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Size-exclusion chromatography in multidimensional separation schemes for proteome analysis
J. Biochem. Biophys. Methods 56 (2003) 141 – 152 www.elsevier.com/locate/jbbm
Size-exclusion chromatography in multidimensional separation schemes for proteome analysis Paolo Lecchi *, Anand R. Gupte, Ricardo E. Perez, Lyubov V. Stockert, Fred P. Abramson Department of Pharmacology, The George Washington University School of Medicine and Health Sciences, 2300 I St. N.W., Washington, DC 20037, USA
Abstract Size-exclusion chromatography (SEC) is a separation technique with a relatively low resolving power, compared to those usually utilized in proteomics. Therefore, it is often overlooked in experimental protocols, when the main goal is resolving complex biological mixtures. In this report, we introduce innovative multidimensional schemes for proteomics analysis, in which SEC plays a practical role. Liquid isoelectric focusing (IEF) was combined with SEC, and experimental results were compared to those obtained by two-dimensional polyacrylamide gel electrophoresis (2DPAGE), well-established techniques relying upon similar criteria for separation. Additional experiments were performed to evaluate the practical contribution of SEC in multidimensional chromatographic separations. Specifically, we evaluated the combination of SEC and ion exchange chromatography in an analytical scheme for the mass spectrometric analysis of protein-extracts obtained from bacterial cultures grown in stable isotope enriched media. Experimental conditions and practical considerations are discussed. D 2003 Elsevier Science B.V. All rights reserved. Keywords: Size-exclusion chromatography; Rotofor; Proteomics; Mass spectrometry; CRIMS; Stable isotopes
1. Introduction Combining different separation strategies such as: size-exclusion chromatography (SEC), ion exchange chromatography (IE), reversed phase (RP), or other chromatographic Abbreviations: ACN, acetonitrile; CRIMS, chemical reaction interface mass spectrometry; HFBA, heptafluorobutyric acid; IE, ion-exchange chromatography; IEF, isoelectric focusing; MW, molecular weight; PAGE, polyacrylamide gel electrophoresis; RP, reversed-phase chromatography; SEC, size-exclusion chromatography; TFA, trifluoroacetic acid. * Corresponding author. Tel.: +1-202-9942947; fax: +1-202-9942870. E-mail address: [email protected] (P. Lecchi). 0165-022X/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0165-022X(03)00055-1
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techniques, is a common approach to maximally disperse proteins expressed in a biological system [1,2]. These experimental protocols exploit the specific separation mechanisms of each type of chromatography. SEC is widely utilized in protein purification. However, if compared to other separation techniques, it provides limited resolving power. A graphical exemplification of this statement is that a chromatogram produced by SEC usually does not contain more than just a few fully resolved peaks. Therefore, common SEC applications are those not requiring high resolving power such as desalting or fractionation. From a conceptual point of view, a ‘‘size-dependent’’ separation method such as SEC is more than ‘‘low resolution but biologically friendly’’ chromatography. Indeed, SEC is a comprehensive technique able to classify even heterogeneous mixtures of biopolymers according to a universal parameter (i.e., molecular size). In principle, SEC might find application for the analysis of every class of biological molecules because among the features carried by a molecule, size is one that is always present. For this general quality, combining SEC with detectors sharing the same ‘‘universal’’ characteristics generates ‘‘comprehensive’’ analytical schemes able to cover a broad range of molecular weights (MWs) in a structure-independent way [3,4]. Recent technical achievements such as: high throughput analytical schemes, more accurate and sensitive mass spectrometers, increasingly powerful computers, and more exhaustive databases, have made possible a new approach to the analysis of biomolecules. For every class of biological molecules, an ‘‘-omics’’ definition that characterizes a particular comprehensive analytical approach has been reported [5]. In any ‘‘-omics’’ analytical scheme, the contribution of a universal separation method could be an important means to evaluate even very heterogeneous mixtures. Proteomics is the most tangible example of such a comprehensive analysis. The combination of two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) with mass spectrometry (MS) is the most common analytical scheme in proteomics [6]. Briefly, the high resolving power of 2D-PAGE allows separation of a protein mixture into ‘‘individual spots’’ in a flat gel. Ideally, a single protein is isolated from a specific spot of interest and then digested to peptides. The identification of proteolytic fragments (and hence of the precursor protein) is achieved by MS, usually by matching the MS results with an available database. Although 2D-PAGE and MS are not directly connected, fully integrated technology that combines the two techniques via robotic systems are now commercially available. Even with recent improvement [7], 2D-PAGE as the only separation device for protein analysis has many drawbacks and intrinsic limitations [8]. To achieve a wider dynamic range of analysis, obtain better reproducibility and increase analytical throughput, several multidimensional chromatographic separations have been proposed as alternatives to 2D-PAGE [9]. When multiple separations are combined, a key factor in achieving the highest resolving power is to select two (or more) separation techniques with ‘‘orthogonal’’ characteristics (i.e., featuring un-overlapped separation mechanisms). Only a few applications of SEC for proteomics have been reported. Opiteck et al. [10,11] suggested a multidimensional scheme where several SEC columns (up to 12) were connected to fractionate whole bacterial protein extracts; the fractions collected after SEC were then automatically separated by reversed-phase HPLC and analyzed off-line by
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MALDI-TOF. This scheme reported a separation capacity adequate to resolve all the proteins expressed by a simple living organism such as Escherichia coli. However, it is not sufficient for more complex biological systems with a higher number of expressed proteins. Given that a biological system could synthesize a huge number of proteins, the complete resolution of a dynamic entity such as a proteome is truly a challenge for any kind of multidimensional separation scheme reported so far. Therefore, besides higher resolution, new analytical concepts will be required to deal with the complexity of the problem. For our experimental procedures, we evaluate the combination of several separation techniques. Also, we demonstrate that the use of stable isotopes as an ‘‘additional dimension’’ facilitates the analysis of selected proteins even in the presence of an overwhelming amount of interfering material.
