Anal. Chem. 2003, 75, 1819-1827
Elution-Modified Displacement Chromatography Coupled with Electrospray Ionization-MS: On-Line Detection of Trace Peptides at Low-Femtomole Level in Peptide Digests Rong Xiang, Csaba Horva´th, and James A. Wilkins*
Department of Chemical Engineering, Yale University, New Haven, Connecticut 06520-8286
Elution-modified displacement chromatography (EMDC) was employed to achieve peptide separations with high efficiency. On-line ESI-MS and ESI-MS/MS measurements showed enrichment and detection of kemptide, a protein kinase A peptide substrate, at low femtomole levels when it was added as a trace marker component to a tryptic digest of bovine serum proteins or to a human growth hormone peptide digest at concentration ratios between 1:105 and 1:106. In another EMDC separation, five peptides were detected in a mixture containing 20 fmol of human growth hormone tryptic digest mixed with the bovine serum protein digest. We found that EMDC facilitated rapid detection and sequence analysis of trace peptides at levels of ∼0.5 fmol/µL in complex peptide mixtures with a wide dynamic concentration range. Accordingly, the detection of kemptide by EMDC was found to be 3-4 orders of magnitude more sensitive than that attained in conventional linear elution chromatography separations performed with the same peptide loads. Kemptide was phosphorylated in vitro and was detected along with its neutral loss product in peptide mixtures at low femtomole levels. EMDC enabled both detection and amino acid sequence determination on trace levels of phosphorylated and other posttranslationally modified peptides, suggesting that the technique may be useful for proteomics applications where detection and analysis of trace level peptides are problematic. Posttranslational protein modifications are fundamentally important in the control of cellular functions. For instance, phosphorylation by specific protein kinases represents an important control mechanism in a variety of cellular responses to stimuli, both chemical and physical. Changes in cellular posttranslational modification patterns have been found in numerous studies comparing protein phosphorylation patterns evaluated by 2-dimensional gel electrophoresis in diseased versus normal states.1,2 Moreover, the detection and precise chemical characterization of these modifications is of central importance to a more complete understanding of cellular behavior. However, the sensitivity and * To whom all correspondence should be addressed. Tel: (203) 432-4373. Fax: (203) 432-4360. Email:
[email protected]. (1) Hunter, T. Cell 2000, 100, 113-127. (2) Haskell, M. D.; Slack, J. K.; Parsons, J. T.; Parsons, S. J. Chem. Rev. 2001, 101, 2425-2440. 10.1021/ac026232t CCC: $25.00 Published on Web 03/20/2003
© 2003 American Chemical Society
selectivity of many current technologies are inadequate primarily because of the wide concentration range of proteins encountered in cell extracts. Therefore, much attention has recently been focused on the development of analytical techniques to enhance the detection and characterization of phosphorylation sites on protein molecules.3-5 Such studies are difficult as a result of several factors: 1. Phosphorylations often occur on a relatively small percentage of any given protein in the cell in response to a particular stimulus. 2. There is often more than a single phosphorylation site per protein molecule. 3. The amounts of phosphoproteins themselves are low. 4. Phosphoproteins and phosphopeptides are often present as a small percentage of total peptide content in complex mixtures and are often detected by labeling cells with AT32P.6 In addition to 2-dimensional gel electrophoresis and single or multidimensional elution chromatographic schemes, several methods have been developed recently to address the detection problem posed by the presence of these low-abundance species in complex mixtures encountered in cellular extracts or in column fractions.4,7 All based on affinity chromatography, these methods fall into three general categories. In one set of methods, phosphopeptides are labeled specifically by chemical modification followed by attachment of affinity tags.4,5 This is followed by affinity isolation using group-specific adsorbents. These methods are rather time-consuming and also may not allow detection of all phosphopeptides. In the second group, immobilized metal affinity chromatography (IMAC) using either iron or gallium is employed.3,8 Although these methods in some cases offer a faster and more direct approach, their specificity is open to question.9 Recently, a promising modification was introduced to the IMAC technique to reduce nonspecific binding by conversion of peptides to their methyl ester derivatives.3 In the third set of methods, antibodies specific for phosphoserine, -threonine, and -tyrosine (3) Ficarro, S. B.; McCleland, M. L.; Stukenberg, P. T.; Burke, D. J.; Ross, M. M.; Shabanowitz, J.; Hunt, D. F.; White, F. M. Nat. Biotechnol. 2002, 20, 301-305. (4) Oda, Y.; Nagasu, T.; Chait, B. T. Nat. Biotechnol. 2001, 19, 379-382. (5) Zhou, H. L.; Watts, J. D.; Aebersold, R. Nat. Biotechnol. 2001, 19, 375378. (6) Haystead, T.; Garrison, J. In Protein phosphorylation: a practical approach; Hardie, D., Ed.; Oxford University Press: Oxford, 1999; pp 1-31. (7) Goshe, M. B.; Conrads, T. P.; Panisko, E. A.; Angell, N. H.; Veenstra, T. D.; Smith, R. D. Anal. Chem. 2001, 73, 2578-2586. (8) Posewitz, M. C.; Tempst, P. Anal. Chem. 1999, 71, 2883-2892. (9) McLachlin, D. T.; Chait, B. T. Curr. Opin. Chem. Biol. 2001, 5, 591-602.
