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Keywords:

  • Gel electrophoresis;
  • serum proteomics;
  • sample preparation;
  • protein fractionation;
  • TCA precipitation

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND SUPPLIES
  4. EXPERIMENTAL PROCEDURE
  5. SAFETY ISSUES
  6. RESULTS
  7. DISCUSSION
  8. CONCLUSIONS
  9. REFERENCES
  10. Supporting Information

Two-dimensional gel electrophoresis (2DGE) remains an important tool in the study of biological systems by proteomics. While the use of 2DGE is commonplace in research publications, there are few instructional laboratories that address the use of 2DGE for analyzing complex protein samples. One reason for this lack is the fact that the preparation of samples for 2DGE is a complex and difficult process that can commonly yield gels of poor quality and resolution. In this experiment, we use a serum-based sample to mitigate many of the sample preparation issues that occur in cell-based sample preparations and incorporate a protein precipitation method that was developed to address the problem of high-abundance proteins and dynamic range in serum proteomics research. By focusing on 2DGE apart from many other facets of proteomic experimental design, students have the opportunity to gain fruitful experience in the use of this workhorse proteomics technique. This simplified focus also makes this exercise accessible to biochemistry instructors who are not active in proteomics; the requisite techniques may require some new equipment (i.e. an isoelectric focusing apparatus), but this exercise focuses on using familiar techniques (primarily electrophoresis) to cross the threshold of a new field, proteomics.

The application of proteomics has expanded in recent years to areas ranging from human clinical diagnostics to microbial metabolism. Although the frontiers of proteomics research are more typically associated with advances in mass spectrometry in terms of resolution and throughput speed, there is still an important role to be played by two-dimensional gel electrophoresis (2DGE) [1]. In terms of cost and access, 2DGE still represents the most broadly accessible technology for proteomics and it remains an essential tool for proteomic profiling. Despite the seemingly ubiquitous nature of 2DGE within proteomics research, the technique is seriously under-represented in the undergraduate laboratory curriculum. A recent literature search reveals only a single citation that provides students with a first-hand introduction to 2DGE [2]. With the development of immobilized pH gradient (IPG) technology [3], 2DGE has also become a far more reliable and precise tool for protein analysis. This being said, however, the technique is still subject to numerous variables and pitfalls that can add to the complexity of implementing its use in either undergraduate teaching or research laboratories.

When applied to cell-based systems, 2DGE experiments require significant sample preparation work to ensure gels of desirable quality. Issues of sample preparation can include cell homogenization, protein solubility, removal of contaminants (e.g. RNA, DNA, and glycans), high ionic strength, and proteolytic degradation. The experimental ramifications of these issues can be clearly seen in the recent proteomic analysis of the pathogenic fungus, Penicillium marneffei [4]. The culture of P. marneffei cells requires growth on solid potato dextrose agar, isolation of conidia (spores) by centrifugal filtration, inoculation and growth in liquid media, collection of cells, homogenization by mechanical shearing, and a final isolation of proteins using trichloroacetic acid (TCA) precipitation over a cumulative timeframe of no less than 9 days. The amount of time and the number of steps exemplified by this particular cell preparation protocol illustrates how poor gel results are likely to be the product of sample preparation issues rather than any intrinsic problem within the actual 2DGE experiment.

To provide a simplified introduction to 2DGE, we have developed a laboratory based upon the use of serum instead of cellular preparations. Serum proteomics is currently being investigated as a means for early clinical detection of cancer and other diseases [5]. Unlike cellular preparations, serum exists as fully soluble protein sample which eliminates the need to optimize any cell lysis or protein solubilization procedures as well as reducing the number of preparation steps leading up to 2DGE analysis. Also, the naturally high protein concentration in serum ensures that serum samples can be diluted or reconstituted in any medium appropriate for 2D gels while still providing ample quantities of protein for detection in the resulting gels. One of the current impediments to the use of serum proteomics as a diagnostic tool is the fact that the serum proteome is dominated by albumin. In some cases, albumin can constitute as much as 40% of the total protein mass within serum, which has the effect of obscuring many lower abundance proteins in 2DGE [6]. Fractionation of serum samples constitutes a major focus within serum proteomics as researchers work towards using the detection of low abundance clinical markers for cancer diagnosis.