2. Material and methods 2.1. Protein extraction The whole protein extract from E. coli (strain BL21) was used to test the separation schemes proposed. Briefly, 200 ml of bacterial culture was grown overnight in M9 minimal essential medium [1] containing glucose (Sigma, St. Louis, MO) or 13C6glucose (Spectra Stable Isotopes, Columbia, MD) as the only source of carbon. After centrifugation of bacterial culture (10,000 g for 10 min), the pellets obtained were suspended in 5 ml of water. Suspended cells were disrupted by sonication at 4 jC (30 s, three times) and allowed to equilibrate for 4 h at 4 jC after the addition of Tris – HCl (to a final concentration of 20 mM pH 7). The cell lysate was then centrifuged (15,000 g for 30 min) and the supernatant was filtered through a 0.22-Am filter and finally dialyzed overnight against water using 7-kDa cut-off dialysis cassettes (Pierce, Rockford, IL). Five milliliters of a solution containing approximately 2 mg/ml of proteins was obtained. 2.2. Two-dimensional separation Protein extracts were fractionated by liquid isoelectric focusing (IEF) on a Rotofor preparative cell (Bio-Rad, Hercules, CA). Before each analysis, the electrophoretic cell was pre-focused for 30 min with 50 ml of a solution containing 2% ampholyte pH 3 –10 (Bio-Rad). A sample of 4 ml of the protein solution was loaded into the electrophoretic cell and separated at a constant 12 W for 3 h (i.e., until a stable voltage was reached). Twenty fractions were collected and stored at 4 jC for further separations. Aliquots from all fractions except 1, 2, 19, and 20 were analyzed by SEC using a TSK G3000SWXL 7 300 mm column (TosoHaas, Montgomeryville, PA). The mobile phase was KH2PO4 50 mM and NaCl 200 mM in water at pH 6.5/methanol 9/1. The flow was 0.4 ml/min. In a different set of experiments, aliquots from the same 16 fractions were analyzed by RP with a C-18, 4.6 150-mm column (218TP5415 Vydac, Hesperia, CA). The mobile phase was a linear gradient of water and acetonitrile (ACN; both containing 0.1% trifluoroacetic acid, TFA) from 20% to 60% in 40 min. All these analysis were
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Fig. 1. Combination of isoelectric focusing (IEF) and HPLC of an E. coli protein extract (i.e., a liquid-based analog of 2D-PAGE). In both panels, the Y-axis represents the IEF separation. Only IEF fractions from 3 to 18 (noted here as 1 – 16) were further analyzed by SEC (top) or by RP (bottom). The dispersion obtained from the chromatographic separation (either SEC or RP) is on the X-axis (time) and the Z-axis is a 12-color transformation of the intensity of the chromatographic peaks (see Material and methods for further details).
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performed using an HPLC System Gold (Beckman Coulter, Fullerton, CA) with UV detection set at 220 nm. Results obtained from these two analyses were plotted in 3-D contour graphs using Slide Write Plus (Advanced Graphic Technologies, Encinitas, CA). This program only has 12 color zones; therefore, the relative intensities of the chromatographic profiles (reported on the z-axes in Fig. 1) had to be manipulated to show what is a much wider range of intensities. After some experimentation, the fourth-root of the intensities was found to sufficiently narrow the dispersion of the data to allow an effective presentation. 2.3. Multidimensional chromatography Three milliliters of a soluble protein extract obtained from E. coli grown in 13Clabeled M9 medium (prepared as described above) were concentrated by evaporative centrifugation (Savant, Holbrook, NY) to a final protein concentration of approximately 5 mg/ml. Three aliquots, 250 Al each, of this preparation were fractionated with three distinct chromatographic techniques: RP, IE and SEC using a UV detector at 280 nm. Analytical conditions for this first chromatographic dimension are reported in Table 1. Selected fractions obtained from the first dimension and spiked into a constant amount of unfractionated unlabeled protein extract were analyzed with a second chromatographic technique. For this second dimension, different chromatographic approaches were tested, including reversed-phase with four different columns: Poros R1/10 and R2/10 (PerSeptive Biosystems, Framingham, MA) Vydac 218 TP5415, and Micra NPS ODS101.5 (Eichrom, Darien, IL). Weak anion exchange chromatography with a TSKDEAE NPR column (TosoHaas) was also tested. Chromatographic conditions for this second dimension are described in Table 2. The detector for the final dimension was chemical reaction interface mass spectrometry (CRIMS) [12], an isotope-selective detection method. Briefly, in CRIMS analysis all the species eluted from the chromatographic separation are combined with a reactant gas and transformed to a predictable set of low MW reaction products in a microwave powered cavity. Products of the chemical reaction are compounds related to the isotopic composition of the analytes. For example, when SO2 is used as the reactant gas, atoms Table 1 Analytical parameters for the first chromatographic dimension Parameter
Anion-exchange
Reversed phase
Size-exclusion
Column
Biorad DEAE-5 PW, 7 100 mm CH3COONH4 20 mM
Vydac TP214510, 10 250 mm 0.1% HFBA in water
(two) TSK G3000-SWXL, 7.8 300 mm CH3COONH4 200 mM in water/ACN 87/13
CH3COONH4 2 M (both in water/ACN 87/13) 2 min at 100% A, linear to 100% B in 33 min 7 min at 100% B 1 ml/min
0.1% HFBA in ACN
Mobile phase A Mobile phase B Gradient
Flow
5 min at 10% B, linear to 35% B in 15 min linear to 75% B in 25 min 3 ml/min
Isocratic 0.25 ml/min
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Table 2 Analytical parameters for the second chromatographic dimension Parameter
Reversed phase
Anion exchange
Column
(1) Poros R1/10—2.1 30 mm (2) Poros R2/10—2.1 30 mm (3) Vydac 218 TP5415—4.6 150 mm (4) Micra NPS-ODS101.5—4.6 33 mm 0.1% TFA in water 0.1% TFA in ACN
TSK-DEAE NPR-4.6 x 35 mm
Mobile phase A Mobile phase B Gradient
Flow
10 min at 15% B, linear to 35% B in 10 min linear to 65% B in 30 min 1 ml/min (0.3 ml/min for column #4)
CH3COONH4 20 mM CH3COONH4 2 M (in water/ACN 9:1) 5 min 100% A, linear to 25% B in 15 min linear to 100% B in 10 min 0.3 ml/min
of 12C are transformed to 12CO2 and detected at m/z 44. By analogy, the detected as 13CO2 at m/z 45.
