Extensive Deuterium Back-Exchange in Certain Immobilized Pepsin

Jan 28, 2006 - When pepsin immobilized on a POROS support was used for online digestion, back-exchange was within the expected range and was similar t...
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Anal. Chem. 2006, 78, 1719-1723

Extensive Deuterium Back-Exchange in Certain Immobilized Pepsin Columns Used for H/D Exchange Mass Spectrometry Yan Wu, Suma Kaveti,† and John R. Engen*

Department of Chemistry, University of New Mexico, Albuquerque, New Mexico 87131-0001

Pepsin digestion prior to mass analysis increases the spatial resolution of hydrogen exchange mass spectrometry experiments. Online digestion with immobilized pepsin is advantageous for several reasons including better digestion efficiency. We have found that certain immobilized pepsin columns cause substantial deuterium back-exchange, rendering the data unusable. When pepsin immobilized on a POROS support was used for online digestion, back-exchange was within the expected range and was similar to the back-exchange of deuterated peptides produced by in-solution pepsin digestion. However, when pepsin immobilized onto selected polystyrenedivinylbenzene supports was used for online digestion with the same system, deuterium loss was extremely high. The effect seems linked to the properties of the solid support used to conjugate the pepsin. Hydrogen exchange mass spectrometry has been widely used to study protein structure and dynamics.1-3 In a typical hydrogen exchange experiment, the protein of interest is labeled with deuterium, the labeling reaction is quenched by reducing the pH and temperature, and the mass of the labeled sample is measured with mass spectrometry. The mass of the whole protein can be measured or the masses of peptides that are created after enzymatic digestion of the labeled protein can be measured. Whole protein analysis only provides limited information such as the overall amount of deuterium that was incorporated at each exchange in time. When the labeled protein is digested into peptides (as first described by Rosa and Richards4 and later adapted to mass spectrometry analyses5,6), the spatial resolution is significantly enhanced, thereby allowing localization of deuterium exchange to particular parts of protein. To minimize the loss of deuterium during analysis, enzymatic digestion is carried out at low temperature (0 °C) and pH (usually pH 2.5-2.6).5 An acid protease that is active under these * To whom correspondence should be addressed. Phone: (505) 277-4226. Fax: (505) 277-2609. E-mail: [email protected]. † Current address: Lerner Research Institute, Cleveland Clinic Foundation, 9500 Euclid Ave., NC-10, Cleveland, OH 44195. (1) Hoofnagle, A. N.; Resing, K. A.; Ahn, N. G. Annu. Rev. Biophys. Biomol. Struct. 2003, 32, 1-25. (2) Eyles, S. J.; Kaltashov, I. A. Methods 2004, 34, 88-99. (3) Wales, T. E.; Engen, J. R. Mass Spectrom. Rev. 2006, 25, 158-170. (4) Rosa, J. J.; Richards, F. M. J. Mol. Biol. 1979, 133, 399-416. (5) Zhang, Z.; Smith, D. L. Protein Sci. 1993, 2, 522-531. (6) Smith, D. L.; Deng, Y.; Zhang, Z. J. Mass Spectrom. 1997, 32, 135-146. 10.1021/ac0518497 CCC: $33.50 Published on Web 01/28/2006

© 2006 American Chemical Society

Figure 1. Online pepsin digestion setup, modified from ref 8. Sample is loaded into the loop of the injector (1) and driven through a pepsin column with pump C. Peptic peptides are trapped and desalted on a peptide trap. The position of a switch valve (2) is flipped to put the trap inline with pumps A and B, and the peptides are eluted from the trap. Separation takes place in a C18 column, and the eluant is directed into a mass spectrometer. Pump A is 0.05% TFA in H2O, pump B is 0.05% TFA in ACN, and pump C is 0.1% formic acid in H2O.

