Selective Enrichment of Low-Abundance Peptides in Complex

Anal. Chem. 2002, 74, 3933-3941

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Selective Enrichment of Low-Abundance Peptides in Complex Mixtures by Elution-Modified Displacement Chromatography and Their Identification by Electrospray Ionization Mass Spectrometry James A. Wilkins,† Rong Xiang,† and Csaba Horva´th*

Department of Chemical Engineering, Yale University, New Haven, Connecticut 06520-8286

Trace components were selectively enriched and detected in the tryptic digest of recombinant human growth hormone using elution-modified displacement chromatography, a hybrid technique combining features of elution and displacement chromatography. Based on the retention behavior of sample components in the elution mode, rapid and selective trace enrichment and high-resolution separation was achieved in a single step by utilizing appropriate combinations of an eluent such as aqueous acetonitrile with the displacer. Mass spectral and chromatographic analysis of displacement zones revealed up to 400-fold enhancement of the concentration of some low-abundance sample components. Potential application of this technique in proteomics to augment the sensitivity of LC-MS and 2-D gel electrophoretic approaches for the detection of biologically important low-abundance species is discussed.

In the postgenomic era, the elucidation of the structure, interactions, and posttranslational modifications of cellular proteins has taken center stage in the effort to understand cellular functions in normal and diseased states. The analytical demand imposed by the diversity and the wide concentration range of components in proteomics has created a compelling need for new separation technologies with high sensitivity and specificity. Techniques such as two-dimensional gel electrophoresis or elution chromatographic schemes coupled to mass spectrometry are widely used.1,2 * To whom all correspondence should be addressed. Tel: (203) 432-4357. Fax: (203) 432-4360. E-mail: [email protected]. † These authors contributed equally to the work. 10.1021/ac025752l CCC: $22.00 Published on Web 07/20/2002

© 2002 American Chemical Society

Because of its ability to resolve thousands of proteins twodimensionally in a single analytical run, 2-D gel electrophoresis together with mass spectrometry has become a central analytical technology in proteomics today.3 However, it may not have the sensitivity to reveal low-abundance protein species in complex samples.4 It was recently estimated on the basis of codon bias index analysis that more than half of all proteins in the yeast proteome are not detectable by 2-D gel analysis.4 ICAT technology uses affinity chromatography in addition to elution chromatography to specifically enrich biotin/isotope-labeled peptides.5 ICAT labeling can provide quantitative information about differential protein expression by mass spectral assay of d0/d8-labeled cysteinecontaining peptide ratios.5 A similar technique has been introduced for the detection of differential protein phosphorylation.6 Because concentrations of cellular proteins can vary by 5 orders of magnitude,4 labeled peptides derived from low-abundance proteins can easily be missed when these techniques are used given the losses inherent in the use of multiple chromatographic steps. This problem was recently addressed.7 It has been shown, for instance, that phosphorylation may occur in only a small fraction of target protein molecules.8 These modifications may be impossible to (1) Washburn, M. P.; Wolters, D.; Yates, J. R. Nat. Biotechnol. 2001, 19, 242247. (2) Pandey, A.; Mann, M. Nature 2000, 405, 837-846. (3) Rabilloud, T. Proteomics 2002, 2, 3-10. (4) Gygi, S. P.; Corthals, G. L.; Zhang, Y.; Rochon, Y.; Aebersold, R. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 9390-9395. (5) Gygi, S. P.; Rist, B.; Gerber, S. A.; Turecek, F.; Gelb, M. H.; Aebersold, R. Nat. Biotechnol. 1999, 17, 994-999. (6) Goshe, M. B.; Veenstra, T. D.; Panisko, E. A.; Conrads, T. P.; Angell, N. H.; Smith, R. D. Anal. Chem. 2002, 74, 607-616. (7) Gygi, S. P.; Rist, B.; Griffin, T. J.; Eng, J.; Aebersold, R. J. Proteome Res. 2002, 1, 47-54.

