point of their origin. It is hoped that this membrane may be used to simplify MS detection of peptides following SDS-PAGE separation of proteins. It is also hoped that inclusion of this membrane into an experimental scheme that efficiently links SDS-PAGE separation with MS detection will further enable accurate, rapid and reasonably large-scale identification of proteins originating from complex proteomic samples. The ability to accurately inventory and quantify the protein component of a given biological system often remains a challenge to bioanalytical science. Generally, the biggest obstacles to success are sample complexity and the dynamic range of component concentrations.1−4 Large-scale proteomic experiments that attempt to measure and relate the proteins that compose a biological system must rely on separation science5−7 to distribute and often concentrate the various components prior to detection. Without separation, and often several dimensions of separation, the capability of current detectors to distinguish the various components is overwhelmed and rendered incomplete. The detector of choice for many proteomics studies is the mass spectrometer. The reasons for this include the analytical traits of accuracy, sensitivity and speed in the identification of protein and peptide species. Current mass spectrometers can routinely detect nanomolar (10−9) concentrations of material with low ppm mass accuracy at subsecond MS and MS/MS acquisition times and this enables peptide and subsequent protein identification of 100s to 1000s of proteins on a time scale of hours.8−12 © XXXX American Chemical Society

Received: October 24, 2014 Accepted: February 24, 2015

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gained in the gel separation; (4) capture and concentrate these peptides in solution ready for detection by mass spectrometry. The following study highlights the use of MALDI mass spectrometry for peptide detection followed by protein identification by peptide mass fingerprinting. These techniques were chosen because they are well suited to demonstrate the utility of our digestion/capture membrane in the workflow under examination and because they are tools available to us. Regardless, it is important to note that, after elution from the membrane, peptides may be readily analyzed by either electrospray ionization (ESI)14 or MALDI15 mass spectrometry. They may also be stored, further processed, further separated, or detected by another means to best suit the objectives of the experiment.

peptides. Additionally, in samples possessing a large dynamicrange in concentration, constituent peptides from abundant proteins tend to permeate the entire analytical space.23,24 This blanketing effect of peptides from the abundant proteins is often the primary reason why peptides from low abundance proteins or transient protein post-translational modifications are not detected. “Top-down” schemes are those where sample manipulation, separation and detection all take place at the level of intact proteins. Keeping the protein’s primary structure intact is an attractive quality of “top-down” schemes because valuable information regarding variant forms and post-translational modifications is preserved. Modified protein isoforms have been identified in cancer,25−27 Alzheimer’s disease,28 diabetes,29,30 and arthritis,31 and these variants have been proposed as biomarkers and active agents in disease propagation. Therefore, protocols that enable the discernment and identification of these features are often necessary to meet research objectives. Since its introduction by Laemlli32 about 40 years ago, SDSPAGE has remained one of the most popular methods for separating protein mixtures due to its high resolving power, reproducibility, and simplicity.33−35 Unfortunately, however, the interface between gel separation of proteins and mass spectrometry detection of peptides is not efficient.36−38 Typically, the gel must be stained to determine protein locations and then protein(s) of interest are excised. Next, the gel itself is mechanically or chemically disrupted, so that added enzyme can access the protein(s) within.39,40 After incubation to allow for protein digestion, the resulting peptides must be purified (e.g., solid phase extraction, liquid chromatography) to remove gel debris, buffers, and salts in order to optimize mass spectrometric analysis. While gel staining procedures have been optimized to offer reasonable sensitivity (∼1 ng for silver stain and ∼100 ng for Coomassie blue)41 selective band excision leaves sections of gel unanalyzed. The steps involved in sample digestion and isolation from the gel are largely manual, timeconsuming, and are often incomplete and imprecise. The “molecular scanner”42−44 allowed gel-separated proteins to be blotted out of the gel through a membrane containing an immobilized enzyme and onto a hydrophobic capture membrane and was devised to allow seamless integration of gel separation and mass spectrometry. In practice, however, with this approach it proved difficult to control the optimal dwell time for proteins within the enzyme membrane necessary to ensure adequate protein digestion. Furthermore, performing MALDI mass spectrometry directly off of the capture membrane (PVDF) was far less sensitive than conventional MALDI performed off a target surface (stainless steel). Consequently, the “molecular scanner” technology is rarely adopted; however, we credit the efforts and innovation behind the “molecular scanner” as the launching point for this study. The approach we report here is based on a membrane that enables the best features of the “top-down” and “bottom-up” proteomic schemes to be incorporated into a single workflow. The bifunctional characteristics implemented into the membrane’s design efficiently bridge the transition between gelbased separation of proteins and the detection of peptides by mass spectrometry. Our objectives were to (1) harness the utility, convenience and resolving power of gel-based intactprotein separations; (2) electrophoretically extract the separated proteins from the gel; (3) digest the resolved proteins into their constituent peptides while maintaining the resolution