In the experiment described herein, students will employ a recently published protocol for the removal of albumin from serum using a TCA precipitation step [7]. Comparison of two-dimensional gels of fractionated samples against an untreated serum sample will demonstrate the effect of sample preparation and will illustrate how dynamic range can play a significant role in our ability to use 2DGE to analyze complex samples. By using serum as the basis for this 2DGE experiment, we have eliminated the time and effort associated with cell culture and homogenization while also simplifying the laboratory to limit the contributions of sample preparation to experimental error in 2DGE.

MATERIALS AND SUPPLIES

  1. Top of page
  2. Abstract
  3. MATERIALS AND SUPPLIES
  4. EXPERIMENTAL PROCEDURE
  5. SAFETY ISSUES
  6. RESULTS
  7. DISCUSSION
  8. CONCLUSIONS
  9. REFERENCES
  10. Supporting Information

All reagents for TCA fractionation were purchased from Sigma-Aldrich. Porcine serum (Sigma-Aldrich, St. Louis, MO) was aliquoted and stored at −20°C. All reagents for electrophoresis (1D and 2D) were purchased from BioRad Laboratories. Rehydration buffer (8 M urea, 4% w/v CHAPS, 15 mM dithiothreitol, 0.15% Bio-Lyte ampholytes (BioRad, Hercules, CA), 0.0001% bromophenol blue) was prepared using ultrapure (18 M Ω-cm) water and stored at 5°C for up to 1 month. Commercial preparations of rehydration buffer have a lower CHAPS concentration that can diminish the resolubilization of proteins from precipitate. Stored rehydration buffer was warmed to room temperature before use.

A comprehensive reagent/equipment list and laboratory protocol handout are available as Supporting Information in the online version of this manuscript.

EXPERIMENTAL PROCEDURE

  1. Top of page
  2. Abstract
  3. MATERIALS AND SUPPLIES
  4. EXPERIMENTAL PROCEDURE
  5. SAFETY ISSUES
  6. RESULTS
  7. DISCUSSION
  8. CONCLUSIONS
  9. REFERENCES
  10. Supporting Information

TCA Fractionation of Albumin from Serum

A 100 μL sample of porcine serum was loaded into a 1.5 mL microcentrifuge tube and precipitated by adding 400 μL of ice–cold 10% TCA/acetone. Mixing of the sample was achieved by briefly vortexing or inverting the microcentrifuge tube. The sample was then incubated at −20°C for 45 minutes. While the precipitated sample was incubated, a separate unprecipitated serum sample was diluted 20-fold in 50 mM phosphate buffer (pH 7.0) and stored on ice. After the incubation, the precipitated sample was centrifuged at 16,000 × g, 4°C, for 20 minutes. The supernatant was transferred to a clean microcentrifuge tube and 1 mL of ice–cold acetone was added to both the precipitate and supernatant fractions. Both fractions were mixed by vortexing or inversion and then incubated on ice for 15 minutes. After incubation, the samples were centrifuged as described above. The supernatant was removed from both the fractions and the tubes were incubated at room temperature for 10 minutes to evaporate any residual acetone. Protein pellets were resolubilized by the addition of rehydration buffer (400 μL for pellet fraction, 100 μL for supernatant fraction) followed by intermittent vortexing until the pellet had fully dissolved. Solubilization could also be achieved by sonication of protein mixture after addition of rehydration buffer.

Isoelectric Focusing of Serum Fractions

Resolubilized protein fractions from the TCA precipitation step should each contain a protein concentration of approximately 5 mg/mL. A 20 μL aliquot of each fraction was added to clean, labeled microcentrifuge tube. An additional 105 μL of rehydration buffer was added to each fraction, resulting in a total protein mass of approximately 100 μg in a total volume of 125 μL. Each diluted fraction was then transferred to a separate well within an isoelectric focusing (IEF) focusing tray. With its protective backing removed, an IPG strip was laid, gel side down over the protein solution, taking care to minimize any bubbles under the IPG strip. Once all IPG strips were placed over their respective samples, each strip was overlaid with enough mineral oil to completely cover the entire sample well. The strips were then loaded into a Protean IEF machine and run using a standard preset program with active rehydration (linear ramp, 20,000 V h focusing step; details can be found in the Supporting Information). Once the IEF was complete, samples were transferred to equilibration trays and stored at −80°C.