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characterize by standard analytical techniques. Various affinity chromatographic techniques can be used in cases where enrichment and characterization of target peptides is desired.9,10 Although immobilized metal affinity chromatography has been employed for concentrating phosphorylated proteins,11,12 in most cases, the specificity of the technique is not satisfactory. Furthermore, detection of low-abundance phosphopeptides is hindered in part because of interference by nonphosphorylated peptides at higher loading levels.8 Affinity techniques using monoclonal antibodies directed against phosphoserine, phosphothreonoine, and phosphotyrosine have been used to successfully detect and characterize certain phosphoproteins.13 However, these antibodies exhibit sequence-limited recognition of phosphorylated residues, and therefore, currently available antibodies fail to recognize phosphorylated residues in some proteins. Review of the current literature1,4 proves that, in proteomics, it is critical to develop more advanced techniques for trace component enrichment and detection and thus to facilitate elucidation of the structure, function, and regulation of cellular proteins. Chromatography plays a key role in proteomics; reversedphase HPLC, sometimes preceded by ion exchange or affinity separation, is routinely used as a sample purification step before mass spectrometry.5 In analytical applications, linear elution has long overshadowed displacement and frontal chromatography, due to its predictability and ease of use for both analytical and preparative applications. Further, this predictability is supported by well-established chromatographic theories.14,15 Nevertheless, linear elution suffers from two major shortcomings. First, by definition, it underutilizes the chromatographic surface in the column, which is therefore easily overloaded, with concomitant loss of efficiency. Second, high-abundance sample components overload the column at concentrations needed to achieve trace component enrichment and this results in untoward bandspreading. This, in turn, makes the low-abundance species hard to detect. In contrast, displacement chromatography relies on high loading levels for efficient operation due to its nonlinear character. However, this mode of chromatography has experienced limited use because of the nonlinear behavior of sample components and relatively long experimental times required for displacement development.

hormone (hGH).17 The authors were able to detect chemically modified trace peptides at levels as low as 0.9% of the unmodified peptide in the hGH digest by using displacement chromatography either on a capillary column17 with on-line fast atom bombardment (FAB) mass spectrometry or on a conventional octadecyl silica column,18 using aqueous cetyltrimethylammonium bromide as the displacer in both cases. Another technique termed “selective” displacement chromatography was shown to achieve selective displacement of more-highly retained proteins by an “induced salt gradient” in ion exchange chromatography.19 The theoretical underpinning of displacement chromatography is well-established, and several thorough reviews have been published.20-24 The capacity of the technique to enrich selectively the concentrations of trace components in mixtures arises from the thermodynamics of the displacement method.25 Unlike elution chromatography, displacement relies on the competition of a displacer with the sample components for binding sites on the chromatographic surface. The displacer is chosen so that it has a higher affinity for the stationary phase than any of the sample components. Because of the high sample loading, the sample components compete with each other for binding sites at the surface of the stationary phase. Under isotachic conditions, all components form adjacent bands in a fully developed displacement train and move with the same velocity, which is determined by the displacer type and concentration. Rather than appearing as peaks, sample components exit the column as a series of adjacent zones as shown in Figure 1B. Each component’s concentration in the zone (Cm) is determined by its affinity for the stationary phase relative to the other components and by the concentration and type of displacer used (Figure 2A). Since the Cm (the concentration in the displacement train) of any component is dictated by the intersection of the operating line (as shown by the dashed line in Figure 2A) with its isotherm, trace components will be concentrated to a level dictated by this intersection (Figure 2B). The concentration of component 3 is very low as illustrated in the hypothetical elution separation of a mixture in Figure 1A. However, because of the intersection of its isotherm with the operating line as shown in Figure 2A, component 3 exits the displacement column as a narrow, concentrated zone as shown in Figure 1B.

DISPLACEMENT CHROMATOGRAPHY Displacement chromatography was introduced by Tiselius,16 and he was the first to point out the potential of displacement for trace enrichment, i.e., to increase the concentration of certain minor sample components. The promise of displacement to enrich low-abundance peptides in a complex mixture was first demonstrated with the tryptic digest of recombinant human growth

ELUTION-MODIFIED DISPLACEMENT CHROMATOGRAPHY (EMDC) The need for high-sensitivity/high-speed analytical methods for proteomics, along with this laboratory’s experience in the pertinent technologies, led us to reexamine the utility of displacement chromatography for trace peptide enrichment in complex

(8) Stensballe, A.; Andersen, S.; Jensen, O. N. Proteomics 2001, 1, 207-222. (9) Lowe, C. R.; Lowe, A. R.; Gupta, G. J. Biochem. Biophys. Methods 2001, 49, 561-574. (10) Hage, D. S. Clin. Chem. 1999, 45, 593-615. (11) Bonn, G. K.; Kalghatgi, K.; Horne, W. C.; Horva´th, C. Chromatographia 1990, 30, 484-488. (12) Luong, C. B. H.; Browner, M. F.; Fletterick, R. J.; Haymore, B. L. J. Chromatogr.-Biomed. Appl. 1992, 584, 77-84. (13) Yan, J. X.; Packer, N. H.; Gooley, A. A.; Williams, K. L. J. Chromatogr., A 1998, 808, 23-41. (14) Snyder, L. R.; Dolan, J. W.; Gant, J. R. J. Chromatogr. 1979, 165, 3-30. (15) Horva´th, C.; Melander, W. J. Chromatogr. Sci. 1977, 15, 393-404. (16) Tiselius, A. Ark. Kemi Miner. Geol. 1943, 16A, 1-11.