Analytical Chemistry, Vol. 75, No. 8, April 15, 2003 1819
Figure 1. Schematic diagram of HPLC coupled to ESI-MS for on-line EMDC. Flow is directed from the HPLC pump through a splitter where ∼90% is directed to waste. The remaining flow goes through the injection valve and into a divert valve where another sample loop (500-µL volume) can be put on-line for EMDC. Flow then travels through the column (0.32 × 150 mm, packed with 5-µm, 300-Å octadecyl silica) and into the ESI interface through a connecting capillary.
are used as specific adsorbents for peptides containing these modified amino acids.10 The latter methods suffer from the antibodies’ differential ability to recognize phosphorylated amino acids in all possible amino acid sequence motifs. We previously showed enrichment of trace peptides in a tryptic peptide digest of human growth hormone using elution modified displacement chromatography, a hybrid technique combining elution and displacement chromatography.11 According to theory, it should be possible to enhance the concentration of most lowabundance peptides using displacement chromatography, a nonlinear chromatographic technique that relies on high loading levels and competition between components for the stationary phase.12 The principal advantages of EMDC over traditional displacement chromatography are that specific areas of a peptide map can be “targeted” for enhancement and that separations can be done in the same general time frame as traditional linear elution separations. The latter advantage thus allows EMDC to be directly compared to linear elution chromatography in terms of its ease of operation and sample throughput.11 In the current study, we demonstrate that EMDC can be scaled down to a level compatible with small sample amounts typically encountered in proteomics experiments and is amenable to direct coupling with electrospray
ionization mass spectrometry. To explore the usefulness of this approach in the broad area of proteomics technologies, we have coupled EMDC with electrospray ionization mass spectrometry (ESI-MS). This configuration allows for direct identification and sequence analysis of low-abundance peptides in protein digests examined. In contradistinction to other enrichment techniques, no pretreatment of samples is necessary, since EMDC relies only on relative stationary phase affinities of the peptides in order to achieve selective enrichment of low-abundance components.11,13 In this report, low femtomole level trace peptides including phosphopeptides were enriched from complex peptide mixtures including a tryptic digest of bovine serum proteins by EMDC. Peptides were detected and sequenced on-line by coupled ESIion trap MSn in a single chromatographic step using both conventional electrospray and nanospray interfaces. In the current study, EMDC is shown to provide a new, more sensitive approach for the enrichment and rapid high-resolution separation of lowabundance components.
(10) Kalo, M. S.; Pasquale, E. B. Biochemistry 1999, 38, 14396-14408. (11) Wilkins, J. A.; Xiang, R.; Horvath, C. Anal. Chem. 2002, 74, 3933-3941. (12) Tiselius, A. Ark. Kemi Mineral Geol. 1943, 16A, 1-11.
(13) Frenz, J.; Horvath, C. In High-Performance Liquid Chromatography Advances and Perspectives; Horvath, C., Ed.; Academic Press: San Diego, 1988; Vol. 5, pp 212-314.
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Analytical Chemistry, Vol. 75, No. 8, April 15, 2003
EXPERIMENTAL SECTION Materials and Reagents. All solvents used were HPLC grade. Trifluoroacetic acid (TFA) was from Aldrich (Milwaukee, WI).
Figure 2. Peptide map of rhGH spiked with kemptide and phosphokemptide. The rhGH tryptic digest (0.25 mg/mL) was mixed with kemptide and phosphokemptide, each at a final concentration of 4 µg/mL. After loading, the column was eluted with a linear gradient (0-50%) of acetonitrile over 20 min. Panel A: total ion chromatogram for the run. Numbered peaks are the kemptides and major rhGH tryptic peptides, which are identified in Table 1. Panel B: selected ion chromatogram for p-kemptide. Panel C: selected ion chromatogram for kemptide. Panel D: total ion scan at 13.2 min showing the singly (m/z 852.43) and doubly charged (m/z 426.82) ions for p-kemptide along with their neutral loss products. Panel E: MS/MS spectrum for kemptide. Panel F: MS/MS spectrum for p-kemptide.
