Anal. Chem. 2003, 75, 1691-1698
Fractionation of Cytosolic Proteins on an Immobilized Heparin Column Kevin Shefcheck, Xudong Yao, and Catherine Fenselau*
Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland 20742
Currently there is great interest in the development of methods to simplify complex protein mixtures for analysis by proteomic strategies. The objective of this study was to develop and evaluate immobilized heparin chromatography to simplify such mixtures and to enrich minor proteins. The method is evaluated with cytosol from human breast cancer MCF-7 cells. This protein mixture was fractionated into three portions and eluted with a stepwise salt gradient. These were characterized by protein analysis, two-dimensional gel electrophoresis, and mass spectrometry, with attention to reproducibility, overlap between fractions, simplification of protein mixtures, and enrichment of low-abundance proteins. It was possible to identify proteins enriched in the fractionated mixtures that were not even detectable in gel arrays of the total cytosol. The method was shown to be suitable for integration with other proteomics strategies. The application of proteomic strategies to studies of human disease states is challenged by recognition that mammalian cells may contain as many as 100 000 proteins1 with a dynamic range variously estimated between 5 and 12 orders of magnitude.1-4 None of the tools presently used in proteomics can handle either of these sample characteristics. As a result, scientists working with mammalian cells have advocated substantial prefractionation of their protein mixtures. Leaders in the field have espoused separation of subcellular organelles for protein analysis,5 and intense ongoing efforts address the development of advanced physicochemical methods for fractionation of proteins.6-8 Both affinity chromatography9 and ion exchange chromatography10 are being actively investigated. Heparin is a natural mixture of linear polymeric sulfated glycosaminoglycans, which binds some proteins strongly, by a combination of affinity binding and ion pairing11-13 In general, * Corresponding author. Phone: 301-405-8616. Fax: 301-405-8615. E-mail:
[email protected]. (1) Hochstrasser, D. F.; Sanchez, J. C.; Appel, R. D. Proteomics 2002, 2, 807812. (2) Aebersold, R.; Goodlett, D. R. Chem. Rev. 2001, 101, 269-296. (3) Anderson, N. L.; Anderson, N. G. Mol. Cell. Proteomics 2002, 1 845-67. (4) Panisko, E. A.; Conrads, T. P.; Goshe, M. B.; Veenstra, T. D. Hematology 2002, 30, 97-107. (5) Bell, A. W.; Ward, M. A.; Blackstock, W. P.; Freeman, H. N. M.; Choudhary, J. S.; Lewis, A. P.; Chotai, D.; Fazel, A.; Gushue, J. N.; Paiement, J.; Palcy, S.; Chevet, E.; Lafreniere-Roula, M.; Solari, R.; Thomas, D. Y.; Rowley, A.; Bergeron, J. J. M. J. Biol. Chem. 2001, 276, 5152-5165. (6) Badock, V.; Steinhusen, U.; Bommert, K.; Otto, A. Electrophoresis 2001, 22, 2856-2864. (7) Fountoulakis, M.; Langen, H.; Gray, C.; Takacs, B. J. Chromatogr., A 1998, 806, 79-291. 10.1021/ac026153h CCC: $25.00 Published on Web 03/01/2003
© 2003 American Chemical Society
topologic regions of basic amino acids in a protein bind to the acidic carbohydrate polymer. Immobilized heparin columns are now available commercially, advertised for use in the analysis of nucleic acid-binding proteins (which are strongly cationic)14-16 and blood coagulants, and there is growing interest17-23 in taking advantage of heparin’s unique combination of affinity binding and ion pairing to fractionate complex protein mixtures. The objective of the present study was to provide a quantitative evaluation of the use of heparin chromatography to separate protein mixtures from the cytosol of human MCF-7 breast cancer cells for subsequent analysis using 