13
C atoms are
3. Results and discussion 2D-PAGE combines isoelectric focusing with a size-dependent separation to generate a gel-based multidimensional separation scheme [13]. In our experiments, we evaluated an analog liquid-based preparative scheme that uses the same two separation mechanisms. To get a closer comparison, analytical results are plotted in a way that resemble a virtual 2DPAGE. We use a contour plot diagram in which, the IEF fractions (i.e., the pH gradient) are reported on the Y-axes and the retention time for the chromatographic separation is on the X-axis. The relative intensities of chromatographic peaks are noted by different colors in the Z-dimension. It is apparent from a graphical representation (Fig. 1, top) that a liquidbased method that combines IEF and SEC does not provide a separation power comparable to an analogous gel-based scheme [14]. If resolution is the only parameter considered, the use of SEC could represent a considerable drawback. This became even more evident when the higher resolving power of an RP separation replaced SEC as the second separation dimension. The lower panel in Fig. 1 shows the analytical results of IEC combined with RP. The number of spots (i.e., the number of peaks in each chromatogram) is noticeably higher in this second diagram. The substantially different resolution between SEC and RP is emphasized in a more conventional way in Fig. 2 where a direct comparison between SEC and RP chromatograms obtained from IEF fraction 9 is shown. This sample is dispersed over 6 min in several partially resolved peaks in SEC while RP gave a dispersion of well-resolved peaks over more than 20 min. Repeated injections of the fractions separated by liquid IEF provided very reproducible results. For the IEF separation, we used a range of pH between 3 and 10. However, the very acidic and very basic fractions do not contain proteins and were not further analyzed. We note the separation capacity of the first dimension could be improved by using a narrower range of pH. In any case, separations obtained with the Rotofor-IEF apparatus will provide no more than 20 discrete fractions. That is much lower than the number of bands achievable on a
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Fig. 2. Illustrative comparison between RP and SEC chromatograms (panels A and B, respectively). Both chromatograms were obtained from two aliquots of IEF fraction 9.
gel-based IEF. In addition, some precautions should be taken to avoid protein precipitation inside the electrophoretic cell. To overcome this major limitation, several remedies have been proposed [15]. Most of them are aimed at increasing protein solubility by addition of chaotropic agents, detergents, or simply by increasing the ampholyte concentration. Despite poor resolution, this liquid-based IEF-SEC separation has the ability to fractionate milligrams of proteins according to predictable chemico-physical parameters (i.e., isoelectric point and molecular size). If compared to a flat 2D-PAGE, this 2Dchromatography has a wider dynamic range of analysis. The huge range of protein expression levels in a biological system could span several orders of magnitude. In view of the limited dynamic range of analysis of 2D-PAGE, detecting proteins expressed at very low levels is one of the main challenges of proteomics [8]. A liquid-based scheme such as
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IEF-SEC could represent an alternative analytical method with the ability to enrich in predictable fractions proteins expressed in very low amounts. As another strategy involving multidimensional chromatography for proteomics, we have added the use of isotopic labels. We reported earlier that SEC is a general, sizedependent separation technique suitable for a structure independent analysis [3,16]. To evaluate the separation capacity and to explore new schemes to facilitate the identification of selected compounds, we used an isotopic selective detector. Metabolic labeling with stable isotopes is an established experimental approach for differential proteomics analysis [17,18]. We have explored selective detection of stable isotopes as another ‘‘dimension’’ by which the complexity of a proteome can be simplified. Our approach begins with a uniformly stable-isotope labeled protein extract from E. coli, captures short sections of a first-dimension HPLC separation, spikes those sections into an unlabeled but comparable E. coli extract, and then compares the combination of another type of HPLC separation combined with CRIMS for isotopeselective detection. In this sequence, the chromatogram of the enriched isotope represents two-dimensional dispersion and allows a comparison of the effectiveness of various 2-D HPLC schemes to resolve complex proteomes. In this set of experiments, CRIMS is seen as an ‘‘isotopic filter’’ intervening between the chromatographic separation of biopolymers and their MS identification. The ‘‘CRIMS filter’’ greatly reduces the complexity of the biological sample by monitoring only those molecules that contain an isotopic (2H, 15N, 13 C, 18O) or elemental tag (S, P, and Se). Different chromatographic techniques were used to fractionate mixtures of 13C-labeled proteins. Fig. 3 shows a comparison between SEC, IE, or RP separation of three aliquots of the same preparation obtained from E. coli grown in labeled medium. RP as a first chromatographic dimension consistently showed an overwhelming amount of unretained material. Instead, SEC was particularly effective in providing wide dispersion and cleaner fractions for the subsequent analytical separation. The best results were obtained by using weak anion-exchange chromatography with a short non-porous column (TSK-DEAE NPR) as the second dimension. The analyses of two selected 30-s fractions obtained from SEC (indicated by arrows in Fig. 3) are shown in Fig. 4. The chromatography was performed as described in the experimental section using CRIMS as an isotope specific detection system. In chromatographic terms, the top trace shows a one-dimensional chromatogram of the E. coli mixture and the