A Desalting Approach for MALDI-MS Using On-Probe Hydrophobic

and the Department of Chemistry, University of Georgia, 220 Riverbend Road, Athens, Georgia 30602-4712. One of the problems encountered in preparing ...
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Anal. Chem. 1997, 69, 4716-4720

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A Desalting Approach for MALDI-MS Using On-Probe Hydrophobic Self-Assembled Monolayers Adam H. Brockman, Brian S. Dodd, and Ron Orlando*

Complex Carbohydrate Research Center, the Department of Biochemistry and Molecular Biology and the Department of Chemistry, University of Georgia, 220 Riverbend Road, Athens, Georgia 30602-4712

One of the problems encountered in preparing samples for matrix-assisted laser desorption/ionization (MALDI) analysis is the presence of nonvolatile salts in the sample. This difficulty is often exacerbated by the necessity to prepare the sample in the appropriate sample-to-matrix ratio. This paper reports a probe surface derivatization method that greatly simplifies this sample preparation process. By constructing self-assembled monolayers of octadecyl mercaptan (C18) on the MALDI probe surface, we were able to generate a surface capable of reversibly binding polypeptides via hydrophobic interactions, which in turn, permits the analyte to be easily concentrated and desalted directly on the probe tip. The optimum sample handling conditions for biological preparations usually involve nonvolatile salts. Biologists employ nonvolatile salts in preparing samples for several reasons, including maintenance of a nontoxic environment for cells, stabilization of solvated samples, and maintenance of enzymatic or other biological activity. In addition, many separation strategies used in the isolation of biological molecules require high concentrations of nonvolatile salts and buffers. However, the same salts used to preserve biological activity and optimize sample preparation and handling often cause problems for subsequent analyses, particularly mass spectrometric analytical steps.1 For example, modern polymeric supports have made high-performance ion exchange of peptides and proteins a reality,2 but the salts used to maximize the efficiency of these separations are often not volatile. Therefore, in order for mass spectrometry to be useful in characterizing ion exchange eluents, a subsequent preparative separation must be performed to remove interfering salts from the sample. This second separation step is often costly in terms of sample loss and time. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is one of the most sensitive MS approaches and is probably the most compatible with biological buffers.3-5 Sample preparation for MALDI is performed by drying a mixture of sample and matrix on the probe surface. Analyte ionization is achieved (1) Lundell, N.; Markides, K. J. Chromatogr. 1993, 639, 117. (2) Alpert, A. J.; Andrews, P. C. J. Chromatogr. 1988, 443, 85. (3) Tanaka, K.; Waki, H.; Ido, Y.; Akita, S.; Yoshido, Y.; Yoshido, T. Rapid Commun. Mass Spectrom. 1988, 2, 151. (4) Hillenkamp, F.; Karas, M.; Beavis, R.; Chait, B. T. Anal. Chem. 1991, 63, 1193A. (5) Wang, R.; Chait, B. T. Curr. Opin. Biotechnol. 1994, 5, 77.