2-D gel electrophoresis and mass spectrometry. Criteria included chromatographic reproducibility, return of the detector to baseline between fractions, recovery in each fraction, overall protein recovery, and enrichment of low-abundance proteins. Molecular masses and pI values of proteins in the three fractions were characterized by 2-D gel electrophoresis, which also allowed quantitative assessment of the enrichment of minor components and of the possibility that a protein might show up in more than one fraction. Finally, some proteins in one fraction were identified using mass spectrometry. This confirmed the image overlap procedure and allowed arrays of fractionated material to be related to the laboratory’s master array24 for MCF-7 cytosolic proteins. (8) Fountoulakis, M.; Takacs, M. F.; Takacs, B. J. Chromatogr., A 1999, 833, 157-168. (9) Kelly, M.; McLellan, T.; Rosner, P. Anal. Chem. 2002, 74, 1-9. (10) Harrington, M. G.; Coffman, J. A.; Calzone, F. J.; Hood, L. E.; Britten, R. J.; Davidson, E. H. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 6252-6256. (11) Nader, H. B.; P., D. C. In Heparin: Chemical and Biological Properties, Clinical Applications; Lane, D. A., Lindahl, U., Eds.; CRC Press: Boca Raton, FL, 1989; pp 81-96. (12) Bjork, I.; Lindahl, U. Mol. Cell. Biochem. 1982, 48, 161-182. (13) Burnouf, T. In Plasma and recombinant blood products in medical therapy; Prowse, C. V., Ed.; Wiley: Chichester, U.K., 1992; pp 67-87. (14) Sternbach, H.; Engelhardt, R.; Lezius, A. G. Eur. J. Biochem. 1975, 60, 51-55. (15) Ussery, D. W.; Hinton, J. C.; Jordi, B. J.; Granum, P. E.; Seirafi, A.; Stephen, R. J.; Tupper, A. E.; Berridge, G.; Sidebotham, J. M.; Higgins, C. F. Biochimie 1994, 76, 968-980. (16) Fletcher, C.; Heintz, N.; Roeder, R. G. Cell 1987, 51, 773-781. (17) Fountoulakis, M.; Langen, H.; Evers, S.; Gray, C.; Takacs, B. Electrophoresis 1997, 18, 1193-1202. (18) Fountoulakis, M.; Takacs, B. Protein Expression Purif. 1998, 14, 113-119. (19) Karlsson, K.; Cairns, N.; Lubec, G.; Fountoulakis, M. Electrophoresis 1999, 20, 2970-2976. (20) Ueberle, B.; Frank, R.; Herrmann, R. Proteomics 2002, 2, 754-764. (21) Utt, M.; Nilsson, I.; Ljungh, A.; Wadstrom, T. J. Immunol. Methods 2002, 259, 1-10. (22) Kristoffersen, H. E.; Flengsrud, R. Electrophoresis 2000, 21, 3693-3700. (23) Wheeler, T. T.; Broadhurst, M. K.; Rajan, G. H.; Wilkins, R. J. J. Dairy Sci. 1997, 80, 2011-2019.
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EXPERIMENTAL SECTION Cell Culture and Cytosol Isolation. The MCF-7 cancer cell line was a gift from Prof. K. H. Cowan (Eppley Institute, Omaha, NE). The cell line was cultured with Improved Essential Medium (AATCC, Manassas, VA) containing 5% fetal calf serum (Sigma Chemical Co., St. Louis, MO) and 0.1% penicillin-streptomycin antibiotic solution (Sigma) at 37 °C under a 5% CO2 atm. The cells were grown to confluence and harvested as previously reported.24 The cytosol was extracted following the method of Ramsby and Makowski,25 which uses an ice-cold digitonin solution to lyse the cells. The cytosol protein mixture was frozen at -70 °C for storage. This cytosol fraction is considered to be relatively free of contaminants, based on the identification of ∼75 proteins in its gel array.24 Proteases are inhibited by the presence of phenylmethanesulfonyl fluoride.25 This procedure contains no reducing agents or detergents and was designed25 to provide native protein. Heparin Fractionation. For each fractionation, ∼3.0 mg of cytosol protein (the pooled recovery from 10 culture flasks) was diluted to a final volume of 24.0 mL with 10 mM phosphate buffer, pH 7.4. The sample was loaded onto a 1-mL HiTrap porcine heparin column (Amersham Pharmacia Biotech, Uppsala, Sweden) using