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(17) Frenz, J.; Bourell, J.; Hancock, W. S. J. Chromatogr. 1990, 512, 299-314. (18) Frenz, J.; Quan, C. P.; Hancock, W. S.; Bourell, J. J. Chromatogr. 1991, 557, 289-305. (19) Kundu, A.; Barnthouse, K. A.; Cramer, S. M. Biotechnol. Bioeng. 1997, 56, 119-129. (20) Horva´th, C.; Nahum, A.; Frenz, J. H. J. Chromatogr. 1981, 218, 365-393. (21) Helfferich, F. G. J. Chromatogr. 1993, 629, 95-96. (22) Katti, A. M.; Guiochon, G. A. Adv. Chromatogr. 1992, 31, 1-118. (23) Helfferich, F. G.; Klein, G. Multicomponent Chromatography Theory of Interference; Marcel Dekker: New York, 1970. (24) Frenz, J.; Horva´th, C. In High-Performance Liquid Chromatography Advances and Perspectives; Horva´th, C., Ed.; Academic Press: San Diego, 1988; Vol. 5, pp 212-314. (25) Ramsey, R.; Katti, A. M.; Guiochon, G. Anal. Chem. 1990, 62, 25572565.

Figure 1. Schematic illustration of the chromatographic separation of a four-component mixture. (A) Chromatogram obtained by linear elution. (B) Chromatogram obtained by high-performance displacement chromatography. The magnitude of enrichment can be estimated by comparing (A) and (B).

mixtures. We reasoned that a combination of displacement with elution might allow us to take advantage of their respective strengths. Modification of the traditional displacement mode by the inclusion of selected eluent concentrations in the displacer solution should suppress the isotherms of all components26 including that of the displacer itself; this suppression should increase the velocity of the displacement process. Such mixtures have been used, for instance, to displace various melanotropins.27 At the same time, it was essential to maintain under the modified conditions the capacity of the technique to enrich trace components. This concept is expanded in the hypothetical chromatogram in Figure 3 (compare A and B). The time required for peptide displacement is a major obstacle encountered in traditional peptide displacement experiments where reported times were as long as 3-6 h17 (Figure 3A). The time can be reduced by inclusion of eluent with the displacer without significant sacrifice of trace component enrichment. EMDC offers several other advantages illustrated in Figure 3. To simplify analysis of complex mixtures, some components are eluted (components 1-3 in Figure 3B) ahead of the displacement train, which may allow for higher resolution of components in the displacement zone (components 4-6 in this example). A third group of components is retained past the displacement zone and appears mixed with the displacer (Figure 3B, components 8-10 in this example). This allows selection of a group of components for enhancement and parcels the peptide mixture for easier analysis. In conventional displacement chromatography, all components are either displaced or exit the column mixed with the displacer (Figure 3A). Thus, in traditional displacement, the components are displaced in relatively (26) Sabharwal, A. P.; Chase, H. A. Food Bioprod. Process. 1999, 77, 18-26. (27) Viscomi, G.; Lande, S.; Horva´th, C. J. Chromatogr. 1988, 440, 157-164.

Figure 2. Schematic illustration of the relationship between the adsorption isotherms and the zone heights of the components at a fixed operating line in the fully developed displacement train. Isotherms of components 1-4 are shown along with that of the displacer. The slope of the operating line (dashed line) represents the ratio of the stationary-phase concentration of the displacer over its mobilephase concentration. The operating line connects the origin with the point on the displacer isotherm corresponding to its mobile-phase concentration. The intersection of the operating line with the isotherms of the components determines the position and concentration of each component in the displacement train.