lower trace is the result of the two-dimensional approach, i.e., SEC followed by IEC. As an isotope-selective display, the lower (13C-enrichment) trace shows only those analytes containing the 13C isotopic tag while the upper chromatogram is the 12C trace in which all carbon containing species are detected. Fig. 4 shows that a selectivity based on isotopic ratio can be detected by CRIMS in the presence of an overwhelming amount of interfering material (unlabeled proteins). Even small peaks on the ‘‘enrichment trace’’ (enhanced four times for graphical reasons) are detected in presence of 100 times of unlabeled interfering material (i.e., carbon trace). The good orthogonality of this separation scheme is revealed by the fact that a 30-s fraction obtained from SEC is further dispersed over a 20-min span by the second chromatography. It is important to note that the integration of a peak on a CRIMS trace provides quantitative information of the isotope under evaluation produced by the chemical reaction
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Fig. 3. Analysis of a stable-isotope labeled E. coli protein extract by three different chromatographic methods: Ion exchange chromatography (panel A), reversed phase chromatography (panel B) and size-exclusion chromatography (panel C). The two 30-s bars in the SEC chromatogram (at 70 and 84 min, respectively) indicate the two fractions submitted to a second dimension of HPLC, seen in Fig. 4.
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Fig. 4. Second-dimension HPLC analyses of the two fractions in Fig. 3 taken from SEC and spiked into an unfractionated E. coli extract using IE with CRIMS detection. Panel A shows this extract spiked with an aliquot from the 70-min fraction and panel B shows the extract spiked with an aliquot from the 84-min fraction. In both chromatograms, the upper trace represents the total carbon content and the lower trace represents the ‘‘enrichment trace’’ (i.e., only those species with a 13C/12C isotope ratio different from the natural ratio 1.119%). Because only the spiked component is labeled with 13C, the lower trace isolates those species from the rest of the E. coli extract and shows the effectiveness of the two-dimensional HPLC approach.
(in this case, the amount of 13C that exceeds natural abundance). When an isotope is always present in a monomeric unit of a polymer, there is a linear relation between the amount of the element in the polymer and the MW of this polymer. Thus, using a CRIMS chromatogram, accurate quantitation can be achieved even without knowing the analyte identity, and without requiring any specific standardization procedure. The calibration procedure requires three components: the area of the 13CO2 reaction product for a protein and its fractional enrichments (both obtained by CRIMS analysis). The third parameter, MW of the protein, can be obtained in advance (i.e., retention on a size-exclusion chromatogram), or subsequently (i.e., analysis with ‘‘conventional’’ MS). The same experimental model can be applied to other elements contained in biopolymers, in this
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case different elemental tags have to be used (e.g. 2H, 15N or 18O). The enrichment trace of an HPLC-CRIMS can be quantitatively interpreted when a separation scheme includes SEC. Therefore, in addition to its separation power and loading capacity, the use of SEC in combination with a universal detector provides quantitative information.
4. Conclusions This paper describes innovative separation schemes for proteomics. Even if SEC is a technique with a relatively low resolving power, its very predictable separation mechanism combined with its high loading capacity makes it a useful part of separation schemes for proteomic analysis. In addition to a separation scheme, the selective detection of isotopic tags will help to monitor labeled proteins against a background of interfering material and provide preliminary quantitative information.
Acknowledgements The support from NIH Grant RO1 GM 58623 is acknowledged.
References [1] Coligan JE, Dunn BM, Ploegh HL, Speicher DW, Wingfield PT. Current protocols in protein science. New York: John Wiley & Sons Inc; 1998. p. 8.0.1 – 8.6.12. [2] Harris ELV, Angal S. Protein purification methods. A practical approach series. Oxford, England: IRL Press; 1990. p. 175 – 306. [3] Wen J, Arakawa T, Philo JS. Size-exclusion chromatography with on-line light-scattering, absorbance, and refractive index detectors for studying proteins and their interactions. Anal Biochem 1996;240:155 – 66. [4] Lecchi P, Abramson FP. Size exclusion chromatography – chemical reaction interface mass spectrometry: ‘‘a perfect match’’. Anal Chem 1999;71:2951 – 5. [5] Lederberg J, McCray AT. ‘‘Ome sweet omics’’—a genealogical treasury of words. Scientist 2001;15:8. [6] Shevchenko A, Jensen ON, Podtelejnikov AV, Sagliocco F, Wilm M, Vorm O, et al. Linking genome and proteome by mass spectrometry: large-scale identification of yeast proteins from two-dimensional gels. Proc Natl Acad Sci U S A 1996;93:14440 – 5. [7] Lilley KS, Razzaq A, Dupree P. Two-dimensional gel electrophoresis: recent advances in sample preparation, detection and quantitation. Curr Chem Biol 2002;6:46 – 50. [8] Gygi SP, Corthals GL, Zhang Y, Rochon Y, Aebersold R. Evaluation of two-dimensional gel electrophoresis-based proteome analysis technology. Proc Natl Acad Sci U S A 2000;97:9390 – 5. [9] Liu H, Lin D, Yates J. Multidimensional separations for protein/peptide analysis in the post-genomic era. BioTechniques 2002;32:898 – 911. [10] Opiteck GJ, Jorgenson JW, Anderegg RJ. Two-dimensional SEC/RPLC coupled to mass spectrometry for the analysis of peptides. Anal Chem 1997;69:2283 – 91. [11] Opiteck GJ, Ramirez S, Jorgenson JW, Moseley AM. Comprehensive two-dimensional high-performance liquid chromatography for the isolation of overexpressed proteins and proteome mapping. Anal Biochem 1998;258:349 – 61. [12] Abramson FP. CRIMS: Chemical Reaction Interface Mass Spectrometry. Mass Spectrom Rev 1994; 13:341 – 56. [13] O’Farrell PH. J Biol Chem 1975;250:4007.