1D SDS–PAGE of Serum Fractions

As a quick validation of fractionation and concentration, Laemmli gel separation was performed on the serum fractions. To prepare samples for gel-loading, 8 μL of each protein fraction was added to 22 μL of 1×-loading buffer within a separate 1.5 mL microcentrifuge tube. Sample tubes were then vortexed briefly, incubated at 100 °C for 2 minutes and centrifuged to collect sample at the bottom of the sample tube. Samples and molecular weight marker solution were then loaded onto a precast Laemmli gel as shown in Fig. 1. Gels were run under constant voltage (200 V) for ∼45 minutes until the dye front reached the bottom of the gel. Once complete, each gel was washed three times in distilled H2O and stained with BioRad BioSafe gel stain. Images of the resulting gels could then be obtained using a flatbed scanner or gel imaging system.

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Figure 1. One-dimensional gel of serum fractions produced from TCA precipitation. A 10% SDS–PAGE gel was loaded with 7 μg of diluted crude serum (Lane 2), TCA pellet fraction (Lane 3), and TCA supernatant fraction (Lane 4) along with molecular weight markers (Lane 1). All molecular weights are provided in kDa.

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SDS–PAGE of IPG Strips

IPG strips from the IEF experiment were removed from the −80°C freezer and allowed to thaw to room temperature. Strips were then treated with 2.5 mL of equilibration buffer I for 10 minutes with gentle shaking. Equilibration buffer I was then removed from the strips and replaced with 2.5 mL of equilibration buffer II. Strips were incubated in equilibration buffer II for another 10 minutes with gentle shaking. Strips were then dipped in SDS–PAGE running buffer and loaded onto a precast, 10% polyacrylamide slab gel (1-mm thickness). The strip was overlaid with prewarmed overlay agarose, which was then allowed to cool until hardened. Once the overlay agarose was hardened, the gel was run at a constant voltage (200 V) for ∼45 minutes until the dye front reached the bottom of the gel. The resulting gels were then stained and imaged as described above.

SAFETY ISSUES

  1. Top of page
  2. Abstract
  3. MATERIALS AND SUPPLIES
  4. EXPERIMENTAL PROCEDURE
  5. SAFETY ISSUES
  6. RESULTS
  7. DISCUSSION
  8. CONCLUSIONS
  9. REFERENCES
  10. Supporting Information

Trichloroacetic acid is a highly corrosive solid that poses a significant irritant threat from exposure to skin or from inhalation of powder. Gloves should be worn to prevent contact when handling TCA. Solid TCA is very hygroscopic which makes the handling and weighing of solid TCA problematic. Rather than working with solid TCA, a stock 100% (mass/volume) solution can be prepared by adding 227 mL of distilled H2O to 500 g of TCA. The resulting solution can easily be diluted to the desired final concentration.

Although precast gels are suggested for this laboratory, some individuals may prefer to cast their own polyacrylamide gels as a supplement to the described experiment. Unpolymerized acrylamide is a known neurotoxin and should be handled with gloves. A dust mask is highly recommended when acrylamide is handled in its solid, powder form. Due to the high voltages/currents used, care should also be taken during the running of electrophoresis experiments to prevent accidental shocks or short circuits.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND SUPPLIES
  4. EXPERIMENTAL PROCEDURE
  5. SAFETY ISSUES
  6. RESULTS
  7. DISCUSSION
  8. CONCLUSIONS
  9. REFERENCES
  10. Supporting Information

One-dimensional SDS–PAGE provides clear evidence of the removal of albumin from the total serum fraction (Fig. 1). The TCA supernatant fraction is composed almost exclusively of albumin (Fig. 1, Lane 4). The absence of albumin from the TCA pellet fraction (Fig. 1, Lane 3) does not, however, appear to have resulted in any significant differences between the TCA pellet fraction and the original whole serum (Fig. 1, Lane 2). Thus, it appears that within the limits of the 1D electrophoresis experiment, the TCA precipitation has successfully removed albumin from serum without any additional perturbations to the serum protein composition.