an AKTA purifier FPLC system (Amersham Pharmacia). The column was then washed with a solution of 15 mM NaCl, 10 mM phosphate, pH 7.4, and the flow-through was collected until the detector (280 nm) returned to baseline (∼43 mL). A weak binding-protein fraction was eluted with a solution of 170 mM NaCl, 10 mM phosphate, pH 7.4, until the detector returned to the baseline (∼23 mL). Strong-binding proteins were eluted with 2 M NaCl, 10 mM phosphate, pH 7.4. The detector returned to baseline after ∼4 mL was eluted. This gradient was adapted from the procedure of Ta et al.,26 with special attention to separating aldolase exclusively into the stronger binding fraction. The fractions were stored at -20 °C Protein Quantitation. Protein quantitation was carried out using the BCA protein assay kit (Pierce, Rockford, IL). Concentration was calculated using Excel Software (Microsoft Corp., Redmond, WA). Desalting. The flow-through and weak-binding fractions were concentrated to ∼4.5 mL on a vacuum centrifuge. The strongbinding fraction was processed without concentration. Solutions were loaded 1 mL at a time onto a 5-mL HiTrap desalting column (Amersham Pharmacia) and eluted in 10 mM ammonium bicarbonate. Each sample was then dried and redissolved in gel rehydration solution. An aliquot adjusted to a concentration of 60 mg of protein in 310 µL was applied to the IPG strip for 2-D gel electrophoresis. 2-D Gel Electrophoresis and Imaging. Gel electrophoresis was carried out in two dimensions following a published procedure24 and stained with Sypro Ruby Red fluorescent gel stain (BioRad, Hercules, CA). The same amount of protein (60 µg) was used for each 2-D analysis. Each gel was digitally imaged using the Fluor-S Multi-imager (BioRad). Three replicates (24) Hathout, Y.; Riordan, K.; Gehrmann, M.; Fenselau, C. J. Proteome Res. 2002, 1, 435-442. (25) Ramsby, M. L.; Makowski, G. L. In Methods in Molecular Biology; Link, A., J., Ed.; Humana Press Inc.: Totowa, NJ, 1999; Vol. 112, pp 53-66. (26) Ta, T. V.; Takano, R.; Kamei, K.; Xu, X. Y.; Kariya, Y. K.; Hara, S. J. J. Biochem. 1999, 125, 554-559.
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Figure 1. Chromatograms showing three replicate separations of MCF-7 cytosol on the porcine heparin column.
were digitally averaged for each gel to produce a master image. Overlays and quantitative comparisons between two gel master images were made using PDQuest gel imaging software (BioRad). At each step, 26 anchor proteins were manually assigned. Normalization of the gels was done using the total spot density of each gel. Digestion of Protein Spots and Preparation for Mass Spectrometry. Spots were retrieved manually from the gel, treated with iodoacetamide, and incubated with trypsin as reported earlier.24 Peptide extracts were evaporated, redissolved in 0.1% TFA, and desalted using either ZipTips (Waters, Bedford, MA), C18 resin (Millipore), or Poros 50 R2 (PerSeptive Biosystems, Framingham, MA). Mass Spectrometry. Mass spectra of the peptides and tandem mass spectrometric analysis were obtained using a quadrupole time-of-flight instrument (Q-Star/Pulsar, Applied Biosystems, Foster City, CA). A mass map was initially recorded for each spot studied. Two microliters of the peptide solution was loaded into a capillary nanospray tip (Protana, Odense, Denmark).
Figure 2. Composite gel of proteins in the flow-through fraction 1 and in the total cytosol extract. Red circles mark the proteins that were observed in the cytosol extract but were excluded from the flow-through fraction. The blue circles represent the proteins that were only observed in the flow-through fraction and not detected in the cytosol. The green circles indicate proteins found in both.