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[14] Tonella L, Hoogland C, Binz PA, Appel RD, Hochstrasser DF, Sanchez JC. New perspectives in the Escherichia coli proteome investigation. Proteomics 2001;1:409 – 23. [15] Ayala A, Parrado J, Machado A. Use of Rotofor preparative isoelectrofocusing cell in protein purification procedure. Appl Biochem Biotechnol 1998;69:11 – 6. [16] Lecchi P, Abramson FP. Analysis of biopolymers by size-exclusion chromatography mass spectrometry. J Chromatogr, A 1998;828:509 – 13. [17] Oda Y, Huang K, Cross FR, Cowburn D, Chait BT. Accurate quantitation of protein expression and sitespecific phosphorylation. Proc Natl Acad Sci U S A 1999;96:6591 – 6. [18] Pasa-Tolic L, Jensen PK, Anderson GA, Lipton MS, Peden KK, Martinovic S, et al. High throughput proteome-wide precision measurements of protein expression using mass spectrometry. J Am Chem Soc 1999;121:7949 – 50.
Size-exclusion chromatography in multidimensional separation schemes for proteome analysis Paolo Lecchi *, Anand R. Gupte, Ricardo E. Perez, Lyubov V. Stockert, Fred P. Abramson Department of Pharmacology, The George Washington University School of Medicine and Health Sciences, 2300 I St. N.W., Washington, DC 20037, USA
Abstract Size-exclusion chromatography (SEC) is a separation technique with a relatively low resolving power, compared to those usually utilized in proteomics. Therefore, it is often overlooked in experimental protocols, when the main goal is resolving complex biological mixtures. In this report, we introduce innovative multidimensional schemes for proteomics analysis, in which SEC plays a practical role. Liquid isoelectric focusing (IEF) was combined with SEC, and experimental results were compared to those obtained by two-dimensional polyacrylamide gel electrophoresis (2DPAGE), well-established techniques relying upon similar criteria for separation. Additional experiments were performed to evaluate the practical contribution of SEC in multidimensional chromatographic separations. Specifically, we evaluated the combination of SEC and ion exchange chromatography in an analytical scheme for the mass spectrometric analysis of protein-extracts obtained from bacterial cultures grown in stable isotope enriched media. Experimental conditions and practical considerations are discussed. D 2003 Elsevier Science B.V. All rights reserved. Keywords: Size-exclusion chromatography; Rotofor; Proteomics; Mass spectrometry; CRIMS; Stable isotopes
1. Introduction Combining different separation strategies such as: size-exclusion chromatography (SEC), ion exchange chromatography (IE), reversed phase (RP), or other chromatographic Abbreviations: ACN, acetonitrile; CRIMS, chemical reaction interface mass spectrometry; HFBA, heptafluorobutyric acid; IE, ion-exchange chromatography; IEF, isoelectric focusing; MW, molecular weight; PAGE, polyacrylamide gel electrophoresis; RP, reversed-phase chromatography; SEC, size-exclusion chromatography; TFA, trifluoroacetic acid. * Corresponding author. Tel.: +1-202-9942947; fax: +1-202-9942870. E-mail address: [email protected] (P. Lecchi). 0165-022X/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0165-022X(03)00055-1
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techniques, is a common approach to maximally disperse proteins expressed in a biological system [1,2]. These experimental protocols exploit the specific separation mechanisms of each type of chromatography. SEC is widely utilized in protein purification. However, if compared to other separation techniques, it provides limited resolving power. A graphical exemplification of this statement is that a chromatogram produced by SEC usually does not contain more than just a few fully resolved peaks. Therefore, common SEC applications are those not requiring high resolving power such as desalting or fractionation. From a conceptual point of view, a ‘‘size-dependent’’ separation method such as SEC is more than ‘‘low resolution but biologically friendly’’ chromatography. Indeed, SEC is a comprehensive technique able to classify even heterogeneous mixtures of biopolymers according to a universal parameter (i.e., molecular size). In principle, SEC might find application for the analysis of every class of biological molecules because among the features carried by a molecule, size is one that is always present. For this general quality, combining SEC with detectors sharing the same ‘‘universal’’ characteristics generates ‘‘comprehensive’’ analytical schemes able to cover a broad range of molecular weights (MWs) in a structure-independent way [3,4]. Recent technical achievements such as: high throughput analytical schemes, more accurate and sensitive mass spectrometers, increasingly powerful computers, and more exhaustive databases, have made possible a new approach to the analysis of biomolecules. For every class of biological molecules, an ‘‘-omics’’ definition that characterizes a particular comprehensive analytical approach has been reported [5]. In any ‘‘-omics’’ analytical scheme, the contribution of a universal separation method could be an important means to evaluate even very heterogeneous mixtures. Proteomics is the most tangible example of such a comprehensive analysis. The combination of