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MATERIAL AND METHODS Sheets (8′ × 10′ × 3 mm) of A/C glass filter paper were purchased from Pall Scientific (Lancing, MI), silane compounds from Gelest Inc. (Morrisville, PA) and TPCK treated trypsin from Worthington Biochemical (Lakewood, NJ). Tris-glycine 4−20% SDS-PAGE gels and associated gel loading, running and transfer buffers, and SimpleBlue SafeStain were purchased from Life Technologies. Immobilon membrane was purchased from Millipore Inc. (Bedford, MA). Bovine serum albumin (BSA), cytochrome-C, 25% glutaraldehyde solution, and all experimental solvents, buffers, and salts were purchased from Sigma-Aldrich, St. Louis, MO. Mass spectrometry instrumentation was developed in-house at SimulTOF Systems Inc. (Sudbury, MA). Membrane Construction. Prior to modification, glass fiber filter paper was immersed in a 1:1 mixture of 50% aqueous sulfuric acid/30% hydrogen peroxide for 30 min, followed by sequential rinses in DI H2O/CH3OH/DI H2O and air-dried overnight. Silane Solutions and Application. Solution 1 (enzyme immobilization): 2% 3-aminopropyltriethyoxysilane, 2% hydroxymethyltriethoxysilane, 1% N,N′-bis-[(3triethoxysilylpropyl)aminocarbonyl]poly(ethylene oxide), 1% bis(triethoxysilyl)methane, 5% glacial acetic acid, 10% ethanol, and 79% H2O. Solution 2 (hydrophobic peptide capture): 5% n-octyltrimethoxysilane, 1% bis-(triethoxysilyl)methane, 1% bis(triethyoxysilyl)hexane, 10% H2O, 5% glacial acetic acid, 20% ethanol, and 58% n-butanol. Silane solutions were applied sequentially by airbrush (350 Model, Badger) to opposite sides of the glass fiber filter paper. The application air pressure was 20 psi and the nebulizer was set to apply a fine mist in a 2 in. swath at a distance of 6 in. from the target. The glass fiber filter paper has a coarse surface on one side and comparatively smooth surface on the other. To ensure good surface contact between membrane and gel, and to keep track of the proper membrane orientation, solution 1 was applied to the smooth side of the glass paper. For the construction of a 150 mm2 membrane of type A/C glass fiber filter, two 2.5 mL aliquots of each solution were sprayed in an alternating, serial manner. Between each application, a 30 min drying time at room temperature (RT) was allowed. After the final application, the membrane was placed in an oven at 85 °C for 2 h. Finally, the membrane was rinsed with CH3OH, then H2O, and placed back in the oven for an additional hour. The membrane was then incubated in a solution of 50% 100 mM Na2HPO4 (pH 5.7), 25% glutaraldehyde, and 25% CH3OH for 2 h at RT. Immobilization of Trypsin. Silanized glass fiber filter paper was wetted with CH3OH and immersed in 100 mM B