A 900-V spray voltage was applied to the tip. Tandem mass spectrometry was performed using the same spray voltage. Collision energy was manually ramped from 10 to 50 eV, depending on the size and composition of the peptide in question. For the most part, doubly or triply charged ions were manually selected for tandem mass spectrometric analysis. These CID spectra were used to search the protein database to identify proteins. Bioexplore Tool software (Applied Biosystems) was used to search for human proteins the Swiss-Prot and NRDB databases. RESULTS AND DISCUSSION The chromatogram recorded during three heparin fractionations are shown in Figure 1. Subsequent fractionations had similar profiles. The stepwise elution with three salt concentrations was adapted from the method of Ta et al.,26 with the requirement that baseline detection be reestablished between each fraction in order to ensure no or minimal overlap of proteins between two fractions. It can be seen in Figure 1 that reproducibility was satisfactory. The three protein fractions were assayed as discussed in the Experimental Section. Table 1 summarizes the recoveries of protein in each fraction in the three experiments. The recoveries do not reflect the chromatograms in Figure 1, where phenylmethanesulfonyl fluoride and other contaminants affect the
Table 1. Protein Recovery (%) in Three Fractionation Experimentsa expt
fraction 1
fraction 2
fraction 3
total recovery
1 2 3
23 ( 0 21 ( 2 27 ( 5
29 ( 3 28 ( 0 18 ( 2
28 ( 3 23 ( 1 20 ( 2
81 ( 5 72 ( 1 65 ( 2
a
n ) 3 for all entries.
detector response, but were determined by the protein-specific method detailed in the Experimental Section. Total recoveries are also presented in the table, with an average of 73%. On average, 24% of the protein applied to the column was recovered in the flow-through fraction without binding. Another 25% was recovered in the weak-binding fraction, and 24% (average) in the final, strong-binding fraction. As Table 1 indicates, overall recovery of protein decreased through sequential experiments. No additional protein was recovered when the column was washed with 20% ethanol or with 5 M NaCl, 10 mM phosphate buffer solution. Desalting was carried out on a commercial size exclusion column after protein precipitation was found not to remove enough salt. Dialysis was also evaluated, but it caused considerable protein Analytical Chemistry, Vol. 75, No. 7, April 1, 2003
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Figure 3. Composite gel of proteins in the weak-binding fraction 2 and in the total cytosol extract. Red circles mark the proteins that were observed in the cytosol extract but were excluded from the weak-binding fraction. The blue circles represent the proteins that were only observed in the weak-binding fraction and not detected in the cytosol. The green circles indicate proteins found in both.
precipitation. Recovery of protein from the desalting column was routinely found to exceed 90%. Two-dimensional gel electrophoresis was used to examine each fraction to determine the simplification of the mixture and enrichment of low-abundance proteins. The possibility of proteins being present in two sequential fractions was also evaluated. Three gel patterns were digitized and averaged (see Experimental Section) to create master gels for the total cytosol protein mixture and for each fraction eluted from the heparin column. Composite gels were constructed digitally by overlaying the array of proteins visible in the total cytosol master gel and the arrays of proteins visible in master gels from each fraction, using commercial software. Approximately the same amount of protein was used in each 2-D experiment, from cytosol or from one of the heparin column fractions. The master gel image of the flow-through fraction (fraction 1) is overlaid on that of the total cytosol master gel (see Experimental Section) in Figure 2. In the cytosol master gel 390 spots were visible, while the flow-through fraction displayed 234 spots. Red circles indicate the proteins that are present in the cytosol and not detected in this first fraction. The 99 spots circled in green are observed in both gels. Eighty-six of these are enriched in fraction 1, relative to their abundance in the cytosol mixture, with 33 enriched ∼4-fold. Ten spots are observed with 1694
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lower intensity. Five spots appear with the same coordinates as spots that are also detected in the second, weak-binding fraction. These could indicate a 2% overlap between the two fractions; however, the proteins in the spots may be different and the overlap minimal. Most striking, 133 protein spots (57%) are visible in the master gel of fraction 1 (marked in blue in Figure 2), which are not detectable at all in the master gel of the more complex cytosol mixture. These unique spots are mostly acidic and have low molecular masses. They range in abundance over 2 orders of magnitude and appear to have been significantly enriched by fractionation on the heparin column. It should be noted that fraction 1 contains more low-abundance and more acidic proteins than the two other fractions. When both the common spots and the unique spots are considered, 93% of the spots detected in its master gel are enriched compared to their abundances in the total cytosolic mixture. The overlay of master images of the weak-binding fraction (fraction 2) and the total cytosol extract is shown in Figure 3. The master gel of this second fraction contained 181 spots. The red circles mark the proteins that are present in the total cytosol, but excluded in this fraction. Fraction 2 has 80 spots in common with the cytosol extract, indicated in green. Seventy of these spots were found to occur with a relatively greater
Figure 4. Composite gel of proteins in the strong-binding fraction and in the total cytosol extract. Red circles mark the proteins that were observed in the cytosol extract but were excluded from the strong-binding fraction. The blue circles represent the proteins that were only observed in the strong-binding fraction and not detected n the cytosol. The green circles indicate proteins found in both.