narrow bands and typical run times for peptides are long. In EMDC, components exit the column by three mechanisms: elution, displacement, and mixed with the displacer. Run time with the same sample is less than 1 h. In contrast, control over the traditional displacement process is limited to selection of displacer type and concentration for a particular stationary phase. EXPERIMENTAL SECTION Materials and Reagents. All solvents used were HPLC grade. Trifluoroacetic acid (TFA) was from Aldrich (Milwaukee, WI). Benzyldimethyldodecylammonium bromide (BDDAB; >99%) was from Fluka (St. Louis, MO). Recombinant human growth hormone was a gift from Genentech, Inc. (South San Francisco, CA). This protein, although in lyophilized form, had been stored for ∼15 years in the refrigerator. This probably accounts in part for the occurrence of some of the chemically modified minor peptide species observed in our study. Bovine pancreatic trypsin (TPCKtreated), malantide (RTKRSGSVYEPLKI), tri leucine (3L) and R mating factor peptide (YHYLQLKPGQPMY) were obtained from Sigma (St. Louis, MO). Peptide Digest of rhGH. rhGH was dissolved in 0.1 M TRISHCl, pH 8.3, at a protein concentration of 2.5 mg/mL and was Analytical Chemistry, Vol. 74, No. 16, August 15, 2002

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Figure 3. Chromatograms illustrating schematically the separation by traditional (A) and eluent-modified (B) displacement chromatography.

treated with bovine pancreatic trypsin (TPCK-treated to reduce chymotryptic activity) at 37 °C at a molar trypsin/rhGH ratio of 1:100 (w/w) for 2 h essentially as described previously.17 After the initial incubation, additional trypsin was added (1:100) and incubation was continued for 2 h more. The reaction was stopped by addition of 0.1 N HCl until the pH reached 5.0. The hydrolyzate was stored at 2-8 °C and was filtered through a 0.22-µm Millipore Durapore membrane prior to use. Elution Chromatography. All experiments were performed using an HP model 1090 HPLC instrument (Agilent, Palo Alto, CA) equipped with a diode array detector. Data were collected and analyzed using HP Chemstation software (Agilent). In some experiments, on-line fluorescence measurements were made using a model HP 1046A fluorescence detector (Agilent). In all experiments, a 2.1 × 250 mm Supelco Discovery wide-pore octadecyl silica (300 Å) column was used at a flow rate of 0.5 mL/min at 50 °C. The particle size of the stationary phase was 5 µm. In Figure 4A, a simple mixture of oligoleucine (3L) and malantide was made. Five microliters of an aqueous mixture containing 0.1% (v/v) TFA, 5.6 mg/mL 3 L, and 22.4 µg/mL malantide (a 14-amino acid, tyrosine-containing peptide) was loaded onto the column and eluted with a linear 0-50% acetonitrile (ACN) gradient over 10 min at 50 °C. For tryptic peptide mapping, the column was equilibrated with solvent A (0.1% TFA in water). The peptide digest (2.5-12.5 µg) was loaded, and the column was eluted with a linear gradient of ACN containing 0.1% TFA (solvent B). The gradient generated by the HP 1090 system ran from 0 to 50% solvent B in 20 min followed by a regeneration step for 0.5 min at 70% solvent

B and a return to initial conditions. A representative chromatogram is shown in Figure 6A. Peptides were identified by mass spectrometry as described below. Elution-Modified Displacement Chromatography of Peptide Mixtures. EMDC was performed using the 2.1 × 250 mm octadecyl silica column described above. For the simple peptide mixture described in Figure 4B, the column was equilibrated in 5% aqueous ACN with 0.1% (v/v) TFA. The load was 1.4 mg of 3L and 5.6 µg of malantide. The displacer was 10 mg/mL BDDAB containing 10% ACN. For EMDC of the hGH peptide digest, the column was preequilibrated with the starting buffer (see figure captions). One milliliter (2.5 mg) of the hGH tryptic digest was loaded onto the column as described in the figure captions. Immediately after loading, the displacer solution (10 mg/mL BDDAB) containing the desired acetonitrile concentration was pumped into the column at a flow rate of 0.1 mL/min. As a first approximation, the acetonitrile concentration in the displacer mixture was determined by examination of the hGH elution chromatogram along with calculation of the acetonitrile concentration adequate to elute the first components of interest in a typical window. The windows concept is explained in Results and Discussion. A window was found to have a width of ∼2.5 min (see Figure 8). Experiments are currently underway to determine methods to accurately predict a priori the eluent concentration necessary to precisely select a specific window. The effluent of the column was monitored at 260 nm, and in some cases, fluorescence was monitored using an online detector. Excitation and emission wavelengths were set at