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solution as a reference. In some cases, multipoint internal calibration was performed using additional masses of deduced peptides from the peptide mass fingerprint. Following protein digestion and peptide capture, membrane sections were washed in water and then eluted in 75% CH3CN/0.1% TFA. A 1 μL aliquot of eluate was then mixed with 1 μL of matrix (10 mg/ mL α-cyanohydroxycinnamic acid in 75% CH3CN/0.1% TFA) and spotted for MALDI analysis. Peptide Mass Fingerprinting (PMF). After internal calibration, spectra were submitted to a peptide mass fingerprinting program integrated into the SimulTOF software derived from ChemApplex (Parker et al, 2004, Parker and Hattan, manuscript in preparation, 2014).46,47 This software is designed to take advantage of the chemistry of tryptic digests acquired from MALDI mass spectrometers. Proteins receive scores weighted according to the percentage of the total arginine-containing tryptic peptides that are accounted for in the spectra. The database used derives from a SwissProt FASTA file reconfigured into sqlite3 (see http://www. sqliteexpert.com/download.html) and is configured so that the user can select any combination of organisms. This database also contains sets of commonly encountered contaminant proteins like trypsin and human keratins. The program iteratively subtracts assigned peaks from the peak list, and can therefore provide the user with evidence for many mutually consistent protein identifications. In favorable cases, as many as 20 proteins can be identified correctly from a single MALDI mass spectrum. For the present study, PMF results were obtained from searching spectra obtained from gel fractions against a database containing the proteome of yeast (Saccharomyces cerevisiae; 6582 proteins) and the available contingent of the proteomes of human, mouse, rat, cow, Escherichia coli, Bacillus subtilis, and Dictyostelium discoideum in addition to yeast (69459 proteins in total). The proteins reported as positively identified represent only the yeast proteins that scored higher than hits to any of the other species in a given fraction.

Na2HPO4 (pH 5.7) with 25% CH3OH. Once equilibrated, the membrane was placed in the same solution containing trypsin (0.5 mg/mL) and sodium cyanoborohydride (1 mg/mL) at a ratio of ∼0.2 mL of trypsin solution per cm2 of membrane, covered, and allowed to react overnight in a shaker rotating at 60 rpm. Following reaction, the membrane was washed with 50% CH3OH to remove noncovalently attached trypsin and sequentially rinsed for 30 min each in (50 mM Tris pH 8.0, 10% CH3OH)/(50 mM Tris pH 8.0, 1 M NaCl, 10% CH3OH)/(50 mM Tris pH 8.0, 10% CH3OH). After the final rinse, the membrane was patted with a paper towel until moist, placed in a zip-lock plastic bag, and stored in a refrigerator at 4 °C until use. Membrane Characterization. A hole-punch was used to create discs of consistent size (25 mm2) at random locations on the membrane surface. To test consistency in protein digestion, 10 pmol of reduced and alkylated BSA was added to 10 separate membrane discs that had been equilibrated in transferblot buffer. The discs were placed in individual tubes and allowed to incubate at RT for 4 h. After the allotted time, the digestion was quenched by addition of 50 μL of 0.1% TFA. To test peptide binding capacity, membrane discs were spotted with a 1, 5, 10, or 20 μL aliquot of a 1 pmol/μL reduced, alkylated, and digested BSA sample (10 sections for each quantity). The sample was applied under suction so that it was readily pulled through the membrane. A PVDF membrane was placed behind the glass fiber membrane to capture any peptides that did not bind and to determine the point of breakthrough and maximum binding capacity. To test the speed of protein digestion, 1 pmol of cytochrome C was placed on 18 holepunch sections of membrane, and the sample was allowed to incubate for different amounts of time (5 min, 0.5, 1, 2, 4, and 16 h). After the allotted time, the digestion was quenched by addition of 50 μL of 0.1% TFA. Electrophoresis. Electrophoresis experiments were performed on an Invitrogen XCell SureLock Mini-Gel system using Novex tris-glycine gradient gels (4−20%), SDS running buffer (10×), SDS sample buffer (2×), and transfer buffer (25×). Samples were diluted 1:1 in SDS sample buffer, sonicated for 10 min and applied in 10 μL aliquots. Gels were run at a constant voltage of 125 V for 1.5 h. After SDS-PAGE separation, gels were equilibrated in transfer blot buffer for 10 min. Electro-transfer blotting was carried out on the same system using the XCell II Blot Module. Blots were assembled as outlined in the XCell II manual, except for the insertion of our functionalized glass fiber membrane between the separation gel and the PVDF capture membrane. The PVDF membrane was kept in place as a means of checking for the complete capture of proteins/peptides by the glass fiber membrane. Blots were run at a constant voltage of 25 V for 1.5 h. After blotting, the glass fiber membrane was cut vertically along the lines of the lanes of the gel, then horizontally into ∼2 mm strips. Mass Spectrometry. Mass spectra were acquired on a high performance MALDI-TOF spectrometer built in-house at SimulTOF Systems45 and operated in reflector mode to achieve a 14.8 m flight path. The instrument was operated at a laser frequency of 1 kHz, a scan speed of 1 mm/s and an operational gas pressure of 2 × 10−8 Torr. Typically, one nsec time bin spectra averaged from about 10000 shots were collected from each sample, in bundles of 500 shots that were combined at the level of the digitizer. Spectra were internally calibrated by using synthetic peptide RfffR, where f equals pentafluorophenylalanine, which was added to the matrix