Benzyldimethyldodecylammonium bromide (>99%) was from Fluka (St. Louis, MO). Recombinant human growth hormone (rhGH) was a gift from Genentech, Inc. (So. San Francisco, CA). This protein, although in lyophilized form, had been stored for ∼15 years in the refrigerator. This accounts for the occurrence of some of the chemically modified minor peptide species observed in our study. Bovine pancreatic trypsin (TPCK-treated), bovine serum (lyophilized), protein kinase A, ATP, cyclic AMP, dithiothreitol, iodoacetamide, and kemptide (LRRASLG) were obtained from Sigma (St. Louis, MO). Peptide Digest of rhGH. As described previously,11,14 rhGH was digested 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. Peptide Digest of Bovine Serum Proteins. Lyophilized bovine serum was reconstituted to its original volume (1 mL) with water. The sample was diluted 1:10 with digestion buffer (0.1 M NH4HCO3, pH 7.8) to a protein concentration of ∼6 mg/mL. The sample was reduced by incubation at 37 °C for 1 h following the addition of dithiothreitol to a final concentration of 10 mM. After reduction, iodoacetamide was added to a final concentration of 50 mM to alkylate all available free thiols, and the sample was incubated for 1 h at 37 °C. Bovine pancreatic trypsin was then added at a ratio of 1:50 (w/w) to serum protein (final trypsin concentration 0.12 mg/mL), and the sample was incubated at 37
°C for 2 h to hydrolyze serum proteins. After verification of hydrolysis by reversed-phase HPLC (data not shown), buffer components were removed, and the serum peptides were exchanged into 95% aqueous acetonitrile containing 0.1% TFA on a 1-mL solid-phase extraction cartridge following the manufacturer’s instructions for solvent volumes (Oasis HLB, Waters Corp., Medford, MA). Briefly, the column was equilibrated with 1 mL of 0.1% trifluoroacetic acid (TFA) in water. The peptide sample (0.6 mg) was loaded onto the column followed by 1 mL of the same buffer. Peptides were eluted with 1 mL of aqueous 95% acetonitrile containing 0.1% TFA. This sample was vacuum-dried in a Speed Vac (Savant Instruments, Holbrook, NY), and peptides were suspended in 0.02% TFA in water in their original volume. Recovery of peptides was quantitative as measured by reversedphase HPLC (data not shown). Phosphorylation of Kemptide. Kemptide, a model protein kinase A substrate,15 was phosphorylated using cyclic AMPdependent protein kinase according to a procedure outlined for malantide.16 Elution chromatography and EMDC. Experiments described in Figures 2-6 were performed using the instrument that (14) Frenz, J.; Bourell, J.; Hancock, W. S. J. Chromatogr. 1990, 512, 299-314. (15) Denis, C. L.; Kemp, B. E.; Zoller, M. J. J. Biol. Chem. 1991, 266, 1793217935. (16) Malencik, D. A.; Anderson, S. R. Anal. Biochem. 1983, 132, 34-40.
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Figure 3. Overloaded elution chromatography of the rhGH digest containing a low concentration of kemptide. rhGH digest (0.25 mg/ mL) was mixed with a low concentration of kemptide (final concentration, 0.4 µg/mL). Panel A: total ion chromatogram. Panel B: ion chromatogram for kemptide at m/z 772.6. Panel C: ion chromatogram at m/z 769.5 to demonstrate background noise in this region.
is schematically illustrated in Figure 1. The Surveyor HPLC pump and autosampler/oven are products of ThermoFinnigan (San Jose, CA). The output of the HPLC pump was split (1:80) in order to attain the desired flow rate for elution or EMDC. Flow was directed from injection valve 1 to a second valve (injection valve 2), where a sample loop (500 µL) was installed for use in EMDC (see below). Flow was then directed to the column whose outlet was linked via a fused silica capillary directly to the electrospray ionization (ESI) interface of a ThermoFinnigan LCQ ion trap mass spectrometer for on-line analysis of the column effluent. Instrument control, data collection, and analysis were performed using Excalibur software from ThermoFinnigan. For experiments using the ESI interface (Figures 2-6), a 0.32 × 150-mm Supelco Discovery wide-pore octadecyl silica (300 Å, 5 µm particle size) column was used at a flow rate of 5 µL/min at 50 °C. For tryptic peptide mapping, the column was equilibrated with solvent A (0.02% TFA in water). The peptide digest (0.25 µg) was loaded along with the two marker peptides kemptide and phosphokemptide (e.g., Figure 2, and the column was eluted with a linear gradient of acetonitrile (ACN) containing 0.02% TFA (solvent B). The gradient generated by the Surveyor 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. EMDC in Figures 2-6 was performed using the 0.32 × 150mm octadecyl silica column described above. In the current experiments, the column was equilibrated in 3% aqueous ACN with 0.02% (v/v) TFA. The injection valve positions for EMDC are illustrated in Figure 1B. Using the valve arrangement shown, 25 µg of the rhGH tryptic digest containing various amounts of a trace peptide was loaded into the sample loop of injection valve 1 (position 1) at a flow rate of 2.5 µL/min. Injection valve 1 was then switched to the inject mode (position 2, Figure 1B) by the autosampler. After 5 min, the flow rate was reduced to 1.25 µL/ min, and injection valve 2 was manually switched to position 3 in 1822 Analytical Chemistry, Vol. 75, No. 8, April 15, 2003
Figure 4. EMDC of kemptide/rhGH mixture at a 1:300 ratio. The column was equilibrated with aqueous 3% ACN containing 0.02% TFA at 50 °C. The column was operated as described in the Experimental Section. Panel A: total ion chromatogram. Panels B, C, D, E, and G: selected ion chromatograms for rhGH peptides. Panel F: selected ion chromatogram for kemptide (m/z 772.6); zone displaced at 34.77 min. Ion current measured at ∼9 min in the chromatogram represents interference from T12 (m/z 773.4) shown in panel B. Panel H: selected ion chromatogram for the displacer BDDAB showing the position of the displacement front (m/z 304.4).