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by focusing a pulse of a laser light onto the sample/matrix preparation. More than one theoretical description for the subsequent ionization processes are presently being considered. In a prominent theory, the biological molecule is thought to be embedded in the desorption volume of the matrix. This large and nonvolatile molecule is susceptible to entrainment into the resulting plume of plasma caused by the laser pulse.6 This entrainment is followed by the transfer of protons and energy from the ionized matrix. If the sample-to-matrix ratio is not proportioned correctly or if an interfering nonvolatile salt is present in the preparation in sufficient quantities, entrainment and energy transfer to the biological molecule will be inhibited. An alternative theory explaining the detrimental effects of nonvolatile salts is that they exclude the biological molecules from the crystal lattice of the matrix. In this case, the biological molecules would not be desorbed/ionized in the matrix plume generated by the laser pulse. Therefore, although MALDI is a very powerful technique in the analysis of biological molecules, the method may become challenging under typical biological conditions. In many instances the salt concentration in a MALDI preparation is brought to an inhibitive level by bringing the analyte’s concentration to a level suitable for MALDI analysis. Although the mass detection limit of MALDI lies in the attomole to nanomole range, materials are deposited on the probe surface in microliter volumes. Therefore, the analyte’s concentration needs to be in the picomolar to millimolar range, which often necessitates a preconcentration procedure (typically evaporation of the solvent) to optimize the sample-to-matrix ratio. However, if a small amount of salt is present in the original sample preparation, it can be magnified to inhibitive levels upon concentration of the sample. This is particularly true for small peptide samples that are not amenable to dialysis. Therefore, although samples with physiological salt concentrations may not have excluded the possibility of direct MALDI analysis when the analyte is present at a high concentration, samples requiring preconcentration are another matter. Consideration of sample-to-matrix ratio and salt concentration is thus necessary in order to optimize the ionization processes of MALDI. Several sample preparation techniques that aid the analyst in overcoming these limitations have appeared in the literature. The first of these methods relies upon a thin film of matrix that is deposited in acetone prior to deposition of the peptide sample.7 (6) Vertes, A.; Irinyi, G.; Gijbels, R. Anal. Chem. 1993, 65, 2389. (7) Vorm, O.; Mann, M. J. Am. Soc. Mass. Spectrom. 1994, 5, 955. S0003-2700(97)00650-1 CCC: $14.00

© 1997 American Chemical Society

The peptide sample is then deposited on top of the matrix film and desalted using aqueous organic acids. Other investigators have used this matrix film approach to good advantage by eluting avidin-bound biotinylated peptide materials directly onto the matrix underlayer.8 The captured eluent could then be washed directly on the MALDI probe surface. Using microscopic examination of matrix/peptide films following preparation, a recrystallization method was developed that incorporates peptide molecules to a greater degree than salt contaminates, which allows salts to be selectively washed away from the adhering crystals to a large extent.9 Samples can also be freed of salt contaminants by depositing them onto polymeric membranes, such as porous polyethylene (PE) and poly(vinylidine difluoride) (PVDF).10 Salts and detergents can be washed from the sample that binds to the membrane, after which, the membrane is attached the MALDI probe, the matrix deposited onto the membrane, and the sample analyzed. All of these methods have become incorporated into the arsenal of sample preparative methods of use to the MALDI analyst and have been of great assistance in battling the potential problems involved with MALDI analysis of unknown peptide samples. Self-assembled monolayer (SAM)11-13 technologies have recently been widely applied to MALDI preparative methods. The first application of SAM-based technologies to MALDI-MS was based on the covalent attachment of antibodies directly onto a MALDI probe surface.14,15 This method enabled the analyst to carry out affinity separations directly on the MALDI probe surface with no elution step. Subsequent to this, SAMs have been used to attach enzymes directly onto the MALDI probe.16 In 1996, we improved upon our original work with probe-based affinity separations by using covalently linked dextran molecules that are capable of drastically increasing the number of molecules covalently bound to the surface.17,18 The most recent application of SAMs in MALDI-MS involves the use of a UV-absorbing SAM as the MALDI matrix.19 This application is an appealing advance in MALDI sample preparation methodology, as it may offer a MALDI process more amenable to automation. The growing marriage between MALDI and SAM technologies promises to generate a number of exciting fast separation and automated technologies that should be very useful in bioanalysis in the years to come. We report here the use of a hydrophobic SAM as a sample preparative device. This simple monolayer of octadecyl mercaptan on gold is used as a desalting device, which offers a significant improvement in salt tolerance in the preparation of peptides for (8) (9) (10) (11) (12) (13) (14)

(15) (16) (17) (18) (19)