concentration in fraction 2, and 50 of these are enriched by at least 25% due to the fractionation. Abundances of 17 of the enriched proteins are increased greater than 4-fold. Seven spots (4%) were found with the same coordinates from the weak-binding fraction and the third, strong-binding fraction. These spots may be overlapping proteins or nonidentical proteins with coincident coordinates. The master gel of fraction 2 contains 101 protein spots, 56%, that are not visualized at all in the array of the total cytosol extract. These are shown in blue in the overlay in Figure 3. Some of these protein spots are newly visible due to the removal of higher intensity spots in the cytosol extract. Among the spots visualized from fraction 2, 94% have been enriched relative to the total cytosol mixture. Thus, simplification of the mixture, removal of some abundant proteins, and enrichment of many others has been achieved. Sixty micrograms of protein from the desalted strongbinding fraction 3 was also characterized by 2-D gel electrophoreses, and the gel was compared to that of 60 µg of protein from the cytosol. The composite of these two gels is shown in Figure 4. The gel of the third (strong-binding) fraction displays 157 spots. Again, many cytosol proteins, indicated in red, have been excluded from this heparin fraction. Eighty-one of the 89
proteins detected in both mixtures (indicated in green) have been enriched by at least 25% in the heparin fraction and 41 of these by at least 4-fold. Six spots (4%) that occur in both master gels are not found to be enriched and occur as part of streaks in the gels. The strong-binding fraction contributes 68 new spots, 43%, that are not visualized in the cytosol extract. These are indicated in blue in Figure 5 and tend to be clustered at neutral pI and in the higher mass range of the master gel. Counting those present in both arrays and these unique spots, 95% of the spots detected in the strong-binding array are enriched relative to the cytosol array. About 25% of the total cytosolic protein was not recovered during the fractionation procedure (Table 1). Elutions with 20% ethanol and with 5 M NaCl, 10 mM phosphate buffer did not recover any more protein. Some proteins may have been covalently bound on the column, while others may have precipitated out of the salt solutions. An analysis of the digitized gels revealed characteristics of the cytosolic proteins lost during fractionation. Figure 6 shows 148 spots in the cytosol that were not qualitatively accounted for in any of the three fractions. Although no firm trends are discerned, many of these proteins are neutral or basic and of mid to high mass. Analytical Chemistry, Vol. 75, No. 7, April 1, 2003
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Figure 5. Two-dimensional gel array of strong-binding proteins, with spots circled in blue that are not seen in the cytosol array.
In a point-by-point comparison, matched sets of 18 spots were excised from the gels holding the strong-binding fraction and the total cytosol protein. These were digested with trypsin and analyzed by peptide mapping27 and in tandem mass spectrometry28,29 experiments. Database searching allowed the proteins to be identified, usually based on analysis of microsequences from two to five peptides. The proteins are listed in Table 1. This exercise confirmed that heparin fractionation can be successfully incorporated into this laboratory’s proteomics strategies. Twelve proteins were identified in common, from both the fraction and cytosol arrays (Table 2), and these were then used among the digital “anchor spots” to overlay the two arrays. These may also serve as anchor spots to relate the heparin fractions to a master gel being constructed in this laboratory24 for MCF-7 cytosol proteins and to studies in other laboratories. A second set of proteins was identified only in spots cut from the strongbinding fraction gel (Table 1). In some cases, faint spots at corresponding positions were visible in the cytosol gel, but insufficient protein was recovered for identification. The sixth protein presents an interesting example, because it could not be (27) Henzel, W. J.; Billeci, T. M.; Stults, J. T.; Wong, S. C.; Grimley, C.; Watanabe, C. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 5011-5015. (28) Mann, M.; Wilm, M. Anal. Chem. 1994, 66, 4390-4399. (29) Yates, J. R., 3rd; Speicher, S.; Griffin, P. R.; Hunkapiller, T. Anal. Biochem. 1993, 214, 397-408.