The effect of sample fractionation can be seen clearly in the 2D gels shown in Fig. 2. Although each of the three gels shown has been loaded with comparable amounts of total protein, there is a clear difference in number of spots that can be resolved in the fractionated samples versus the original serum. Visual determination of resolved protein spots for the TCA pellet fraction (Fig. 2a) yields a spot count that is more than double the number of spots found in the original serum sample (Fig. 2b). Additional computer analysis of the gels using PDQuest (BioRad) yields a total protein spot count of 94 spots in the TCA pellet fraction versus 15 spots detected in the total serum sample. The removal of albumin from the serum (Fig. 2b) both uncovers spots that lay beneath the original albumin loci (Fig. 2c) as well as causing an increased relative representation of proteins that lie well outside of the albumin loci. A 2D gel of the TCA supernatant fraction shows the full scope and breadth of the impact that albumin exerts on the overall quality of the serum 2D gels. Based upon a cursory measurement of the dimensions in the gel in Fig. 2c, the various isoforms of albumin span a pI range of nearly 1.5 pH units and a range of molar masses of over 20 kDa to influence nearly 20% of the overall resolution area of the gels described.

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Figure 2. Two-dimensional gels of serum fractions produced from TCA precipitation. IEF separation of proteins was achieved using 7 cm IPG strips, pH 4–7 and the second dimension was run on a 10% polyacrylamide mini gel. (a) Proteome of the pellet (albumin-depleted) fraction of the TCA precipitation. (b) Proteome of unfractionated serum. (c) Proteome of supernatant (albumin) fraction of the TCA precipitation.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND SUPPLIES
  4. EXPERIMENTAL PROCEDURE
  5. SAFETY ISSUES
  6. RESULTS
  7. DISCUSSION
  8. CONCLUSIONS
  9. REFERENCES
  10. Supporting Information

In contrast to the full scope of proteomics technology that is represented in earlier study [2], this experiment focuses exclusively on presenting 2DGE as a stand-alone experiment. This simplification of the proteomic paradigm allows for a broader level of participation due to reduced cost and time. In this context, the power of 2DGE as an introduction to proteomics lies in its ability to provide an intuitive, visual result that can be examined further by computer analysis or simple visual markup. 2DGE results can be generated with a relatively modest investment of equipment beyond what can be found in a typical undergraduate biochemistry teaching laboratory.

When used within the broader scope of proteomics research, 2DGE typically plays a complimentary role to mass spectrometry in the identification of proteins associated with specific biological phenomena. The collection of proteomic data also requires a great deal of sample preparation as well as some type of digital gel image analysis. With recent technical advances, such as immobilized pH gradient strips and precast polyacrylamide gels, 2DGE is often represented as a simple, surefire methodology with the ability to yield flawless gels on a regular basis. While 2DGE may indeed be a straightforward technology, 2D gels will only be as good as the sample preparation that precedes them and establishing the appropriate sample preparation requires a significant investment of time and energy. The vast majority of technical problems that are associated with poor gel quality have to do with sample conditions prior to gel separation [1]. Accordingly, 2DGE is poorly represented in the science education literature despite its importance in proteomics research and its purported simplicity. Thus, if students are to learn the use of 2DGE in manageable and practical manner, the troubleshooting aspects of sample preparation need to be simplified to manageable levels.