Schriemer, D. C.; Li, L. Anal. Chem. 1996, 68, 3382. Xiang F.; Beavis, R. C. Rapid Commun. Mass Spectrom. 1994, 8, 199. Blakledge, J. A.; Alexander, A. J. Anal. Chem. 1995, 67, 843. Bain, C. D.; Evall, J.; Whitesides, G. M. J. Am. Chem. Soc. 1989, 111, 7155. Dubois, L. H. Annu. Rev. Phys. Chem. 1992, 43, 437. Jung, D. R.; Czanderna, A. W. Crit. Rev. Solid State Mater. Sci. 1994, 19, 1. Brockman, A. H.; Hirsh, S.; Kaswan, R.; Orlando, R. Proceedings of the 43rd ASMS Conference on Mass Spectrometry and Allied Topics, Atlanta, GA, May 21-26, 1995. Brockman, A. H.; Orlando, R. Anal. Chem. 1995, 67, 4581. Nelson, R. W.; Dogruel, D.; Krone J. R.; Williams, P. Rapid Commun. Mass Spectrom. 1995, 9, 1380. Brockman, A. H.; Orlando, R. Proceedings of the 44th ASMS Conference of Mass Spectrometry and Allied Topics, Portland, OR, May 12-16, 1996. Brockman, A. H.; Orlando, R. Rapid Commun. Mass Spectrom. 1996, 10, 1688. Mouradian, S.; Nelson, C. M.; Smith, L. M. J. Am. Chem. Soc. 1996, 118, 8639.

MALDI analysis. We have found the robust nature of these surfaces to be a practical solution for analyzing salty peptide samples by MALDI-MS. EXPERIMENTAL SECTION Our goal in this work was to develop a methodology for the use of C18 SAMs in the MALDI analysis of peptides. Several variables had to be taken into account in the development of this method. The most critical of these involved how to deposit the sample onto the derivatized surface and how to carry out the subsequent separation step. These initial experiments were performed on preparations of renin substrate in a solution of 2 M sodium acetate. Finally, a true unknown was explored following fractionation of peptides from a parasite- (Trypanosoma cruzi) infected heart extract. The T. cruzi peptide mixtures were fractionated using a phosphate buffer gradient before analysis to provide an example of a peptide separation in a nonvolatile buffer. In the following discussion MALDI, reversed-phase high-performance liquid chromatography (RP-HPLC), and SAM methodologies are summarized. In addition, the protocol with which we prepared the renin substrate and T. cruzi samples is outlined. MALDI data were collected using an HP LDI1700 XP linear time-of-flight mass spectrometer (Hewlett-Packard Inc., Palo Alto, CA). The repeller was operated at 30 kV, and the intermediate extraction lens was operated at 9 kV. A mass gate was employed to deflect ions below mass-to-charge ratio (m/z) 800. The matrix was sinapinic acid, which was recrystallized and purified by flash chromatography. The matrix was a saturated solution of 80% aqueous acetonitrile (ACN; J. T. Baker Inc., Phillipsburg, NJ) with 0.05% trifluoroacetic acid (TFA; Aldrich, Milwaukee, WI). “Conventional” MALDI refers to a typical MALDI preparation in which the analyst mixes a volume of matrix and a volume of sample and then dries the resulting mixture onto the probe surface. This method is the technique to which we compare our method using C18 surface technology. This procedure is referred to throughout the text as conventional MALDI. We were cautious in preparing suitable reagents before constructing SAMs in our laboratory. Aqua regia was made by mixing 5.4 mL of nitric acid (∼70%, J. T. Baker) and 24.6 mL of hydrochloric acid (37%, Aldrich). Octadecyl mercaptan was purchased from Aldrich and used as received. Ethanol was also purchased from Aldrich, dried over a slurry of aluminate, deaerated with helium, and filtered on a 45 µm diameter pore size nylon membrane. High-purity gold was sputtered onto an aqua regiaetched probe tip using a common commercial apparatus. Care must be taken in this step not to scorch the tips, as this contaminates tip preparation. The tip itself is a disposable circle manufactured for us by Hewlett-Packard that can be fastened onto an insertable probe following sample preparation. This tip provides a surface with eight raised “mesas” similar to the typical Hewlett-Packard MALDI probe. The SAM was constructed by preparing a 1 mm solution of the octadecyl mercaptan in the dry, deaerated ethanol (2.87 mg in 10 mL). The solution was gently heated and sonicated in a Cole-Parmer (Vernon Hills, IL) sonicator to rapidly dissolve the thiol. Batches of gold-sputtered disposable probe tips were allowed to remain in the thiol solution overnight (Figure 1). The following day, the tip was rinsed with ethanol followed by water to remove the excess octadecyl mercaptan. After air-drying, the steep contact angle of 1 µL of water could be observed on each mesa surface. Analytical Chemistry, Vol. 69, No. 22, November 15, 1997