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Table 2. Proteins Identified from Strong-Binding Fraction 3 pI
mass (Da)
4.75 4.94 5.29 4.94 4.94 5.37 5.37 6.42 8.39 8.30 8.30 8.39 5.33 6.61 6.46 6.44
49 758 50 158 41 737 84 542 84 542 70 898 70 898 95 207 39 289 44 596 44 596 39 289 46 489 36 683 39 324 55 805
7.01 7.57
24 683 57 746
protein tubulin β-1 chain tubulin R-1 chain actin cytoplasmic-1 (β-actin) heat shock protein 90 R heat shock protein 90 R heat shock cognate 71-kDa protein heat shock cognate 71-kDa protein elongation factor-2 fructose-biphosphate aldolase A phosphoglycerate kinase-1 phosphoglycerate kinase-1 fructose-biphosphate aldolase A translation initiation factor 4A2 fructose-1,6-bisphosphatase fructose biphosphate aldolase C inosine-5′-monophosphate dehydrogenase 2 GTP binding protein RAN pyruvate kinase
detected in cytosol yes yes yes yes yes yes yes yes yes yes yes yes no no no no no no
identified in the cytosol. However, it is purified and enriched in fraction 3 sufficiently for excision and identification. Enlarged corresponding sections from the two gels are shown in Figure 7. No protein could be identified by analysis of the mixture
Figure 6. Two-dimensional gel array of proteins in the total cytosol extract, with proteins circled in purple that were not detected in the three fractions collected.
Figure 7. Enlargements of the same section of the two-dimensional gel arrays of proteins (left) in the total cytosol extract and (right) in the strong-binding fraction. On the lower right is the tandem mass spectrum from a tryptic peptide recovered after the spot was digested in situ.
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shown on the left. The spot shown on the right, from fraction 3, was identified as inosine-5′-monophosphate dehydrogenase 2, based on the tandem mass spectrum of a tryptic peptide (NLIDAGVDALR) recovered after the spot was digested in situ. This spectrum was used in a successful database search. Inosine5-monophosphate dehydrogenase 2 is reported to play a role in the growth of tumors.30 Smaller proteins are generally eluted in the flow-through fraction, while the third fraction, eluted with high salt concentration, contains mostly proteins in the mid to high molecular weight range. Although it might be expected that elution with a salt gradient would separate proteins according to their pI values, no strong correlation is seen with pI in the progression from one fraction to another in Figures 2-4. Proteins do not need a basic net pI to bind to heparin, but rather, topologic regions of basic residues will suffice.7-9 Affinity binding is also important in fractionation on heparin columns. CONCLUSIONS Reproducible fractionation of complex protein mixtures, such as the cytosol fraction from human breast cancer cells (30) Williams, K.; Chubb, C.; Huberman, E.; Giometti, C. S. Electrophoresis 1998, 19, 333-343.
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studied here, can be obtained on immobilized heparin columns with baseline separation of fractions. Recovery (75% here) is especially good compared to size exclusion chromatography, which in our hands provides recoveries between 20 and 50%. Overlap between the fractions is minimal or zero, again in contrast to separation achieved by size exclusion methods. Striking enrichment is achieved for many proteins. Three hundred proteins were visible in 2-D gel patterns of the three fractions, which could not be detected in the more complex cytosol mixture. This prefractionation technique has strong potential for incorporation into both qualitative and quantitative proteomics strategies. ACKNOWLEDGMENT We thank Dr. Yetrib Hathout for expert advice. The work was supported by grants from the NIH (GM21248 to C.F.) and the NSF (to Dr. Murray Johnston, University Delaware, for multisite training).
Received for review September 20, 2002. Accepted January 22, 2003. AC026153H