two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) with mass spectrometry (MS) is the most common analytical scheme in proteomics [6]. Briefly, the high resolving power of 2D-PAGE allows separation of a protein mixture into ‘‘individual spots’’ in a flat gel. Ideally, a single protein is isolated from a specific spot of interest and then digested to peptides. The identification of proteolytic fragments (and hence of the precursor protein) is achieved by MS, usually by matching the MS results with an available database. Although 2D-PAGE and MS are not directly connected, fully integrated technology that combines the two techniques via robotic systems are now commercially available. Even with recent improvement [7], 2D-PAGE as the only separation device for protein analysis has many drawbacks and intrinsic limitations [8]. To achieve a wider dynamic range of analysis, obtain better reproducibility and increase analytical throughput, several multidimensional chromatographic separations have been proposed as alternatives to 2D-PAGE [9]. When multiple separations are combined, a key factor in achieving the highest resolving power is to select two (or more) separation techniques with ‘‘orthogonal’’ characteristics (i.e., featuring un-overlapped separation mechanisms). Only a few applications of SEC for proteomics have been reported. Opiteck et al. [10,11] suggested a multidimensional scheme where several SEC columns (up to 12) were connected to fractionate whole bacterial protein extracts; the fractions collected after SEC were then automatically separated by reversed-phase HPLC and analyzed off-line by
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MALDI-TOF. This scheme reported a separation capacity adequate to resolve all the proteins expressed by a simple living organism such as Escherichia coli. However, it is not sufficient for more complex biological systems with a higher number of expressed proteins. Given that a biological system could synthesize a huge number of proteins, the complete resolution of a dynamic entity such as a proteome is truly a challenge for any kind of multidimensional separation scheme reported so far. Therefore, besides higher resolution, new analytical concepts will be required to deal with the complexity of the problem. For our experimental procedures, we evaluate the combination of several separation techniques. Also, we demonstrate that the use of stable isotopes as an ‘‘additional dimension’’ facilitates the analysis of selected proteins even in the presence of an overwhelming amount of interfering material.
2. Material and methods 2.1. Protein extraction The whole protein extract from E. coli (strain BL21) was used to test the separation schemes proposed. Briefly, 200 ml of bacterial culture was grown overnight in M9 minimal essential medium [1] containing glucose (Sigma, St. Louis, MO) or 13C6glucose (Spectra Stable Isotopes, Columbia, MD) as the only source of carbon. After centrifugation of bacterial culture (10,000 g for 10 min), the pellets obtained were suspended in 5 ml of water. Suspended cells were disrupted by sonication at 4 jC (30 s, three times) and allowed to equilibrate for 4 h at 4 jC after the addition of Tris – HCl (to a final concentration of 20 mM pH 7). The cell lysate was then centrifuged (15,000 g for 30 min) and the supernatant was filtered through a 0.22-Am filter and finally dialyzed overnight against water using 7-kDa cut-off dialysis cassettes (Pierce, Rockford, IL). Five milliliters of a solution containing approximately 2 mg/ml of proteins was obtained. 2.2. Two-dimensional separation Protein extracts were fractionated by liquid isoelectric focusing (IEF) on a Rotofor preparative cell (Bio-Rad, Hercules, CA). Before each analysis, the electrophoretic cell was pre-focused for 30 min with 50 ml of a solution containing 2% ampholyte pH 3 –10 (Bio-Rad). A sample of 4 ml of the protein solution was loaded into the electrophoretic cell and separated at a constant 12 W for 3 h (i.e., until a stable voltage was reached). Twenty fractions were collected and stored at 4 jC for further separations. Aliquots from all fractions except 1, 2, 19, and 20 were analyzed by SEC using a TSK G3000SWXL 7 300 mm column (TosoHaas, Montgomeryville, PA). The mobile phase was KH2PO4 50 mM and NaCl 200 mM in water at pH 6.5/methanol 9/1. The flow was 0.4 ml/min. In a different set of experiments, aliquots from the same 16 fractions were analyzed by RP with a C-18, 4.6 150-mm column (218TP5415 Vydac, Hesperia, CA). The mobile phase was a linear gradient of water and acetonitrile (ACN; both containing 0.1% trifluoroacetic acid, TFA) from 20% to 60% in 40 min. All these analysis were
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Fig. 1. Combination of isoelectric focusing (IEF) and HPLC of an E. coli protein extract (i.e., a liquid-based analog of 2D-PAGE). In both panels, the Y-axis represents the IEF separation. Only IEF fractions from 3 to 18 (noted here as 1 – 16) were further analyzed by SEC (top) or by RP (bottom). The dispersion obtained from the chromatographic separation (either SEC or RP) is on the X-axis (time) and the Z-axis is a 12-color transformation of the intensity of the chromatographic peaks (see Material and methods for further details).