order to put its sample loop, which contained the displacer/ acetonitrile mixture, in-line. The displacer in these experiments was 10 mg/mL benzyldimethyldodecylammonium bromide (BDDAB) containing 5% ACN. The column was regenerated offline by performing three gradient elutions at 80 °C at a flow rate of 5 µL/min as follows. The column was equilibrated at 30% solvent B for 15 min followed by a 30-90% solvent B gradient over 15 min. It was then held at 90% solvent B for another 15 min. ESI-MS Analysis. The column effluent was analyzed on-line with a ThermoFinnigan LCQ ion trap instrument operated in the positive ion mode using a steel-sheathed fused-silica capillary as the electrospray tip operated at a voltage of ∼5 kV and capillary temperature of 300 °C. The sheath gas flow was set up at 35-50 units for elution chromatography and 95 units for EMDC. To “catch” narrow zones produced in EMDC, the microscan rate was changed in some cases to a value of 1. MS/MS data were collected in the data-dependent mode with parameters set at 772.6 Da for the singly charged ion of the kemptide molecule and 427.3 Da for the doubly charged phosphokemptide molecule. The collision energy was set at 35%. Nanospray-MS Analysis. For the EMDC experiments described in Figures 7 and 8, we used a capillary column with an
Figure 5. Selected ion scans and an MS/MS spectrum from EMDC separations of kemptide/rhGH tryptic digest mixtures. Kemptide was mixed with the rhGH tryptic digest (2.5 mg/mL) at the indicated ratios, and the mixtures were separated by EMDC as described in Figure 4. The weight ratios and times for maximum ion current at m/z 772.6 are given for each separation along with the total mass of kemptide loaded. Panel A: kemptide/rhGH, 1:30 000; time, 41.67 min.; 50 fmol loaded. Panel B: kemptide/rhGH, 1:300 000; time, 36.83 min.; 5 fmol loaded. Panel C: isotope profile of kemptide ions obtained in the 50-fmol run. Panel D: MS/MS spectrum of kemptide from the 5-fmol run.
integral spray tip (Picofrit, 8-µm tip, New Objective, Woburn, MA). The column was 75-µm i.d. and 15 cm in length and was packed with the same stationary phase described for the 320-µm-diameter column above. Flow from the Surveyor pump was split prior to entering the “divert/inject” valve (fitted with a 5-µL sample loop) on the LCQ Deca instrument in order to achieve the desired flow rate of 300 nL/min. The latter valve was used to supply both sample and displacer solution to the column. Flow was directed to the column, which was mounted in the electrospray interface supplied by Thermofinnigan. For EMDC, the column was operated at a flow rate of ∼150 nL/min; the displacer concentration was 10 mg/mL, and the acetonitrile concentrations were 5% in Figures 7 and 10% in Figure 8. Spray voltage (1.5 kV) was applied to the inlet of the capillary column. The heated capillary temperature was set at 300 °C. RESULTS AND DISCUSSION In a series of elution experiments, we mixed the rhGH tryptic digest (0.25 mg/mL) with kemptide (LRRASLG) and its phosphorylated derivative (LRRApSLG; p-kemptide) at final concentrations of 4 µg/mL each (a 1:3 wt/wt ratio of the kemptides to rhGH peptides) and loaded 1 µL of the mixture (containing 5 pmol of kemptide and 25 ng of rhGH digest) onto the C18 column. Kemptide was phosphorylated in vitro as described in the Experimental Section. Panel A in Figure 2 shows a total ion chromatogram from a linear elution chromatography experiment performed as described in the Experimental Section. Twelve major peaks were detected in the chromatogram. Along with the
Table 1. Major Ions in Elution Chromatogram of Figure 2, Panel A no.