Figure 4. Illustration of the EMDC enrichment of malantide, a tyrosine-containing peptide, which is a trace component in a mixture with oligoleucine. (A) Elution chromatography of the 3L/malantide mixture (1, fluorescence at excitation 275 nm, emission 302 nm; 2, absorbance at 214 nm). (B) EMDC of the mixture. Solid line, absorbance at 260 nm; dashed line, fluorescence measured as in (A). 3936

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Figure 5. Schematic of the trace enrichment process by eluent modified displacement chromatography. (A) Selection of windows A-C of interest from the source chromatogram of the multicomponent mixture under consideration. The appropriate displacer strength is chosen as described in the text. (B) EMDC is performed, and fractions (a-c) are collected. (C) The fractions are analyzed to evaluate enrichment of trace components. The peak areas of components 1-9 are measured and compared to their peak areas in source chromatogram obtained prior to EMDC.

295 and 350 nm for measurement of intrinsic tryptophan fluorescence of R mating factor peptide and 275 and 302 nm for intrinsic tyrosine fluorescence of malantide. Fractions (0.5 min each) were collected following the increase in absorbance at 260 nm. Fraction collection was continued throughout the run. Following the

appearance of the displacer front (detected by a large stepwise increase in A260, the column was regenerated over a period of ∼30 min using several cycles of washing with 100% solvent A followed by 95% solvent B. Each wash step was performed at a flow rate of 0.5 mL/min and was continued until a stable baseline at 260 nm Analytical Chemistry, Vol. 74, No. 16, August 15, 2002

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Figure 6. Eluent-modified displacement chromatography of the tryptic hGH digest. (A) Peptide map of hGH; the inset shows the window of interest. (B) EMDC chromatogram (solid line, absorbance at 260 nm; dashed line, tryptophan fluorescence at 295-nm excitation, 350-nm emission). (C-H) Elution analysis of EMDC zones. Individual fractions were analyzed by elution chromatography as shown by the solid lines. Dashed lines represent the corresponding time intervals of the hGH peptide map. The column was preequilibrated with the carrier containing 10% (v/v) ACN.

Mass Spectral Analysis of Fractions. Fractions were diluted 1:100 with 50% (v/v) acetonitrile in water and were analyzed using a MassLynx Q-Tof mass spectrometer (Micromass) by direct injection using gold-tipped microcapillaries. Selected components from the spectra were chosen for amino acid sequence analysis by collision-induced dissociation mass spectrometry (CID-MS). To identify individual peptides unambiguously, some of the fractions were rechromatographed by preparative reversed-phase gradient elution chromatography. Individual peptides were sequenced by CID-MS.

Figure 7. Histogramatic comparison of peak areas of hGH peptides in the original peptide map before enrichment (open bars) to the ratios of the same peptides after EMDC enrichment to their original concentrations (hatched bars). Asterisks denote minor peptides enriched by EMDC.

was obtained. Aliquots of the fractions (5 µL) were chromatographed by reversed-phase HPLC as described above and subsequently subjected to ESI-MS analysis. 3938

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RESULTS AND DISCUSSION Demonstration of Trace Enrichment in a Simple Model System. As mentioned in the introduction, displacement chromatography can be used to enrich the concentration of trace components under appropriate conditions. In an experiment designed to demonstrate such trace component enrichment, we used a mixture containing two peptides: On a weight basis, 250 parts of 3L (FW 357.5) was mixed with one part of malantide (FW 1633.9). Figure 4A shows a chromatogram that illustrates the relative elution positions of malantide and the 3L peptide upon gradient elution. The 2.1 × 250 mm octadecyl silica column had been preequilibrated with aqueous 0.1% (v/v) TFA and was eluted

Figure 8. Typical HPLC chromatograms of the fractions from the displaced zones obtained at various concentrations of ACN in the displacer and carrier. Fractions A-F were collected at the following time points (in minutes) during the displacement runs: (A) 46.3; (B) 47 (solid line), 48 (dotted line); (C) 37.2; (D) 32.5; (E) 23; (F) 25.8. Dashed traces in (A-F) represent corresponding time intervals in the hGH peptide map. These results are equivalent to the chromatograms shown in Figure 5C.