conditions is required. Pepsin is a good choice5 and has been used widely in hydrogen exchange mass spectrometry experiments, although other acid proteases have been explored.7 Pepsin is a nonspecific but reproducible enzyme, whose efficiency is greater when the enzyme/protein ratio is increased. Historically, most hydrogen exchange mass spectrometry experiments have been performed with in-solution pepsin digestion.3,6 Pepsin immobilized onto beads has also been used. It was recently shown that immobilized pepsin packed into a conventional stainless steel column is superior to pepsin digestion in solution.8 Immobilized pepsin packed into a column allows protein digestion to be carried out online. Online digestion typically requires an additional pump to drive the protein through the pepsin column and a switch valve to divert the trapped peptides onto an analytical column for separation (Figure 1). Other online pepsin digestion systems have also been reported.9,10 There are multiple advantages to online digestion versus insolution digestion (we will hereafter refer to digestion in solution (7) Cravello, L.; Lascoux, D.; Forest, E. Rapid Commun. Mass Spectrom. 2003, 17, 2387-2393. (8) Wang, L.; Pan, H.; Smith, D. L. Mol. Cell. Proteomics 2002, 1, 132-138. (9) Rist, W.; Jorgensen, T. J.; Roepstorff, P.; Bukau, B.; Mayer, M. P. J. Biol. Chem. 2003, 278, 51415-51421. (10) Rist, W.; Rodriguez, F.; Jorgensen, T. J.; Mayer, M. P. Protein Sci. 2005, 14, 626-632.

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with nonimmobilized pepsin as “in-solution digestion”. However, it should be kept in mind that solution digestion with pepsin immobilized onto beads is also considered digestion in solution). With online digestion, the peptic peptides are concentrated and desalted before being separated by the analytical column. Because the pepsin is immobilized, it never enters the analytical column as it does with in-solution digestion. With online digestion, the local concentration of pepsin is much higher than it typically is in in-solution digestion, meaning that the digestion efficiency is better. As a result, the same level of digestion can be completed in a shorter period of time with less deuterium loss during analysis. The experimental parameters of online digestion are also easier to control, making the reproducibility of protein digestion better than in-solution digestion. To immobilize pepsin on a stationary phase, different supports can be used such as agarose11 or Sepharose beads.12 POROS, a stationary phase used for perfusion chromatography, was first utilized for online digestion of deuterium-labeled proteins.8 POROS particles have large flow-through channels which, in part, permit much faster biomolecule separation than conventional LC. In POROS beads, a base matrix of cross-linked poly(styrenedivinylbenzene) (PS-DVB) is coated with a cross-linked polyhydroxylated polymer and activated with aldehyde. Aldehyde groups on the surface of the polymer react with primary amines in proteins to create a highly stable secondary amine linkage between the support and the protein (in this case, pepsin). In contrast, the support matrix of a commercially available pepsin column from Orachrom Inc. is composed entirely of cross-linked PS-DVB. PS-DVB is a widely used chromatography matrix for the separation of biomolecules including peptides, proteins, and oligonucleotides. The matrix is utilized in the three different forms: totally nonporous particles, particles with a bimodal pore size distribution, and monolithic column beds.13 Monolithic column beds have rapid mass transfer and enable highly efficient peptide and protein separations. In the Orachrom column, pepsin is covalently tethered to the surface of monolithic, uniformly hydrophilic polymer-based beads with proprietary coupling technology. We have determined that the commercially available pepsin columns from Orachrom Inc. cause large deuterium backexchange when compared with columns containing pepsin immobilized on a POROS support. We demonstrate the deuterium back-exchange originates in the pepsin column itself and is likely a result of the solid support used to immobilize the pepsin. EXPERIMENTAL SECTION Materials. Horse heart cytochrome c, bradykinin, porcine pepsin (1:60 000 grade), trifluoroacetic acid (TFA, spectrophotometric grade), and formic acid (A.C.S. reagent) were obtained from Sigma Chemical Co. (St. Louis, MO) and used without further purification. HPLC grade water and acetonitrile (ACN) were obtained from Burdick & Jackson (Muskegon, MI). A plasmid encoding dihydrofolate reductase (DHFR) with a 6xHis purification tag was kindly provided by Professor Carla Koehler, (11) Kurimoto, E.; Harada, T.; Akiyama, A.; Sakai, T.; Kato, K. J. Biochem. (Tokyo) 2001, 130, 295-297. (12) Tomono, T.; Suzuki, T.; Tokunaga, E. Biochim. Biophys. Acta 1981, 660, 186-192. (13) Walcher, W.; Toll, H.; Ingendoh, A.; Huber, C. G. J. Chromatogr., A 2004, 1053, 107-117.