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RESULTS Figure 1 illustrates construction of the membrane with photographs demonstrating the effectiveness of the procedure. Figure 1A depicts the hydrophobic side of the membrane showing droplets of H2O from 40 to 1 μL that readily bead upon contacting this surface. Figure 1B shows effective enzyme immobilization by the staining of bound trypsin with Ponceau S dye. As shown in Figure 1B, the immersion of the membrane into the Ponceau S solution results in staining to a depth of ∼1.5 mm into the membrane. Figure 2 shows the overlay of 10 MALDI mass spectra resulting from the parallel digests of a 10 pmol (10 μL of 1 pmol/μL) of BSA. Figure 2 shows the peptide region (m/z 900−2500) and two blow-ups of the peaks at m/z 927 and 1567. The inset in Figure 2 shows an example of the holepunched membrane and ∼6 mm diameter sections used for membrane characterization experiments. The spectra in Figure 2 demonstrate both qualitative and quantitative precision in protein digestion with a relative standard deviation of 13% in the measured signal intensity across the range m/z 900−2500 calculated for the 10 replicates. Figures 3−5 outline a peptide binding capacity study. Peptides (previously digested BSA at 1 pmol/μL) were added to the membrane to produce four load quantities (i.e., 1, 5, 10, and 20 pmol). A total of 10 replicates were prepared at each C

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Figure 1. Membrane construction: (A) Surfaces of 3 mm thick A/C glass filter paper are differentially treated with silane chemistry to produce a bifunctional membrane. One side of the membrane is coated to enable enzyme immobilization; the other is treated to create a hydrophobic web. (B) Photographs show sections of membrane after immersion in Ponceau S dye. The red surface indicates even staining by immobilized trypsin, whereas the pink surface indicates less staining of the hydrophobic surface. The cross section shows the evenness of the membrane layers.