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Figure 2. Conventional MALDI spectrum of 1 µg/mL renin substrate in 200 mM sodium acetate.

Figure 1. Mechanistic illustration of SAM formation and subsequent separation.

The C18 surfaces were first tested using a commercial synthetic sample of renin substrate (Sigma, St. Louis, MO). The testing was done by placing a 1 µL aliquot of the peptide solution directly onto a mesa. Specifically, a 1 µL quantity of 1 ng/mL peptide solution was deposited onto a C18-derivatized mesa and allowed to bind for a few minutes. The salt was then washed from this picogram of peptide material by soaking the probe tip in a beaker of 1% TFA. Any TFA solution remaining on the mesa was allowed to air-dry, after which 1 µL of matrix was added to the surface and homogenized with the released peptide. A MALDI spectrum of the sample was then acquired. Several controls were performed on the C18 surfaces. These involved methanol washing to test the surface for memory effects, a conventional MALDI preparation of the salty peptide, and a control using an underivatized surface. The results of this initial experiment with renin substrate suggested that the incubation time should be extended to (∼8 h) for all experiments and that the entire tip should be incubated directly in the sample. Renin substrate solutions of 1 µg/mL and 1 ng/mL were thus made up in 1 mL aliquots of a 2 M sodium acetate buffer. In addition to the sodium acetate experiment, a 1 µg/mL solution of renin substrate in 6 M urea was analyzed by the C18 method to test the effects of chaotropes. In each experiment, the tips were placed directly in the sample solution and incubated overnight. The following day, the tips were rinsed and analyzed by MALDI as described. The MHC class-I peptide extract was obtained by infecting five immunodeficient mice with 1000 bloodform trypomastigotes following standard level 2 safety protocols. The infected mouse hearts were explanted 21 days postinfection. The hearts were homogenized in 1% TFA solution in a dounce homogenizer, similar to previously published methods of tissue extraction.20 The homogenate was sonicated and remained in the 1% TFA solution for several hours to maximize peptide extraction and lysis through the tissue. The homogenate was precleared by centrifugation at 3000 rpm, and the supernatant was then ultracentrifuged at 33 000 rpm (roughly 100000g). The ultracentrifuged supernatant was (20) Wu, M. X.; Tsomides, T. J.; Eisen, H. N. J. Immunol. 1995, 154, 4495.