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performed using an HPLC System Gold (Beckman Coulter, Fullerton, CA) with UV detection set at 220 nm. Results obtained from these two analyses were plotted in 3-D contour graphs using Slide Write Plus (Advanced Graphic Technologies, Encinitas, CA). This program only has 12 color zones; therefore, the relative intensities of the chromatographic profiles (reported on the z-axes in Fig. 1) had to be manipulated to show what is a much wider range of intensities. After some experimentation, the fourth-root of the intensities was found to sufficiently narrow the dispersion of the data to allow an effective presentation. 2.3. Multidimensional chromatography Three milliliters of a soluble protein extract obtained from E. coli grown in 13Clabeled M9 medium (prepared as described above) were concentrated by evaporative centrifugation (Savant, Holbrook, NY) to a final protein concentration of approximately 5 mg/ml. Three aliquots, 250 Al each, of this preparation were fractionated with three distinct chromatographic techniques: RP, IE and SEC using a UV detector at 280 nm. Analytical conditions for this first chromatographic dimension are reported in Table 1. Selected fractions obtained from the first dimension and spiked into a constant amount of unfractionated unlabeled protein extract were analyzed with a second chromatographic technique. For this second dimension, different chromatographic approaches were tested, including reversed-phase with four different columns: Poros R1/10 and R2/10 (PerSeptive Biosystems, Framingham, MA) Vydac 218 TP5415, and Micra NPS ODS101.5 (Eichrom, Darien, IL). Weak anion exchange chromatography with a TSKDEAE NPR column (TosoHaas) was also tested. Chromatographic conditions for this second dimension are described in Table 2. The detector for the final dimension was chemical reaction interface mass spectrometry (CRIMS) [12], an isotope-selective detection method. Briefly, in CRIMS analysis all the species eluted from the chromatographic separation are combined with a reactant gas and transformed to a predictable set of low MW reaction products in a microwave powered cavity. Products of the chemical reaction are compounds related to the isotopic composition of the analytes. For example, when SO2 is used as the reactant gas, atoms Table 1 Analytical parameters for the first chromatographic dimension Parameter
Anion-exchange
Reversed phase
Size-exclusion
Column
Biorad DEAE-5 PW, 7 100 mm CH3COONH4 20 mM
Vydac TP214510, 10 250 mm 0.1% HFBA in water
(two) TSK G3000-SWXL, 7.8 300 mm CH3COONH4 200 mM in water/ACN 87/13
CH3COONH4 2 M (both in water/ACN 87/13) 2 min at 100% A, linear to 100% B in 33 min 7 min at 100% B 1 ml/min
0.1% HFBA in ACN
Mobile phase A Mobile phase B Gradient
Flow
5 min at 10% B, linear to 35% B in 15 min linear to 75% B in 25 min 3 ml/min
Isocratic 0.25 ml/min
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Table 2 Analytical parameters for the second chromatographic dimension Parameter
Reversed phase
Anion exchange
Column
(1) Poros R1/10—2.1 30 mm (2) Poros R2/10—2.1 30 mm (3) Vydac 218 TP5415—4.6 150 mm (4) Micra NPS-ODS101.5—4.6 33 mm 0.1% TFA in water 0.1% TFA in ACN
TSK-DEAE NPR-4.6 x 35 mm
Mobile phase A Mobile phase B Gradient
Flow
10 min at 15% B, linear to 35% B in 10 min linear to 65% B in 30 min 1 ml/min (0.3 ml/min for column #4)
CH3COONH4 20 mM CH3COONH4 2 M (in water/ACN 9:1) 5 min 100% A, linear to 25% B in 15 min linear to 100% B in 10 min 0.3 ml/min
of 12C are transformed to 12CO2 and detected at m/z 44. By analogy, the detected as 13CO2 at m/z 45.
13
C atoms are
3. Results and discussion 2D-PAGE combines isoelectric focusing with a size-dependent separation to generate a gel-based multidimensional separation scheme [13]. In our experiments, we evaluated an analog liquid-based preparative scheme that uses the same two separation mechanisms. To get a closer comparison, analytical results are plotted in a way that resemble a virtual 2DPAGE. We use a contour plot diagram in which, the IEF fractions (i.e., the pH gradient) are reported on the Y-axes and the retention time for the chromatographic separation is on the X-axis. The relative intensities of chromatographic peaks are noted by different colors in the Z-dimension. It is apparent from a graphical representation (Fig. 1, top) that a liquidbased method that combines IEF and SEC does not provide a separation power comparable to an analogous gel-based scheme [14]. If resolution is the only parameter considered, the use of SEC could represent a considerable drawback. This became even more evident when the higher resolving power of an RP separation replaced SEC as the second separation dimension. The lower panel in Fig. 1 shows the analytical results of IEC combined with RP. The number of spots (i.e., the number of peaks in each chromatogram) is noticeably higher in this second diagram. The substantially different resolution between SEC and RP is emphasized in a more conventional way in Fig. 2 where a direct comparison between SEC and RP chromatograms obtained from IEF fraction 9 is shown. This sample is dispersed over 6 min in several partially resolved peaks in SEC while RP gave a dispersion of well-resolved peaks over more than 20 min. Repeated injections of the fractions separated by liquid IEF provided very reproducible results. For the IEF separation, we used a range of pH between 3 and 10. However, the very acidic and very basic fractions do not contain proteins and were not further analyzed. We note the separation capacity of the first dimension could be improved by using a narrower range of pH. In any case, separations obtained with the Rotofor-IEF apparatus will provide no more than 20 discrete fractions. That is much lower than the number of bands achievable on a
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Fig. 2. Illustrative comparison between RP and SEC chromatograms (panels A and B, respectively). Both chromatograms were obtained from two aliquots of IEF fraction 9.