name
sequence
[M + H]+
1 2 3 4 5 6 7 8 9 10 11 12
phosphokemptide kemptide T13 T20-21 T15 T19 T18-19 T17-18-19 T8 T2 T1 T11
LRRApSLG LRRASLG TGQIFK IVQCRSVEGSCGF FDTNSHNDDALLK VETFLR DMDKVETFLR KDMDKVETFLR SNLELLR LFDNAMLR MFPTIPLSR DLEEGIQTLMGR
852.47 772.59 693.40 1401.66 1489.65 764.43 1253.50 1381.66 844.51 979.52 1061.59 1361.65
p-kemptide and kemptide (peaks 1 and 2 respectively), the identities of the rhGH peptides (peaks 3-12) are shown in Table 1. Selected ion chromatograms for p-kemptide (m/z 852.5), eluting at 13.10 min; and kemptide (m/z 772.6), eluting at 13.28 min, are shown in panels B and C, respectively. Panel D shows a total ion scan at 13.10 min. Both the singly (852.43 Da) and doubly (426.82 Da) charged p-kemptide ions as well as the neutral loss products arising from loss of H3PO4 from each of these charged species at m/z 754.49 and 377.93, respectively, are seen. Panels E and F in Figure 2 show data-dependent MS/MS analysis obtained from the collision-induced dissociation (CID) of kemptide and p-kemptide, respectively. These spectra show fragments expected from these two peptides, including (in the case of p-kemptide) fragments with dissociated HPO3 groups. The fragmentation patterns of the Analytical Chemistry, Vol. 75, No. 8, April 15, 2003
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Figure 6. EMDC on a phosphokemptide/rhGH tryptic digest mixture containing 40 fmol of phosphokemptide. Phosphokemptide was mixed with rhGH tryptic digest at a ratio of 1:37 500, and the mixture was separated by EMDC as described in Figure 4. Panel A: selected ion chromatogram for the doubly charged p-kemptide ion at m/z 427.26. Panel B: selected scan from the LCQ showing detection of the doubly charged ion at m/z 427.26, along with its neutral loss product [M - HPO3 - H2O + 2H]2+. Panel C: MS/MS on the doubly charged p-kemptide ion at m/z 427.26.
Figure 7. Detection and sequence analysis of kemptide from a mixture with bovine serum peptides. A 10-fmol portion of kemptide was mixed with 1.5 µg of serum peptides, and the mixture was separated by EMDC on a 75-µm reversed-phase column. Panel A: ion scan showing detection of the kemptide ion at m/z 772.1. Panel B: MS/MS analysis of kemptide obtained during the run.
kemptides, which contained a number of N- and C-terminal fragments as well as some internal fragments, were useful in evaluating data obtained from EMDC runs described below. Overloaded Elution Chromatography of Kemptide/rhGH Mixture. In the next series of experiments, kemptide was mixed at a 1:30 wt ratio (10-fold lower than that used in Figure 2) with the rhGH peptide digest (0.4 µg/mL kemptide + 0.25 mg/mL rhGH digest). Initially, elution chromatography was performed by loading 1 µL of the mixture. Kemptide could not be detected by ESI-MS in the experiment because of ion suppression by coeluting peptides (data not shown). The effect of ion suppression in elution chromatography, which has been reported by many 1824 Analytical Chemistry, Vol. 75, No. 8, April 15, 2003
authors, became quite obvious at this concentration ratio.17,18 We attempted to detect kemptide in the 1:30 peptide mixture by loading 10-fold more (10 µL) onto the capillary column to achieve the same loading amount of kemptide as in Figure 2. Figure 3A shows a total ion chromatogram from this overloaded linear elution separation. While the peak shape of the major peptides was obviously suboptimal in this experiment, the kemptide ion was still not detectable (Figure 3B). In fact, the ion current measured at 772.6 (the m/z of kemptide) was indistinguishable from that obtained for an irrelevant m/z value (769.5), which was (17) Nelson, M. D.; Dolan, J. W. LC-GC Eur. 2002, 15, 73. (18) Law, B.; Temesi, D. J. Chromatogr., B 2000, 748, 21-30.
Figure 8. Total ion chromatogram of an EMDC separation of bovine serum peptides mixed with trace rhGH peptides. A 20-fmol portion of rhGH tryptic digest was mixed with bovine serum peptides (1.5 µg). The mixture was loaded onto a 75-µm reversed-phase column, and separation was performed by EMDC as described in the Results and Discussion Section. Arrows indicate positions of rhGH peptide zones detected, as verified by data-dependent MS/MS. Peptides are identified by their position in the rhGH sequence.14 The peptides detected were as follows: T12, m/z 773.20; T13, m/z 693.40; T15, m/z 1489.65; T20-21, m/z 1401.66; and T19, m/z 764.43.