The immediate benefits of using blood serum or plasma as the system of choice are threefold. Firstly, proteomic analysis of cellular or tissue samples requires additional time and effort for optimal cell/tissue culture, collection, and homogenization/lysis. Each of these additional steps must be optimized to fully maximize the amount of protein extracted from the cellular sample. Variations in yield at any of these steps can greatly affect the overall quality and completeness of any resulting 2D gels. In contrast, serum requires no isolation steps and can be directly subjected to 2DGE. Secondly, because 2DGE is largely limited to the separation of solubilized proteins, incomplete solubilization of cellular lysates can cause a host of problems including streaking and poor resolution. Analysis of serum proteins circumvents this issue due to the fact that serum is itself fully constituted by soluble proteins. The concentration of protein in serum is also high enough to allow for its dilution into appropriate rehydration media while still allowing for the detection of protein spots in the resulting gels using inexpensive but less sensitive dyes such as Coomassie brilliant blue.

Lastly, the fractionation of serum proteins presents students with an authentic and contemporary issue in proteomics research. To serve as a diagnostic tool, serum proteomics research needs to address the serious impediment presented by the high abundance of serum albumin. The removal of albumin from serum enhances the relative abundance of minor constituents in serum, both by the removal of potential overlapping caused by albumin and by enriching the relative quantities of all other proteins in serum when albumin is subtracted from the sample. When the described TCA precipitation is used as a simple and straightforward sample preparation step, the resulting 2D gels demonstrate a clear, well-defined enhancement. The results are a clear illustration of the how sample preparation can affect the resolution and quality of 2D gels.

This laboratory was originally designed and tested as the sole wet laboratory component for a course in Proteomics and Molecular Modeling. Students in this course were part of a biotechnology/bioinformatics program and were not required to have any prior experience with biochemistry lecture or laboratory before participating. These students worked in pairs and the time frame for the completion of this experiment spanned four 2-h laboratory periods. A complete schedule for the various laboratory periods is presented in Table I. The first laboratory period is dedicated to the TCA precipitation procedure, and the second period is focused on the preparing and initiation of the IEF separation of the resulting fractions. Period three focuses upon the 1D SDS–PAGE experiment, whereas the fourth period is devoted to the second dimension of the 2DGE experiment. For students with prior exposure to basic biochemistry techniques, these periods could potentially be condensed to three or possibly two periods. The loading of the IEF could potentially be performed immediately after the protein fractionation and both the Laemmli and second dimension gels could be performed concurrently, if sufficient equipment were available. In the absence of computer-based gel analysis software, 2D gels can be compared visually by printing images of the corresponding gels onto transparencies and overlaying the images to manually differentiate between the fractions and the crude serum.

Table I. Timetable of laboratory activities by class period
Laboratory periodTimeProcedureNotes
  1. TCA, trichloroacetic acid; 1DGE, one-dimensional gel electrophoresis.

10:00Add 10% TCA/acetone to serumIncubate at −20 °C for 45 min
 0:10Prepare diluted unprecipitated serum and store for later use 
 0:45Centrifuge precipitated sample16,000 × g, 4 °C, 20 min
 1:05Transfer the supernatant to a clean tube 
 1:10Add ice–cold acetone to the supernatant and to the pellet. Mix by inversionAfter mixing, let stand on ice for 15 min
 1:30Centrifuge both the samples16,000 × g, 4 °C, 20 min
 1:50Decant the supernatantAir dry to evaporate acetone
 2:00Add rehydration bufferStore at 4 °C until next laboratory period
20:00Mix 20 μL of each fraction with 105 μL of rehydration bufferApproximately 100 μg in a total volume of 125 μL
 0:10Transfer samples to the isoelectric focusing tray 
 0:30Lay a pH 4–7 IPG strip gel side down on the sample in the tray 
 0:45Overlay each strip with mineral oil to cover the sample well 
 1:00Isoelectric focusingRequires ∼18 h; students or instructor return at the conclusion to remove the strips and place them in a −80 °C freezer until period 4
30:00Prepare samples for 1DGEMix 8 μL of each protein fraction with 22 μL of 1×-loading buffer and boil for 5 min
 0:15Apply samples to the 1D gelStandards on either end
 0:30Initiate electrophoresisConstant voltage (200 V)
 1:15Terminate electrophoresis 
 1:20Wash each gel 3× with distilled water 
 1:30Stain with BioRad BioSafe stainStudents or faculty can remove stain and destain with distilled water after 45–60 min. Gels are then imaged with a flatbed scanner or gel imaging system
40:00Thaw IPG strips 
 0:10Treat IPG strips with equilibration buffer I10 min
 0:20Treat IPG Strips with Equilibration buffer II10 min
 0:30Dip IPG strips in SDS–PAGE running buffer and apply to a gelOverlay the IPG strip with agarose until it hardens (about 10 min)
 0:45SDS–PAGE45 min at 200 V
 1:20Wash each gel 3× with distilled water 
 1:30Stain with BioRad BioSafe stainStudents or faculty can remove stain and destain with distilled water after 45–60 min. Gels are then imaged with a flatbed scanner or gel imaging system