with an ACN gradient at 50 °C. Malantide is a trace component and elutes with a retention time of 8.15 min, as shown by arrows on the chromatogram. Trace 2 was obtained by measuring absorbance at 214 nm whereas trace 1 was obtained by monitoring the fluorescence of the column effluent with excitation and emission wavelengths set at 275 and 302 nm, respectively. In a displacement experiment, 0.25 mL of the mixture containing 1.4 mg of 3L and 5.6 µg of malantide in aqueous 0.1% (v/v) TFA was injected into the column. The mixture was displaced with aqueous 10% (w/v) BDDAB containing 10% (v/v) ACN. By monitoring the malantide’s intrinsic fluorescence due to the tyrosine residue, its behavior during displacement could be monitored directly by online fluorescence (Figure 4B). The fluorescence peak was used to establish the position of malantide in the displacement train. From the data shown in Figure 4, a 305-fold enrichment of malantide was calculated, by dividing the fluorescent peak intensity from malantide in displacement (Figure 4B) by that obtained in elution (Figure 4A). This illustrates that displacement chromatography can be used for selective trace enrichment. The Windows Concept. In the previous section, it was shown that EMDC allows displacement of selected components so that enrichment of selected low-abundance species in a peptide map becomes feasible. For samples containing many components, it is appropriate to divide the components from an elution chromatogram into windows, depicted in Figure 5A, which illustrates windows (A-C) in a typical peptide map to be subjected to EMDC (Figure 5B). Selection of the eluent-modified displacer is based on the eluent strength of the mobile phase required for elution of the desired component(s) in the linear elution chromatogram. Accordingly, later-eluting components in the reversed-phase separation require

higher eluent/displacer ratios in EMDC in order to be displaced and thus enriched. In this hypothetical example, fractions (a-c) were collected from the displacement zones in I-III (Figure 5B). Fractions from 5B are analyzed by elution chromatography as shown in Figure 5C: components 1-3 are detected in fractions from chromatogram I, components 4-6 from chromatogram II, and 7-9 from chromatogram III, respectively. Enrichment of minor components (especially 4, 5, and 7) is shown in Figure 5C. Separation of components in the window of interest is enhanced in part because of the relatively wide profile of the displaced zones of major components. This highlights another area where EMDC differs from (and improves upon) conventional displacement chromatography. In conventional displacement, component zones near the displacer front become very concentrated, narrow, and therefore often poorly resolved.17 In EMDC, this problem is eliminated by raising the eluent concentration to a level at which components of interest lie in a window that is optimized for resolution/displacement/enrichment. Another advantage is that relatively hydrophobic components can easily be displaced from the column by appropriate adjustment of the ACN concentration in the displacer. Since the isotherms are suppressed under these conditions, there is a tradeoff between maximal enrichment and the ability to optimally resolve and displace desired sample components. The speed and specificity of displacement in EMDC is controlled by adjustment of the eluent/displacer mixture, which makes the screening of an entire peptide map possible. EMDC of the hGH Tryptic Digest. The tryptic digest of hGH should contain 20 major peptides. Numerous peptide maps using reversed-phase HPLC have appeared in the literature28 along with those in the displacement studies cited above.17 Figure 6A shows Analytical Chemistry, Vol. 74, No. 16, August 15, 2002