interpreted as background noise. The data for m/z 769.5 is shown in panel C. The latter experiments demonstrate an important limitation associated with elution chromatography of complex peptide mixtures containing trace components. As the level of the trace component decreases, more sample must be loaded in order to try to see the desired signal. However because of the nature of mass spectrometry and band-broadening associated with overloading in linear elution separations, trace components are easily obscured because of ion suppression effects. EMDC of Kemptide/rhGH Digest Mixture. We used EMDC to separate a peptide mixture similar to that used in Figure 3. In these experiments, kemptide and the rhGH digest were mixed together at final concentrations of 0.4 µg/mL and 2.5 mg/mL, respectively, because of loading requirements. The kemptide/ rhGH digest ratio in this case was therefore 1:300, a 10-fold lower ratio than that tested in the elution separation in Figure 3, where kemptide was undetectable. The final amounts of kemptide and rhGH digest loaded in this experiment were 5 pmol and 25 µg, respectively. We chose a concentration of 5% acetonitrile to include with the displacer, BDDAB, on the basis of kemptide’s elution behavior observed in Figure 2. The results of the experiment are presented in Figure 4. Panel A of this figure shows the total ion chromatogram of the run, which lasted ∼35 min. A typical EMDC pattern is seen in the experiment in which elution of T12 (a relatively hydrophilic peptide) was seen at 9.09 min (panel B) followed by a large increase in ion current beginning at ∼33 min with the displacement of the T10C1 peptide of rhGH (m/z 537.3, panel C). Successive panels show the displacement zones representing the major peptides displaced under these conditions, including T13 (m/z 693.4, panel D), T20-21 (m/z 1401.4, panel E), and T15 (m/z 1489.7, panel G). The displaced peptides corresponded to early-eluting species from the hGH peptide map (e.g., Figure 2), as expected from the window selected for EMDC. Peptides were well-separated in narrow zones, a phenomenon that
may be related to elution modification. Peptide detection by online mass spectrometry allows accurate profiling of the displacement train and avoids possible distortion of enriched zones caused by collection of fractions.11 The position of the displacer front (BDDAB at m/z 304.4) is shown in panel H of Figure 4. Panel F shows the displacement behavior of kemptide (m/z 772.6), whose major zone emerged at 34.77 min. It was noted that kemptide was displaced between T20-21 and T15, whereas kemptide eluted ahead of both T13 and T20-21 in the elution separation shown in Figure 2. Such changes in displacement order can be caused by crossing of components’ isotherms. In fact, such changes were noted for hGH peptides in our previous paper.11 Some ion current was seen earlier in the chromatogram shown in panel H. We attributed this observation to overlap from the strong signal seen for the elution of T12 (m/z 773.4) at 9.09min (panel B). The signal from 5 pmol of kemptide was easily seen in the EMDC separation, but a 10-fold greater amount was obscured in the elution separation shown in Figure 3. In addition, the time for the EMDC separation (35 min) compares favorably to many elution separations. Detection of Kemptide at Low-Femtomole Level in Mixtures with rhGH Digest. Because of the relative ease of detection of the trace peptide in the rhGH digest shown in Figure 4, successively lower levels of kemptide were mixed with the rhGH digest, and these mixtures were separated by EMDC in the same way. Figure 5 displays the results of these experiments. Panels A and B represent selected ion scans at m/z 772.6 from EMDC separations in which 50 and 5 fmol of kemptide were mixed with the same amount of rhGH digest (25 µg) as that used in the experiment shown in Figure 4. At 5 fmol, kemptide is present at a 1:300 000 ratio to rhGH peptides. This represents a ratio that may be encountered in peptide mixtures from cellular extracts or other complex extracts in which a wide dynamic range of components is present.19 In fact, it has been estimated that the Analytical Chemistry, Vol. 75, No. 8, April 15, 2003
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Table 2. Synopsis of Results from EMDC and Elution Chromatography Experiments component
concn (µg/mL)
loading amt (fmol)
rhGH digest concn (mg/mL)
kemptide kemptide kemptide kemptide p-kemptide
0.4 0.04 0.004 0.0004 0.0034
5000 500 50 5 40
2.5 2.5 2.5 2.5 2.5
kemptide kemptide p-kemptide kemptide kemptide kemptide
4 4 4 0.4 0.4 0.4
5000 5000 5000 500 500 5000
2.5 0.25 0.25 2.5 0.25 0.25
concn ratio a
max. abs intensity (× 106)
S/N
MS/MS ability
300 3000 30 000 300 000 35 000
2.46 5.76 1.79 1.77 5.27
6 5 2 2.5 2.5
yesb yes yes yes yes
30 3 3 300 30 30
e 3.61 2.45 e e e
e 12.5 10 e e e
yesc yes yes no d no no
EMDC
Elution
a The concentration ratio is calculated by dividing the average concentration of the major peptides in rhGH digest by the concentration of the component. Assume the average concentration of the major peptides is 1/20 of the concentration of rhGH digest. b “Yes” means the sequence of the peptide could be identified correctly by assigning the major fragment ions. c In this case, because of the interference of T12 ([M + H]+ ) 773.4), most MS/MS spectra are mixed with fragments of T12 and kemptide ([M + H]+ ) 772.6). d “No” means either no MS/MS spectrum was collected or the ions trapped to do MS/MS were not kemptide. e The component could not be detected by MS.