Figure 3. Peptide binding capacity: Plot of 10 replicates of BSA digest loaded onto the capture/digest membrane at quantities of 1, 5, 10, and 20 pmol. Data points marked purple result from the analysis of PVDF capture membrane for each load (breakthrough is apparent only in spots 91 and 92). Points marked with yellow indicate the analysis of control samples obtained from the 5, 10, and 20 pmol dilutions spotted directly onto the target (no membrane). Inset plot shows the average intensity plotted against the quantity loaded.

load quantity. The samples being loaded (marked yellow) were used as controls and the PVDF membrane used to catch flowthrough peptides (marked purple) was also analyzed. The same PVDF membrane was used to capture flow-through for all 10 replicates at each load quantity; therefore, one section of PVDF membrane was analyzed for the 10 sections of membrane at each load quantity. All samples were spotted in duplicate. The plot in Figure 3 follows the signal of peptides at m/z 927 and 1567 during the course of the experiment. To the first approximation, there is a similar degree of variability in the intensities of the membrane-eluted replicates as between the control samples. Comparison of the averaged signal intensity for the captured/eluted sample with that of the control yielded 88, 81, and 89% sample recovery for 5, 10, and 20 pmol load quantities, respectively. The inset plot shows the total ion current signal for the full peptide region (900−2500 Da) averaged for all replicates and plotted as a function of the quantity loaded. This inset plot shows linearity over the

concentration range examined with a calculated R2 value of 0.9488. The relative standard deviation of measured signal intensity for 900−2500 Da mass range was 22.7, 42.3, 35.5, and 32.0%, respectively, for the 10 replicates at each load quantity. Figure 4 is an overlay of the averaged mass spectra for all data points at each quantity for the peptide region (m/z 900−2500) and blow-ups of the peaks at m/z 927 and 1567. Figure 4 displays the progressive increase in signal with increase in sample load and consistency in the elution profile. Figure 5 shows an overlay of the averaged mass spectra for the replicates at the 20 pmol load with the averaged mass spectra obtained from the analysis of the PVDF membrane placed behind the digestion/capture membrane to capture unbound material (flow-through), again with blow-ups of peaks 927 and 1567 Da. As can be seen in Figure 5, the 927 peak

Figure 2. Reproducible digestion: Overlay of spectra from 10 parallel digests of BSA acquired from random sections of membrane, with an expanded view of the tryptic peptides at m/z 927 and 1567. The inset shows the hole-punch method for creating membrane sections for use in all characterization studies. D

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Figure 4. Linear increase in signal: Overlay of spectral averages for all data points at the four different sample loadings with blow-ups of peaks m/z 927 and 1567 Da to show detail.

Figure 5. Threshold for peptide binding capacity: Overlay of spectral average for 20 pmol load data and PVDF membrane placed behind the membrane to capture unretained peptides, with blow-ups of the m/z 927 and 1567 peaks. At the 20 pmol loading quantity, the m/z 927 peptide begins to break through, while the m/z 1567 peptide remains predominately retained.

sections were stained with SimpleBlue Stain (Coomassie blue) and the PVDF membrane sections with the Ponceau S stain. Section 1 shows the SDS-PAGE separation. Sections 2 and 3 illustrate the quantitative transfer and capture of the gel separated sample by the digestion/capture membrane. Section 4 demonstrates quantitative transfer and capture of the sample onto the PVDF membrane. Figure 8B shows a photograph of stained molecular weight standards (Life Technologies) that were separated by SDS-PAGE and then blotted into our digestion/capture membrane. The photo shows a distinctive band for each protein adsorbed by our membrane and these same protein standards passing through untreated glass fiber paper and adsorbed on the PVDF membrane. The dye outlining the profile of these proteins will seemingly remain in place indefinitely until purposefully eluted by application of an appropriate organic solvent. Figure 9 shows an overlay of MALDI mass spectra resulting from the elution and analysis of five adjacent ∼2 mm sections of digestion/capture membrane used in the yeast lysate