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the hGH peptide map; the chromatogram contained all of the major peptides previously identified in the hGH tryptic map.18 Minor peaks, which represent modified forms of the major peptides, were also present in the chromatogram; for instance, chymotryptic fragments arising from low-level contaminants in the trypsin preparation proper, autolysis products of the proteolytic enzymes, and degradation products arising from aging of the hGH as mentioned above. We analyzed the components in one of the windows in the hGH map using EMDC in the following way. A 2.5-mg sample of the hGH tryptic digest was loaded onto an octadecyl silica column along with a small amount (2.5 µg) of R mating factor, which was used as a tryptophan-containing “marker” peptide. The displacer was an aqueous solution containing 20% (v/v) acetonitrile and 10 mg/mL BDDAB. The acetonitrile concentration in the displacer was chosen as described in the Experimental Section. Figure 6B shows the chromatogram thus obtained. The effluent was monitored at 260 nm and for tryptophan fluorescence. Fractions were collected at 0.5-min intervals starting 17 min after completion of the feed. Under the mobile-phase conditions chosen, several peptides were isocratically eluted in the initial part of the chromatogram. After this elution zone, the chromatogram exhibited a typical of displacement pattern. The last major increase in absorbance occurred in this case at 45 min due to the appearance of the displacer front. A single sharp zone of fluorescence was observed at ∼40 min (dashed trace), representing the displacement of R mating factor. A calculation dividing the maximum fluorescence intensity in the latter zone with that of the R mating factor peptide in the peptide map (Figure 6A, data not shown) indicated a 400-fold enrichment of this peptide by the displacement process. After regeneration of the column, fractions of displaced peptides were analyzed by HPLC as described above. Chromatograms of several fractions are shown in Figure 6C-H. In each panel, the relevant portion of the chromatogram of the EMDC fraction is shown by solid traces and compared with a gradient elution chromatogram of the same portion of the hGH tryptic map as shown by dashed lines. Components in this window (peptides 1-10 eluting between 12.5 and 15.9 min) are highlighted in the inset of Figure 6A. Peptides 1-10 all appear in the fractions analyzed in panels C-H in Figure 6, and we noted that the displacement order for individual peptides often differed from that in elution chromatography (components 1-5), likely indicating crossing of the components’ isotherms. Selective enrichment of minor components 1, 2, 4, 7, 8, and 10 is seen along with components 3, 5, and 6, which were enriched to a smaller degree. Component 9, identified as the T4 peptide, was actually less concentrated in the fraction selected (panel H) than in the original peptide map while minor components 7 and 10 gave enhanced responses. Components 1-10 were identified by ESI-MS and sequenced by MS/MS analysis; the results are shown in Table 1. Among the peptides identified were tryptic fragments of hGH including T1, T10c2, T11, and T4, which have been reported in the literature.17 Other components represent various chymotrypticlike cleavages including component 1, derived from T16; component 4, a combination of T3 and part of T4; and component 7, a (28) Hancock, W. S.; Bishop, C. A.; Prestidge, R. L.; Hearn, M. T. W. Anal. Biochem. 1978, 89, 203-212.

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Table 1. Peptides from the Fractions in Figure 6 Identified by ESI-MS MW

residues

1 742.5 160-165 2 1078.32 1-9 3 1566.48 1-13 3a,b 2403.89 1-20 4 4a,b 5 6 7a 8 9 10c c

sequence

NYGLLY MFPTIPLSR (Met-ox) MFPTIPLSRLFDN (Met-ox) MFPTIPLSRLFDNAMLRAHR (Met-ox) 1471.74 18-29 AHRLHQLAFDTY 1543.81 7-19 LSRLFDNAMLRAH 1062.32 1-9 MFPTIPLSR 1743.9 101-116 SLVYGASDSNVYDLLK 1930.13 99-116 ANSLVYGASDSNVYDLLK 1361.67 117-128 DLEEGIQTLMGR 2342.13 21-39 LHQLAFDTYQEFEEAYIPK 1684.0 WHWLQLKPGQPMY

name part of T16 T1 (Met-ox)

T1 T10c2 T10c2 + AN T11 T4 AMF

a Without MS/MS verification. b Shoulder peak resolved by EMDC. AMF, R mating factor.

T10-derived peptide. Two extended variations of the T1 peptide containing oxidized N-terminal methionine residues (components 2 and 3) were also detected. Component 10 was identified as R mating factor, which was added to the sample to serve as a marker. Figure 7 compares the areas of major and minor components using the data from the experiment presented in Figure 6. In the histogram, open bars represent the area measurements under the peaks of components 1-10 shown in Figure 6A. To determine the relative areas of each of these components, we calculated from data in this experiment the ratio of the area under each peak after EMDC to its original area in the elution map. The hatched bars represent the latter calculations and show the broad potential of EMDC to selectively enrich minor components. For peptides 7 and 8, the ratios were 25- and 20-fold, respectively. As expected, major components were also enriched, but not to the same extent as minor ones; for instance, component 9 (T4) was enriched 9-fold in the fraction analyzed. Using the current data, we were unable to determine the absolute enrichment factors for the unlabeled hGH peptides because the fraction volume (0.05 mL) led to dilution of the narrow zones containing enriched peptides. In the case of R mating factor (peptide 10), the area ratio in the collected fraction was only ∼15-fold; in contrast, the enrichment factor calculated from direct peak fluorescence measurement in the displacement zone was 400-fold. The latter value reflects the true enrichment factor without dilution. Experiments are underway to directly couple EMDC with mass spectrometry so that enrichment of trace components can be measured on-line. We further examined window selection in the hGH map by performing EMDC experiments with various acetonitrile/displacer mixtures containing between 7 and 35% ACN. The hGH digest was loaded as in Figure 6 in each experiment, and the results are summarized in Figure 8. The displacer (BDDAB) concentration in these experiments was 10 mg/mL in all cases; the acetonitrile concentration was varied as shown. The time interval for windows selected using various acetonitrile concentrations is shown in Figure 9 along with an hGH elution chromatogram for reference. This diagram illustrates the overlapping nature of the windows and suggests that enhancement differences with respect to individual components may occur depending upon the window chosen. Because of the nature of the displacement process,