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UCLA. DHFR was purified with Ni-NTA agarose (Qiagen, Valencia, CA) according to the manufacturer’s instructions. The concentration of DHFR was estimated with a Bradford assay, and the mass of DHFR was confirmed by ESI-MS (23 286.5, measured; 23 286.9, theoretical). HPLC Columns and Traps. The HPLC columns that were used include the following: Jupiter Proteo, 4 µm, 90 Å, 1.0 × 50 mm (Phenomenex, Torrance, CA); PLRP-S, 8 µm, 1000 Å, 1.0 × 50 mm (Michrom BioResources, Auburn, CA); Magic C18, 5 µm, 200 Å, 1.0 × 50 mm (Michrom BioResources); Microbore C18, VM-5-C18W-1000, 1.0 × 50 mm (Micro-Tech Scientific, Vista, CA). Micro peptide trap cartridges were purchased from Michrom Bioresources. The self-packed POROS peptide trap contained POROS 10 R2 (Applied Biosystems, Foster City, CA) packed into a 2 × 20 mm refillable guard column obtained from Alltech (Deerfield, IL). Pepsin Columns. The Orachrom StyrosZyme pepsin column (2.1 × 50 mm) was obtained from Orachrom Inc. (Woburn, MA). POROS 20AL media to immobilize pepsin was obtained from Applied Biosystems. Pepsin was immobilized onto POROS as described8 and packed into an empty stainless steel column (2.1 × 50 mm) obtained from Alltech. Hydrogen Exchange. Horse heart cytochrome c was dissolved in H2O at a concentration of 80 pmol/µL (pH 6). Deuterium exchange was initiated by diluting the protein 15-fold with labeling buffer (20 mM potassium phosphate, D2O, pD 7.6). After 2.5 h, an aliquot of 320 pmol was removed and deuterium exchange was quenched by addition of an equal volume of quench buffer (300 mM potassium phosphate, H2O, pH 2.35), frozen on dry ice, and stored at -80 °C until analysis. Undeuterated samples were prepared using an identical procedure except the labeling buffer was replaced with 20 mM potassium phosphate, H2O, pH 7.6. DHFR was deuterium labeled as above, except exchange was quenched after 10 s and 30 min. Bradykinin was maximally deuterated in labeling buffer at 37 °C overnight and was quenched with an equal volume of quench buffer right before injection. Analysis of Deuterium Incorporation. For in-solution digestion, porcine pepsin was dissolved in H2O (pH 5) at a concentration of 2 µg/µL. Five microliters (10 µg) of pepsin solution was added to each quenched sample (320 pmol; 128 µL) and the resultant mixture was incubated at room temperature for 2 min before injection. HPLC separation was carried out with a Shimadzu 10ADVP HPLC. The injector, tubing, trap, and column were immersed in an ice bath to minimize deuterium loss.5 The elution gradient was as follows: 5% ACN for 3 min, 5-15% ACN over 30 s, and ramp to 50% ACN over 6 min. The flow rate was 50 µL/min. 0.05% TFA was added to both mobile phases. Online pepsin digestion was performed essentially as described.8 Protein (320 pmol, 132 µL) was loaded into a 200-µL loop and pushed through the pepsin column by pump C (H2O, 0.1% formic acid) at a flow rate of 200 µL/min (see Figure 1). Residence time in the pepsin column was 1 min, and the resulting peptides were trapped by the inline peptide trap. After an additional 1 min of desalting, the switch valve was moved to put the peptide trap inline with the pumps A and B and HPLC column. The peptic peptides were eluted from the peptide trap and separated by the C18 column with the same gradient described above.

Figure 2. Combined ESI spectrum of all peptides produced during peptic digestion of DHFR by (a) a pepsin column from Orachrom or (b) a column containing pepsin immobilized on POROS media.