Students were not required to submit a formal laboratory report but had the opportunity to critique the results of the experiment and to propose possible improvements or extensions for the use of the technique. The identification of protein spots from 2D gels could be qualitatively obtained by comparing experimental gels to those published in the TCA precipitation article. In making these comparisons, students were also asked to explain any discrepancies that occurred between their gel results and the published versions. Any deviations from the published gels provided an opportunity to more fully examine the relationship between the individual steps of the overall experiment and the quality of the gel produced. By measuring their results against published data, students also gained a sense of the level of rigor required to produce results of a quality that are consistent with acceptable standards of publication.

Additional Experiments

While the focus of this particular experiment is to provide a clear and direct exposure to 2DGE, there are numerous possibilities for further extensions of serum-based 2DGE. In addition to albumin, serum proteomics is also burdened by the high abundance of immunoglobulins in serum. Further fractionation by protein A/G affinity chromatography has been suggested and could serve as a means to coupling protein chromatography to 2DGE [7]. The clinical relevance of this experiment can be further investigated by using 2DGE as a comparative tool between “healthy” and “diseased” samples. Lipoprotein-deficient serum is commercially available and could be used as a model disease sample related to lipoprotein metabolism (high-density lipoprotein biosynthesis) and cardiovascular health [8]. The addition of detailed protein quantitation assays [9] and an examination of various protein staining methods could further expand the boundaries of this experiment to mirror commonly accepted practices in the proteomics research area. Also, the incorporation of mass spectrometric analysis of protein spots would complete the proteomic experience for students [10].

CONCLUSIONS

  1. Top of page
  2. Abstract
  3. MATERIALS AND SUPPLIES
  4. EXPERIMENTAL PROCEDURE
  5. SAFETY ISSUES
  6. RESULTS
  7. DISCUSSION
  8. CONCLUSIONS
  9. REFERENCES
  10. Supporting Information

We have described an entry-level, stand-alone laboratory to instruct students in the use of 2DGE and to clearly illustrate the effect that proper sample preparation can have on the quality of 2D gels. By using serum as the sample of interest, the laboratory has the benefit of limiting the amount of sample preparation to procedures that can be completed in a single laboratory period. Beyond the technical aspects of performing 2DGE, the fractionation of serum for 2DGE analysis has the added significance of mirroring a current area of “cutting-edge” research in the area of biomedical diagnostics. Anecdotal evidence indicates that a significant number of instructional departments have invested in 2DGE equipment in the past few years. This exercise is also designed to enable faculty members, who may not work with proteomics in their research groups, to introduce the practice of proteomics to an introductory biochemistry laboratory course. It is our hope that the availability of this stand-alone 2DGE experiment will expand the adoption of this technology into the biochemistry curriculum and better prepare students to engage in proteomics and its offshoot disciplines in the future.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND SUPPLIES
  4. EXPERIMENTAL PROCEDURE
  5. SAFETY ISSUES
  6. RESULTS
  7. DISCUSSION
  8. CONCLUSIONS
  9. REFERENCES
  10. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. MATERIALS AND SUPPLIES
  4. EXPERIMENTAL PROCEDURE
  5. SAFETY ISSUES
  6. RESULTS
  7. DISCUSSION
  8. CONCLUSIONS
  9. REFERENCES
  10. Supporting Information

Additional Supporting Information may be found in the online version of this article

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BMBD_20353_sm_suppinfo.doc88KSupporting Information

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.