begins to break through at this level indicating that the membrane has reached its peptide binding capacity. Figures 6 and 7 display the results of a time-course digest of cytochrome C using the digestion/capture membrane. Protein was added to the membrane, incubated, and quenched at reaction times of 5 min, 30 min, 1, 2, 4, and 16 h with each time-point measured in triplicate. Figure 6 shows a plot of peptide signal intensity (m/z 900− 2500) as a function of time for all samples and the inset shows the plot of the average signal intensity for the three replicates at each time-point. Figure 7 shows an overlay of mass spectra collected at each time point with blow-ups of two peptides (i.e., m/z 1296 and 1562). Figures 6 and 7 demonstrate that the digestion/capture membrane is efficient at digesting protein, producing a detectable quantity of peptide in ∼5 min, and largely completed digestion in 4 h. Figure 8 shows results from analyses of gels and membranes following the SDS-PAGE workflow outlined above. Figure 8A shows a photograph of a stained SDS-PAGE gel and the PVDF capture membrane after separation and blotting. The PAGE gel E

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Figure 8. Analysis of SDS-PAGE gels and membranes after blotting: (A) Result of gel (Coomassie stain) and PVDF (Ponceau S stain) membrane staining after a blot of an SDS-PAGE separation of yeast lysate. Section 1 shows a lane that was cut from the gel and stained (no blotting) and demonstrates the protein separation. Sections 2 and 3 show results from lanes that were blotted through the digestion/ capture membrane. For sections 2 and 3, a lack of protein bands in the gel and on the PVDF membrane demonstrates that proteins are quantitatively captured by the digestion/capture membrane. Section 4 shows the result of lanes that were separated and blotted through an untreated A/C glass filter paper. This section serves as a control, showing the transfer of proteins out of the gel and their capture on PVDF. (B) Digestion/capture membrane’s ability to capture proteins and maintain resolution of separation. Blotted standards are shown embedded in cap/dig membrane but easily pass through untreated glass membrane.

Figure 6. Time course of enzyme digestion: Plot of the summed signal intensity for peptides in the mass range m/z 900−2500 resulting from membrane digestion of cytochrome C. Sample digestion was quenched at different times in groups of three, as indicated by the color. The inset plot shows the average signal intensity for three replicates at each point plotted against time.

experiment. Figure 9 demonstrates the abundance and diversity of peptides generated by the digestion/capture membrane. Figure 10 displays results for yeast proteins confidently identified by peptide mass fingerprinting in the analysis of a 1D PAGE-gel separation of yeast lysate. Figure 10A displays a bargraph showing the number of proteins identified in each fractions and Figure 10B shows a plot of the average molecular weight of proteins in each fraction plotted as a function of fraction number. In this experiment, 49 nonredundant yeast proteins were identified out of 98 total identifications across all 18 fractions. Of these 49 nonredundant identifications, 29 (59%) are localized to a single fraction and 86% are located in ≤2 fractions. An average of 6 peptides were obtained for each protein identification (high = 12 and low = 3) and it is important to note that there was no redundant peptide usage; in all cases, peptides were allowed assignment to only one protein. The average mass error for all peptides used for protein identifications was 2.3 ppm. Auxiliary files containing peak lists for all gel fractions and a complete list of the yeast proteins

identified as well as the top hit to a nonyeast protein in each fraction are provided as supplemental data. Searching a database that is ∼10-fold larger than the yeast proteome, the false discovery rate for the lowest ranking yeast protein in each fraction that outscores all nonyeast proteins is estimated to be ∼10%. All the most abundant larger proteins were identified from multiple, mostly adjacent slices. This indicates lower resolution of proteins by the PAGE gel in this region. It is possible that some of these proteins were partially degraded. The gel used in this experiment was a 4−20% acrylamide gradient gel and the lower percentage of acrylamide located at the top of gel provides lower resolving power than the higher acrylamide

Figure 7. Mass spectra from digestion time: Spectral averages of the three replicate measurements of peptides eluted from the digestion/capture membrane across the mass range of m/z 900−2400 for the 5 min, 30 min, 1, 2, and 4 h and overnight digestion of cytochrome C with blow-ups of peaks at m/z 1296 and 1562 to show detail. F