dynamic range of cellular protein concentrations may be on the order of 106. Table 2 presents a summary of the EMDC and elution data obtained from ESI-MS with different kemptide/rhGH digest mixtures. Of particular interest in this table is the data on maximum absolute intensities obtained from kemptide when loaded at different levels. The intensity number for the kemptide ion in the displacement train varied by a factor of only 2-3, while its absolute concentration in the mixtures was varied by a factor of 103. This observation agrees well with displacement theory, which predicts that, under isotachic conditions (in which all components migrate at the same velocity in the displacement train), each component’s concentration in the mobile phase is determined by the intersection of the operating line with that component’s isotherm.20 The current results generally lend support to this prediction, although at extremely low levels, band spreading and other nonideal effects, such as shock layer formation, may not allow for optimal efficiency. From the data in Table 2, it is interesting to note that in elution experiments in which kemptide was detectable (5 pmol kemptide mixed at a 1:3 ratio with rhGH digest), its signal intensity was comparable to that obtained in EMDC for levels of kemptide 1000-fold lower, although the signal-to-noise ratio (S/N) in the elution experiment was ∼2-fold higher than in EMDC. Panel C in Figure 5 shows the isotope profile of the kemptide ion at m/z 772.26 detected in the 50-femtomole sample, verifying the detection of the kemptide ion in this mixture. Panel D presents a data-dependent MS/MS scan obtained from the experiment in which 5 fmol of kemptide was mixed with the rhGH digest at the ratio 1:300 000. In this MS/MS spectrum, a number of fragments characteristic of kemptide are identified, including both y and b ions as well as other fragments (compare panel D to Figure 2E). In addition to those peptides shown in Figure 5, a number of other minor peptides, which arose from the rhGH digest itself, were enriched and detected in the displacement train but not detected in elution separations, such as the one shown in Figure 2. Table (19) Corthals, G. L.; Wasinger, V. C.; Hochstrasser, D. F.; Sanchez, J. C. Electrophoresis 2000, 21, 1104-1115. (20) Horvath, C.; Nahum, A.; Frenz, J. H. J. Chromatogr. 1981, 218, 365-393.
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Table 3. Major Ions Not Included in Table 1 but Shown in the Spectra of Figure 5 after Enrichment by EMDC no.
name
sequence
1 2 3 4 5 6 7 8 9
185-189 T20 T20-212+ 141-152+ T152+ T21 136-144 54-64 141-152
SVEGS IVQCR IVQCRSVEGSCGF KQTYSKFDTNSH FDTNSHNDDALLK SVEGSCGF TGQIFKQTY CFSESIPTPSN KQTYSKFDTNSH
[M + H]+ calcd [M + H]+ 478.24 618.43 701.11 728.06 745.61 785.36 1085.62 1181.50 1455.41
478.21 618.34 701.20 728.34 745.35 785.31 1085.56 1181.51 1455.68
3 shows the sequences and molecular weights of some of these peptides, which arose from chymotryptic-like cleavages on one end of the peptides listed. Detection of Phosphokemptide and Its Neutral Loss Products by ESI-MS. As discussed at the beginning of this paper, posttranslational modifications, such as phosphorylation, play a vital role in cellular function but are sometimes difficult to characterize because of their low occurrence in the complex cellular milieu. Kemptide, a peptide whose sequence was derived from phosphorylase, has been used a model substrate for protein kinase A.15 We phosphorylated kemptide in vitro (see Experimental Section) and spiked phosphokemptide (p-kemptide) into the rhGH digest at a ratio of 1:37 500 (40 fmol of p-kemptide added to 25 µg of the rhGH digest). The ion chromatogram for p-kemptide (m/z 427.26 for the doubly charged ion) in the EMDC separation is shown in Figure 6, panel A. p-Kemptide was displaced at 37.65 min in this experiment and was, thus, easily detected in this mixture. This is confirmed by the data shown in panels B and C. Panel B shows a scan from the 37.65-min region of the chromatogram. The doubly charged ion (m/z 427.26) is observed along with its neutral loss product, the [M - HPO3 - H2O + 2H]2+ ion. The other major ions seen in the spectrum are listed in Table 4. All of them are identified as products of chymotrypticlike cleavages of rhGH. Panel C shows the data-dependent MS/ MS data from CID of the p-kemptide ions. Again, a number of p-kemptide-related fragments are clearly visible in this spectrum,
Table 4. Major Ions Not Included in Table 1 but Shown in the Spectrum of Figure 6 after Enrichment by EMDC no.