Mass Analysis. All mass spectrometry was carried out with a QTOF2 (Waters) equipped with a standard ESI source. Myoglobin was infused at the end of each run for mass calibration. All the mass spectral measurements were taken at a capillary voltage of 2.7 kV, cone voltage of 30 V, source temperature of 85 °C, desolvation temperature of 175 °C, and desolvation gas at 500 L/h. Each 2.4-s scan spanned m/z 200-1990 with an interscan delay time of 0.1 s. Spectra were processed with the Masslynx software. RESULTS AND DISCUSSION To compare the digestion efficiency of the commercial pepsin column from Orachrom Inc. with that of the pepsin immobilized on POROS in-house, DHFR was digested and the resulting fragments were analyzed with mass spectrometry. Both columns were operated using identical conditions and provided good reproducibility. The digestion efficiency observed with the Orachrom column (Figure 2a) was similar but not identical to that obtained from pepsin immobilized onto POROS media (hereafter referred to as POROS column) (Figure 2b). The peptic peptides of DHFR obtained with the POROS pepsin column were very similar to those from an in-solution digest (data not shown), but comparatively fewer peptides were obtained with the Orachrom pepsin column. In the combined spectrum of all DHFR peptides from the Orachrom pepsin digestion, there were fewer high-intensity peaks compared with the POROS column digestion. Some of the peaks observed with POROS column digestion (i.e., m/z ) 495.81, 824.12, 921.20) were not found at all or found at much lower intensity in the Orachrom column digestion. These data showed that pepsin immobilization onto POROS using the previously established protocol8 results in either a higher pepsin concentration on the beads, more active pepsin molecules (as a result of orientation effects or the coupling chemistry), or both relative to the commercial Orachrom column. The deuterium recovery obtained using online pepsin digestion should be greater than or equal to that of in-solution digestion. In an optimized system, deuterium recovery of labeled peptic peptides should be 70-90%, depending on the sequence.6 The deuterium level for peptides produced by the Orachrom column was vastly different from that obtained with in-solution digestion or digestion with a POROS column (Figure 3). When deuterated cytochrome c was digested online with the Orachrom pepsin column, the isotope pattern of many of the peptides resembled that of undeuterated peptides rather than the expected deuterated peptides (Figure 3d, left). The isotopic pattern of deuterated peptides should be a Gaussian distribution (as in Figure 3b,c) due to random loss of deuterium in the HPLC step.6 The

deuterium loss was so great in some peptides produced by the Orachrom column that they appeared to have never been exposed to deuterium. Some peptides displayed a less dramatic alteration and contained a mixture of undeuterated peaks and deuterated peaks (Figure 3d, right). These unexpected results were evident regardless of the deuterium labeling time and were obtained with several different Orachrom columns (data not shown). To verify that the atypical deuterium loss observed with the Orachrom column was not isolated to one protein, another protein was examined at different deuterium labeling times. Dihydrofolate reductase was deuterated for 10 s or 30 min. The isotopic distribution of deuterated peptic peptides produced by the Orachrom pepsin column indicated a substantial loss of deuterium at both exchange time points (Figure 4a). Again, this was in stark contrast to the isotopic pattern displayed by the same deuterated DHFR peptide when digested with the POROS column (Figure 4b). The isotope pattern for peptides produced by the Orachrom column indicated a substantial population of molecules with the mass of an undeuterated peptide. The mass of this population did not increase with time while the rest of the population continued to become deuterated in a normal EX2 fashion,14 as indicated by the gradual increase in the mass of the higher-mass envelope (Figure 4a).

Figure 3. Comparison of deuterium levels in cytochrome c peptides with various digestion methods. (a) Undeuterated cytochrome c. (bd) Cytochrome c deuterium labeled for 2.5 h and subjected to (b) in-solution digestion, (c) POROS pepsin column digestion, or (d) Orachrom pepsin column digestion. HPLC separation conditions (column, gradient) were identical in (a-d). Two peptides shown are as follows: m/z ) 694.45 (+2), AGIKKKTEREDL, cytochrome c residues 83-94 and m/z ) 575.85 (+2), IAYLKKATNE, cytochrome c residues 95-104.16

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Figure 4. Comparison of deuterium levels in DHFR peptides after POROS or Orachrom column digestion. DHFR was either undeuterated (UN) or deuterated for 10 s or 30 min prior to digestion by (a) the Orachrom pepsin column or (b) the POROS pepsin column. The peptide shown is as follows: m/z ) 632.85 (+2), TTSSVEGKQNLV, DHFR residues 81-92.