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Figure 9. Complexity of yeast lysate fractions: An overlay of five spectra obtained by the MS analysis of five adjacent fractions of the digestion/ capture membrane used to process SDS-PAGE separated yeast lysate proteins. The parent spectra and blow-up of a peptide-rich region demonstrate the membrane’s ability to digest proteins into a rich mix of peptides while maintaining the spatial integrity of the separation.

membrane was specifically tailored for use with SDS-PAGE separations; however, samples may be applied by other forces. The membrane operates by protein(s) being forced into the membrane on the side containing the immobilized trypsin. As protein(s) pass through this membrane section, they are cleaved into their constituent peptides. This reasonably thick (∼1.5 mm) section of membrane contains enough enzyme to digest microgram quantities of protein standards and accommodate the digestion of protein loads typical of a resolved PAGE-gel protein band. The peptides that result from the digestion then enter into a region of membrane composed of a hydrophobic web. In this region, many peptides become adsorbed to the membrane by hydrophobic interaction.48−50 The ability of the membrane to bind peptides in this manner keeps the peptides spatially arranged. As evident from Figure 8, the stained protein standards broaden slightly upon passage through an inactive membrane of identical thickness to the capture membrane; however, in the presence of a capture membrane the stained protein bands remain sharp indicating minimal diffusion upon capture. This is an important feature because the resolution of the separation is largely retained, similar to procedures used for Western blots, and peptides bind and reside in the location where the parent protein entered the membrane. Having the peptides that derive from a given protein localized helps combat the problem of highly abundant proteins dominating the detection space. Once adsorbed inside the membrane, it is our expectation that the peptides will remain there until their deliberate elution by the application of an organic solvent. After elution, they may be further processed, further separated or manipulated by any means to best suit the objectives of the experiment. For the current study, we chose to cut the membrane into ∼2 mm wide slices orthogonal to the separation axis of the gel. This was done because it created a manageable number of fractions (∼30) that roughly mimics 2× the size of a typical protein band. However, it is important to note that fractions of greater or lesser size can be prepared to better match the resolution of a given separation or to be tailored to best suit the objectives of a given experiment. Another advantage of having peptides from a given protein localized is that it better enables protein identification by peptide mass fingerprinting.51,52 Peptide mass fingerprinting is

Figure 10. (A) Bar graph showing the number of proteins identified in each fraction. (B) Plot of the average molecular weight for proteins in each fraction plotted as a function of fraction number.

concentration further along the gel axis. In other experiments with different samples, proteins as large as myosin (∼1970 amino acids long) were often found in several adjacent fractions.

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DISCUSSION Demonstrated herein is a novel membrane designed to provide a more seamless transition between the SDS-PAGE separation of proteins and the detection of peptides by mass spectrometry. The emphasis of the present study is to describe the construction and characterize the performance of this new membrane and to develop a protocol for its potential use in the analysis of complex proteomic samples. Membrane construction is simple, and all of the necessary constituents are commercially available. The design of the G

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required and there are no adaptations to instrumentation of any kind. The data generated by mass spectrometry analysis of the various fractions is generated from a single MALDI spot per fraction and, for the present experiment, this constitutes only 1/50 of the total sample (1 μL spotted out 50 μL eluted from each fraction). Samples may be concentrated, further separated, modified, reanalyzed, and so on, to best meet the objectives of a given experiment. Further evidence that the membrane is functioning as designed is that the average protein size systematically decreases moving from one fraction to the next (left to right), as expected from SDS-PAGE protein separation (Figure 10B). The proteins identified in the current experiment correspond to well-known abundant yeast proteins,54,55 in particular, ribosomal proteins and glycolytic enzymes. This observation provides further evidence for the accuracy of the protein identifications because most yeast proteins are known to be less abundant. We have developed and demonstrated the utility of a novel membrane designed to enzymatically digest proteins and to retain the resulting peptides close to the point of their origin. The incorporation of this membrane into a proteomic workflow allows practical, reproducible, and efficient MS identification of proteins following SDS-PAGE separation. We believe that implementation of this technology is easy and will appeal broadly to experimentalists in protein science.