Figure 9. hGH tryptic peptide map, showing the positions of different windows created in EMDC by mixing different amounts of ACN with the displacer. The amounts of ACN (as percent (v/v)) added were as follows: 1, 7%; 2, 10%; 3, 15%; 4, 20%; 5, 25%; and 6, 35%.

peptides displaced closest to the displacer front should experience the highest enrichment. Therefore, in order to achieve maximal trace enrichment, the ACN concentration in the displacer solution should be minimized, while still allowing for high resolution. In EMDC, some sacrifice of either enrichment or resolution may be made, depending on the eluent concentration chosen. Figure 9 shows that windows have a width of 2-3 min. Reversed-phase gradient HPLC (Figure 8) analysis of fraction(s) from each of the EMDC windows illustrated in Figure 8 is shown along with an elution chromatogram (dashed line plots) representing the hGH tryptic map. These results demonstrate that EMDC can be used to selectively enrich peptides of widely differing retention times in the hGH tryptic map. They also show the technique’s capacity to expand the resolution of the peptide separation beyond that attainable by elution chromatography. Specifically, Figure 8B (10% ACN) shows EMDC’s resolving power along with the enrichment of two peptides that were relatively poorly resolved in the elution map but were completely resolved in two fractions with retention times between 12.0 and 12.3 min. The earlier of the two peptides (solid trace) was both enhanced in concentration and completely separated from the other peptide (dotted line), which was similarly enhanced. CONCLUSION In comparison to affinity chromatographic techniques, EMDC is a versatile and generally applicable analytical method that makes possible the displacement and enrichment of a wide range of lowabundance species in a complex mixture. This has been demon(29) Shukla, A. A.; Sunasara, K. M.; Rupp, R. G.; Cramer, S. M. Biotechnol. Bioeng. 2000, 68, 672-680. (30) Kundu, A.; Cramer, S. M. Anal. Biochem. 1997, 248, 111-116.

strated in our experiments using the tryptic digest of human growth hormone. Since enrichment and high-resolution separation occur simultaneously in a single EMDC run, the speed of analysis can be much faster than that of conventional displacement techniques. The purity and relatively high concentration of the trace components is a great advantage in mass spectral analysis because simpler mixtures will be presented to the mass spectrometer from the EMDC separation. This will facilitate data interpretation. Experiments are underway with more complex samples such as those encountered in proteomics studies to examine further the utility of EMDC. Because of the flexibility of the technique, a single area or all areas in a peptide map can be readily probed. For instance, this could make detailed analysis of phosphorylation patterns on proteins easier, since important phosphopeptides could be directly targeted for enrichment after identification by radiolabeling. Although multidimensional elution methods allow greater sample concentrations and separation by introduction of highcapacity elution chromatographic steps,7 EMDC may offer an attractive alternative for enrichment of trace-level isotope-labeled peptides (e.g., ICAT) from complex mixtures, because of its ability to selectively enrich the trace components of interest and to increase resolution of those components. Ideally, of course, multiple EMDC separations may be run in parallel, thus allowing high-throughput analysis of complex mixtures. Various chromatographic methods could also be operated in the EMDC mode in a fashion analogous to that described in the current study. It should be possible to use EMDC for trace protein enrichment in other modes29,30 such as ion exchange or hydrophobic interaction chromatography and potentially as an alternative to gel-based separation methods. EMDC should be applicable to a wide variety of situations where trace enrichment is required. These include the analysis of trace components in samples of pharmaceuticals of small or large molecular mass, environmental samples containing multiple trace components, and samples from drug metabolite studies in which low-level metabolites must be identified and analyzed for toxicity monitoring and for regulatory filings. ACKNOWLEDGMENT The authors thank Kathy Stone and Walt McMurray of the W. M. Keck Foundation Biotechnology Resource Laboratory, Yale University School of Medicine, for performing the mass spectral analysis of samples presented in the paper and for help in interpretation of the results and Supelco for donation of the Discovery column used in this work. This work was supported by Grant GM 20993 from the National Institutes of Health, U.S. Department of Health and Human Services. Received for review May 6, 2002. Accepted July 3, 2002. AC025752L

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