The appearance of a bimodal distribution such as that observed with the Orachrom pepsin column (Figure 4a) could be evidence for EX1 kinetics.6,15 In EX1 kinetics, there are typically two populations: those that have not yet unfolded to exchange and those that have unfolded and cooperatively taken up deuterium. The mass spectra of these populations have a bimodal isotope pattern. EX1 kinetic signatures can be induced with destabilizing conditions (denaturants, extreme pH, high temperature), which alter the refolding rate such that the protein is in a unfolded state long enough to uptake multiple deuterons before refolding back to native state (see ref 14 for more details). EX1 kinetics are rare in folded proteins under physiological conditions. In this experiment, both cytochrome c and DHFR were labeled with deuterium under physiological conditions. Patterns characteristic of EX1 kinetics were observed at all time points for all the peptides produced during Orachrom column digestion. The low mass envelope of the bimodal distribution observed upon digestion with an Orachrom column never moved from the position of an undeuterated peptide. The bimodal distribution was not obtained with either an in-solution digestion or an online pepsin digestion with POROS column of the same samples. The unusual mass spectra are therefore likely not the result of EX1 kinetics, but rather deuterium loss in the Orachrom pepsin column. Our later experiments showed this to be true. Initial attempts to isolate the causes of the bimodal distribution were unsuccessful. Equilibration buffers (either in saline or H2O), labeling buffers (with or without salt), quench buffers (either HCl or phosphate buffer, pH 2.3 or 2.6), and the flow rate of the HPLC system (either 30 or 50 µL) were independently changed, but the bimodal pattern remained. To eliminate the possibility of accelerated back-exchange caused by the peptide trap or the HPLC C18 column, different traps and analytical separation columns were coupled with in-solution digestion. For in-solution digestion, the same setup shown in Figure 1 was used, except that the pepsin column was replaced with a stainless steel union. The HPLC gradient was the same as an online digestion. The results are shown in Figure 5 with the same cytochrome c peptide that was (14) Englander, S. W.; Kallenbach, N. R. Q. Rev. Biophys. 1983, 16, 521-655. (15) Engen, J. R.; Smith, D. L. Methods Mol. Biol. 2000, 146, 95-112. (16) Dharmasiri, K.; Smith, D. L. Anal. Chem. 1996, 68, 2340-2344.

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Figure 5. Effect of various HPLC columns and peptide traps on deuterium level. Horse heart cytochrome c was labeled with deuterium for 0 or 2.5 h and digested with pepsin in solution. The peptide shown is the same as that shown in Figure 3. (a) Varying the separation column. Top, undeuterated sample; middle, PLRP-S column; bottom, Jupiter column. The same POROS peptide trap was used. (b) Varying the peptide trap. Top, undeuterated sample; middle, PLRP-S trap; bottom, POROS trap. The same microbore separation column was used.

illustrated in Figure 3. The spectra of the deuterated peptide trapped on a POROS 10 R2 trap and separated with a PLRP-S column (Figure 5a, middle) or a Jupiter C18 column (Figure 5a, bottom) are clearly Gaussian with no peaks at the undeuterated mass. Reversion to an undeuterated form (as in Figure 3d) was not observed. Some other columns (Micro-tech microbore C18 and Michrom Magic C18 column) were also tested and gave similar results. Based on these data, the HPLC column was ruled out as a source of the deuterium loss observed in Figures 3 and 4a. The trap that captures the peptic peptides prior to separation was also not the source of the deuterium loss. When a typical C18 column was used (Micro-tech microbore C18) and the peptide trap varied between a PLRP-S trap (Figure 5b, middle panel) or a POROS trap (Figure 5b, bottom panel), there was no evidence for the deuterium loss seen in Figures 3 and 4a. From all these data, it can be concluded that the unusual isotope pattern obtained with Orachrom pepsin column digestion was not caused by either the HPLC separation column or the peptide trap. In testing the HPLC separation columns as the source of deuterium loss, a PLRP-S column was specifically tested because its packing material, cross-linked PS-DVB, is theoretically the same as the stationary phase of the Orachrom column. The PLRP-S column (Figure 5a, middle) did not cause the deuterium backexchange observed in the Orachrom column (Figures 3 and 4a). We reasoned, therefore, that at least two other factors may be responsible for deuterium back-exchange in the Orachrom column: the actual cleavage reaction of pepsin immobilized on the PS-DVB beads or the way the surface of the PS-DVB material was treated or end-capped (for example, high charge status on the surface) in the Orachrom column. To determine whether the deuterium loss was caused by the pepsin cleavage reaction in the Orachrom column, a highly deuterated peptide (bradykinin) was passed through the Orachrom column with the same system utilized for all the other tests. If the pepsin cleavage reaction caused the deuterium loss seen previously, the deuterium level of the deuterated bradykinin should not have been altered because pepsin will not catalyze any