a powerful and effective protein identification technique capable of identifying multiple proteins from a single MS spectrum, given high mass accuracy. The workflow outlined here is one that is available to us and we highlight it when possible because it enables practical proteomic analysis without the need of either chromatography or MS/MS instrumentation. This workflow can, however, be used in conjunction with subsequent peptide chromatographic separation and MS/MS analysis to deliver further proteome characterization. Regardless, the primary purpose of the present study is to demonstrate the attributes of the digestion/capture membrane. We have demonstrated that the membrane as constructed enables consistent digestion across a 150 mm2 surface area− one that can accommodate common SDS-PAGE mini-gels (∼100 mm2). Incubation time for protein digestion can be varied depending on the experimental conditions and objectives of the experiment, but we have shown that for a protein standard the membrane can produce detectable peptide signals within 5 min. Typically, however, digestion is complete at about 4 h. Analysis of the membrane’s peptide binding capacity indicates that it can accommodate a load of ∼20 pmol of digested protein/25 mm2 area of membrane and for amounts less than ∼20 pmol we have shown that peptide capture is quantitative. It is important to note that in the peptide binding study, the quantities reported refer to the amount of digested protein (i.e., pmol refers to the amount of BSA-protein digested into peptides); therefore, the binding capacity of the membrane should be able to accommodate the amounts of total protein typically loaded onto gels (10−20 μg) because these proteins will be resolved and distributed across the separation space. If the binding capacity is exceeded, the more hydrophobic peptides will be retained whereas the comparatively more hydrophilic peptides will pass through the membrane in accordance with a reversed phase chromatographic binding mechanism. LC-MALDI analyses53 of digested BSA show that the m/z 927 peptide (BSA tryptic peptide 161−167 -YLYEIAR) elutes much earlier in the reversed phase gradient than the m/ z 1567 peptide (BSA tryptic peptide 347−359 -DAFLGSFLYEYSR-), indicating that the former peptide is more hydrophilic. Our results (Figure 7) are consistent with these findings. As demonstrated in the blow-ups of the spectra in the region m/z 927 and 1567, the m/z 927 peak breaks through, while the m/z 1567 peak is retained on the membrane. Analysis of a yeast lysate demonstrates the utility of our membrane approach when applied to the analysis of a complex protein mixture. The results presented in Figure 8 indicate that the membrane is able to quantitatively capture the full array of proteins generated during the SDS-PAGE separation. Figure 9 demonstrates the rich mixtures of peptides generated from the digest of the blotted proteins. The interfraction variability in the peptide mixtures indicates that they originate from different proteins. The peptides eluted from any given fraction originate from protein(s) at that particular location on the gel. By applying the cleavage rules for the enzyme used, and with accurate mass measurements of multiple peptides derived from each protein, it is possible to identify candidate parent protein(s) by peptide mass fingerprinting. Figure 10 and the accompanying auxiliary files demonstrate the feasibility of this workflow and highlight the potential for performing detailed analyses on protein mixtures with a relatively modest amount of experimental equipment. The separation and blotting experiments are performed in accordance with instrument manufacturer protocol and guidelines; no special settings are

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ASSOCIATED CONTENT

S Supporting Information *

Peak tables and protein lists associated with the presented spectra and data analyses. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We gratefully acknowledge Dr. Marvin Vestal and our colleagues at SimulTof Systems for their help in the preparation of this manuscript and their dedication to science. We also gratefully acknowledge Dr. Jane Gale and Dr. Mark Duncan for helpful and insightful review in document preparation.

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DOI: 10.1021/ac503980x Anal. Chem. XXXX, XXX, XXX−XXX