name
sequence
[M + H]+
calcd [M + H]+
1 2 3 4 5
75-78 37-45 2+ 3-8 103-112 37-45
ELLR IPKEQKYSF PTIPLS VYGASDSNVY IPKEQKYSF
530.26 570.42 627.41 1074.31 1138.74
530.33 570.31 627.37 1074.47 1139.61
thus demonstrating the utility of the EMDC technique in the identification of a low-abundance phosphorylated peptide in this complex mixture. Detection of Low-Level Peptides Mixed with SerumDerived Peptides. Mixtures such as cell and tissue extracts containing many different proteins are often encountered in proteomics research. To test EMDC’s ability to enable trace peptide detection and analysis in a highly complex mixture, we prepared a tryptic digest of proteins from bovine serum and mixed trace peptides with it. In the first experiment, shown in Figure 7, 10 fmol of kemptide was mixed with 1.5 µg of serum peptides that were produced by tryptic hydrolysis of serum proteins. Blood is a complex tissue that is known to contain a vast array of proteins at widely different concentrations and that, therefore, represents a formidable analytical challenge. This mixture was separated by EMDC using displacer containing 5% acetonitrile on a 75-µm reversed-phase capillary column with an 8-µm spray tip formed at its outlet. Peptides were identified by spraying the column effluent directly into the mass spectrometer (see Experimental Section). As shown in Figure 7A, kemptide was detected in this mixture, and data-dependent MS/MS (Figure 7B) revealed a fragmentation pattern characteristic of the molecule. Figure 8 shows a total ion chromatogram from an experiment in which 20 fmol of rhGH digest was mixed with 1.5 µg of the serum peptide digest, followed by EMDC of the mixture. In this experiment, the displacer was mixed with 10% acetonitrile in order to enrich peptides in a specific “window” as defined in our earlier paper.11 Arrows in the figure define zones where rhGH peptides were detected. In this experiment, we were able to unequivocally identify and obtain sequence information using data-dependent MS/MS of five rhGH peptides, including T12, T13, T15, T20-21, and T19. The inset in Figure 8 shows an MS/MS scan of the T13 peptide from rhGH obtained during the run. Thus, enrichment and analysis of the above peptides were obtained in a single chromatographic run. These results compare favorably, for instance, with a 2-dimensional LC separation performed on a similar mixture.21 EMDC experiments are currently underway to examine other windows in the mixture by using various eluent concentrations mixed with displacer. CONCLUSION In the current study, we have verified the concept and, thus, extended our earlier work on elution-modified displacement chromatography (EMDC) in several important ways. The technique has been scaled down to a level commensurate with the (21) Wu, S. L.; Amato, H.; Biringer, R.; Choudhary, G.; Shieh, P.; Hancock, W. S. J. Proteome Res. 2002, 1, 459-465.
small sample sizes encountered in proteomics. The technique has been interfaced with electrospray mass spectrometry. This has made on-line detection and identification of enriched peptide zones possible and has increased its sensitivity.11 EMDC is a versatile technique that can potentially be employed in a number of analytical situations in which enrichment of minor components in complex mixtures is desired. The technique may be particularly useful for complex peptide mixtures, for instance, in studies of cell signaling. The wide concentration range of cellular proteins (and therefore, of peptides derived from them) demands powerful enrichment techniques. As mentioned at the beginnng of this paper, various methods, including both affinity and elution chromatographic techniques, have been developed for this purpose. Most of these techniques involve various combinations of steps, including chemical modification, fraction collection, multidimensional chromatographic separation, and other types of sample preparation. EMDC achieves selective enrichment of trace peptides in a single chromatographic separation with no requirement for chemical modification of the peptides. The detection of 5 fmol of kemptide at ratios between 10-5 and 10-6 in the rhGH digest suggests that this technique compares favorably with other technologies for trace peptide enrichment. Experiments in which trace amounts of kemptide (Figure 7) and rhGH peptides (Figure 8) were mixed with a tryptic digest of bovine serum peptides support the notion that EMDC will be useful for the analysis of quite complex mixtures containing components at wide ranges of concentration. EMDC requires prior knowledge of the relative elution position of the peptide(s) of interest; however, if the position is unknown or if separation of a mixture containing several trace peptides with widely different adsorption properties is desired, a scan can be performed in which the eluent concentration in the displacer solution is varied to sample various “windows” in the peptide map as we previously showed.11 In summary, the ultimate goal of this work is to develop a versatile technique that can be used to enrich trace components in a variety of complex mixtures. The current study encourages application of the technique to complex proteomics samples for the identification of important posttranslational modifications, such as phosphorylation. Because of the extreme complexity and wide dynamic range of samples encountered in proteomics studies, new and better tools are needed in searching for low-abundance species, which are often important components of regulatory and signaling cascades. Because of its versatility and relative ease of operation, EMDC therefore potentially represents a powerful tool for the study of these processes. ACKNOWLEDGMENT The authors acknowledge a research collaboration with Thermofinnigan Inc. and the gift of Discovery Wide Pore C18 stationary phase from Dr. Michael Ye of Supelco. This work was supported by Grant no. GM 20993 from the National Institutes of Health, U.S. Department of Health and Human Services.
Received for review October 12, 2002. Accepted February 20, 2003. AC026232T Analytical Chemistry, Vol. 75, No. 8, April 15, 2003
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