gel-based IEF. In addition, some precautions should be taken to avoid protein precipitation inside the electrophoretic cell. To overcome this major limitation, several remedies have been proposed [15]. Most of them are aimed at increasing protein solubility by addition of chaotropic agents, detergents, or simply by increasing the ampholyte concentration. Despite poor resolution, this liquid-based IEF-SEC separation has the ability to fractionate milligrams of proteins according to predictable chemico-physical parameters (i.e., isoelectric point and molecular size). If compared to a flat 2D-PAGE, this 2Dchromatography has a wider dynamic range of analysis. The huge range of protein expression levels in a biological system could span several orders of magnitude. In view of the limited dynamic range of analysis of 2D-PAGE, detecting proteins expressed at very low levels is one of the main challenges of proteomics [8]. A liquid-based scheme such as
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IEF-SEC could represent an alternative analytical method with the ability to enrich in predictable fractions proteins expressed in very low amounts. As another strategy involving multidimensional chromatography for proteomics, we have added the use of isotopic labels. We reported earlier that SEC is a general, sizedependent separation technique suitable for a structure independent analysis [3,16]. To evaluate the separation capacity and to explore new schemes to facilitate the identification of selected compounds, we used an isotopic selective detector. Metabolic labeling with stable isotopes is an established experimental approach for differential proteomics analysis [17,18]. We have explored selective detection of stable isotopes as another ‘‘dimension’’ by which the complexity of a proteome can be simplified. Our approach begins with a uniformly stable-isotope labeled protein extract from E. coli, captures short sections of a first-dimension HPLC separation, spikes those sections into an unlabeled but comparable E. coli extract, and then compares the combination of another type of HPLC separation combined with CRIMS for isotopeselective detection. In this sequence, the chromatogram of the enriched isotope represents two-dimensional dispersion and allows a comparison of the effectiveness of various 2-D HPLC schemes to resolve complex proteomes. In this set of experiments, CRIMS is seen as an ‘‘isotopic filter’’ intervening between the chromatographic separation of biopolymers and their MS identification. The ‘‘CRIMS filter’’ greatly reduces the complexity of the biological sample by monitoring only those molecules that contain an isotopic (2H, 15N, 13 C, 18O) or elemental tag (S, P, and Se). Different chromatographic techniques were used to fractionate mixtures of 13C-labeled proteins. Fig. 3 shows a comparison between SEC, IE, or RP separation of three aliquots of the same preparation obtained from E. coli grown in labeled medium. RP as a first chromatographic dimension consistently showed an overwhelming amount of unretained material. Instead, SEC was particularly effective in providing wide dispersion and cleaner fractions for the subsequent analytical separation. The best results were obtained by using weak anion-exchange chromatography with a short non-porous column (TSK-DEAE NPR) as the second dimension. The analyses of two selected 30-s fractions obtained from SEC (indicated by arrows in Fig. 3) are shown in Fig. 4. The chromatography was performed as described in the experimental section using CRIMS as an isotope specific detection system. In chromatographic terms, the top trace shows a one-dimensional chromatogram of the E. coli mixture and the lower trace is the result of the two-dimensional approach, i.e., SEC followed by IEC. As an isotope-selective display, the lower (13C-enrichment) trace shows only those analytes containing the 13C isotopic tag while the upper chromatogram is the 12C trace in which all carbon containing species are detected. Fig. 4 shows that a selectivity based on isotopic ratio can be detected by CRIMS in the presence of an overwhelming amount of interfering material (unlabeled proteins). Even small peaks on the ‘‘enrichment trace’’ (enhanced four times for graphical reasons) are detected in presence of 100 times of unlabeled interfering material (i.e., carbon trace). The good orthogonality of this separation scheme is revealed by the fact that a 30-s fraction obtained from SEC is further dispersed over a 20-min span by the second chromatography. It is important to note that the integration of a peak on a CRIMS trace provides quantitative information of the isotope under evaluation produced by the chemical reaction
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Fig. 3. Analysis of a stable-isotope labeled E. coli protein extract by three different chromatographic methods: Ion exchange chromatography (panel A), reversed phase chromatography (panel B) and size-exclusion chromatography (panel C). The two 30-s bars in the SEC chromatogram (at 70 and 84 min, respectively) indicate the two fractions submitted to a second dimension of HPLC, seen in Fig. 4.
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Fig. 4. Second-dimension HPLC analyses of the two fractions in Fig. 3 taken from SEC and spiked into an unfractionated E. coli extract using IE with CRIMS detection. Panel A shows this extract spiked with an aliquot from the 70-min fraction and panel B shows the extract spiked with an aliquot from the 84-min fraction. In both chromatograms, the upper trace represents the total carbon content and the lower trace represents the ‘‘enrichment trace’’ (i.e., only those species with a 13C/12C isotope ratio different from the natural ratio 1.119%). Because only the spiked component is labeled with 13C, the lower trace isolates those species from the rest of the E. coli extract and shows the effectiveness of the two-dimensional HPLC approach.
(in this case, the amount of 13C that exceeds natural abundance). When an isotope is always present in a monomeric unit of a polymer, there is a linear relation between the amount of the element in the polymer and the MW of this polymer. Thus, using a CRIMS chromatogram, accurate quantitation can be achieved even without knowing the analyte identity, and without requiring any specific standardization procedure. The calibration procedure requires three components: the area of the 13CO2 reaction product for a protein and its fractional enrichments (both obtained by CRIMS analysis). The third parameter, MW of the protein, can be obtained in advance (i.e., retention on a size-exclusion chromatogram), or subsequently (i.e., analysis with ‘‘conventional’’ MS). The same experimental model can be applied to other elements contained in biopolymers, in this
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case different elemental tags have to be used (e.g. 2H, 15N or 18O). The enrichment trace of an HPLC-CRIMS can be quantitatively interpreted when a separation scheme includes SEC. Therefore, in addition to its separation power and loading capacity, the use of SEC in combination with a universal detector provides quantitative information.
4. Conclusions This paper describes innovative separation schemes for proteomics. Even if SEC is a technique with a relatively low resolving power, its very predictable separation mechanism combined with its high loading capacity makes it a useful part of separation schemes for proteomic analysis. In addition to a separation scheme, the selective detection of isotopic tags will help to monitor labeled proteins against a background of interfering material and provide preliminary quantitative information.
Acknowledgements The support from NIH Grant RO1 GM 58623 is acknowledged.
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