Figure 6. Analysis of deuterium recovery in highly deuterated bradykinin. (a) Undeuterated bradykinin, (b) highly deuterated bradykinin passed through the Orachrom pepsin column, and (c) highly deuterated bradykinin passed through the POROS pepsin column. The +2 charge state is shown.

cleavage of bradykinin as it is already a peptide that cannot be digested further. Surprisingly, the deuterium loss for deuterated bradykinin in the Orachrom column (Figure 6b) was much more than the deuterium loss for deuterated bradykinin that passed through the POROS column (Figure 6c). These results show that the pepsin cleavage reaction in the Orachrom column did not contribute to the large deuterium loss. The deuterium loss appeared to be a function of the properties of the Orachrom column itself, perhaps the stationary phase. Charge effects on the surface of the PS-DVB beads in the Orachrom pepsin column may have caused the accelerated backexchange. A 500 mM concentration of NaCl was used as buffer C in an effort to neutralize surface charges on the stationary phase of the Orachrom column. Deuterium recovery was not improved with 500 mM NaCl in the buffer (Figure 7d). The isotope envelopes of the same peptic peptide deuterated under the same conditions were nearly identical with or without high salt in the buffer (compare Figure 7c and d). The 500 mM salt conditions were not sufficient to neutralize charge groups, if there are any present on the stationary phase of the Orachrom pepsin column. CONCLUSIONS Two immobilized pepsin columns were compared in this work, a commercial column and a column made in-house. The commercial Orachrom column, with proprietary pepsin coupling technology, provided acceptable digestion but was not suitable for hydrogen exchange experiments because it caused accelerated

Figure 7. Effect of high concentrations of NaCl on the deuterium back-exchange in the Orachrom pepsin column. The peptide shown is the same as that shown in Figure 3. (a) Undeuterated cytochrome c. (b-d) Cytochrome c labeled for 2.5 h and digested by (b) pepsin in-solution, (c) the Orachrom pepsin column (same as in Figure 3d, left), or (d) the Orachrom pepsin column with 500 mM NaCl in the mobile phase.

deuterium back-exchange during analysis. Neither the system, the peptide trap, nor the HPLC C18 column played a role in the large deuterium loss. We believe that the surface treatment or the coupling chemistry of the pepsin immobilized onto the PSDVB beads in the commercial column is responsible for the observed deuterium loss. Although POROS beads have a PS-DVB core, they did not cause major deuterium back-exchange. Naked POROS 20AL beads (before any enzyme was coupled to them) did not cause major deuterium loss (data not shown), suggesting either that the polyhydroxylated polymer coating of POROS beads shields the PS-DVB from solution or that the aldehyde coupling reaction eliminated any groups on the PS-DVB in POROS that could cause accelerated back-exchange. An entirely unique species may be present on the surface of the PS-DVB beads in the Orachrom column that is not present on the POROS beads. It is recommended that care be taken when selecting a pepsin digestion column for hydrogen exchange mass spectrometry experiments. ACKNOWLEDGMENT This work was supported by grants from the NIH P20RR016480 and R01-GM070590. Received for review October 14, 2005. Accepted January 4, 2006. AC0518497

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