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then subjected to mass filtration using an Amicon (Beverly, MA) 10 000 MWCO Centriprep filter. The filtrate was lyophilized, and the resulting complex mixture of peptides and other organics was subjected to reversed-phase fractionation using the nonvolatile buffer system. RP-HPLC separations of the T. cruzi heart extract were performed on a Rainin Dynamax HPLC system (Ridgefield, NJ). Data were collected on a Power Macintosh 7500/100 PC (Apple Computers Inc., Cupertino, CA). The reversed-phase column used was a Rainin Microsorb-mv C18 4.6 × 250 mm. The pumps were Rainin SD200 solvent delivery systems with a pressure gauge installed on pump A. A Rheodyne (Cotati, CA) 7725 injector was used with a 1 mL sample loop. A mixing tee was employed to implement gradient mixing. The same gradient was used for all separations and consisted of a linear increase of buffer B from 0 to 60% in 60 min. The gradient was then ramped to 100% buffer B over the course of 10 min and held at 100% B for a final 10 min. The nonvolatile system employs a 20 mm phosphate concentration. Buffer A is composed of aqueous 20 mM phosphate, while buffer B employs 80% ACN as an organic modifier. Care must be taken to ensure that the phosphate is soluble in buffer B throughout the gradient before use, as the phosphate can precipitate when exposed to the organic modifier. Reversed-phase fractionated samples were employed to test the applicability of the C18 surfaces to nonvolatile HPLC eluents. Thirty 2 mL fractions were taken from an automated fraction collector (Rainin) following separations the T. cruzi-infected heart extracts. These fractions were placed into 15 mL polyethylene centrifuge tubes (Corning Inc., Corning, NY), and the ACN was blown off with ultrapure helium (Southeast Air Gas, Athens, GA) in an N-Evap evaporator (Organomation Associates Inc., Berlin, MA). Once the organic modifier was removed, the peptide solution could be exposed to the C18 surfaces overnight to effect peptide binding on the mesas through hydrophobic interactions. RESULTS AND DISCUSSION A series of conventional MALDI experiments was performed to demonstrate the problems encountered with high concentrations of nonvolatile buffers. For example, the analysis of renin substrate (1 µg/mL) from a 200 mM sodium acetate solution (Figure 2) contains a fairly weak signal from the protonated molecule and the spectrum is complicated by multiple sodium adducts. When the concentration of sodium acetate is raised to

Figure 3. MALDI spectrum of 1 pg of renin substrate tetradecapeptide isolated from a single microliter droplet on a C18-derivatized probe tip mesa.

2 M, no signal is obtained from renin substrate using conventional MALDI. These results suggested that sample containing 2 M sodium acetate would be a reasonable model system to develop C18-MALDI methodologies. The most obvious use of a C18-modified probe is to place a 1 µL aliquot of the salty sample solution directly on the derivatized surface. The sample solution would then be allowed to stand for a period of time to enable capture of the peptide on the derivatized probe surface by hydrophobic interaction. After this incubation period, the probe would be washed with deionized water to remove the salts, the matrix applied, and the probe inserted into the MALDI-MS for analysis. The result from such a procedure is shown by the analysis of a 1 µL aliquot containing 1 pg of renin substrate that was dissolved in 2 M sodium acetate (Figure 3). Note that even though the concentration of renin substrate in this solution is 1000 times lower and the nonvolatile buffer concentration is 10 times higher than that used in the conventional MALDI spectrum (Figure 2), the spectrum is free of interfering sodium adducts. Consequently, this approach can be used to analyze samples in small volumes of very concentrated salts. Although this on-probe desalting procedure is successful, these spectra are fairly difficult to obtain since the analyte appears to be localized to a very small region of the probe surface. This characteristic forces the analyst to search the entire probe surface to obtain a few reasonable, single-shot spectra. One possible explanation for this performance is that these conditions permit only a very small amount of the peptide to bind to the modified probe. For instance, the surface tension of the aqueous sample solution causes it to form a near-perfect sphere on the hydrophobic probe, and consequently, only a very small portion of the probe comes into contact with the solution. This small contact area impedes the rate at which the peptides bind to the surface and confines their adsorption to a fairly small region of the probe. Furthermore, the time period in which peptides can diffuse to the modified surface is limited by the rate at which the sample’s solvent evaporates. For instance, a single microliter of an aqueous sample will evaporate in under 10 min. This severely limits the amount of peptide that can be captured via hydrophobic interactions. Consequently, although small volumes can be successfully analyzed on C18 modified probes, we do not recommend this approach for routine analysis. We decided that a better method for using C18-modified probes is to incubate them in the sample solution for an extended period

Figure 4. (A) MALDI spectrum of C18-isolated renin substrate from a 1 µg/mL solution of the peptide in 2 M sodium acetate. (B) MALDI spectrum of C18-isolated renin substrate from a 1 ng/mL solution of the peptide in 2 M sodium acetate.

(∼8 h) to overcome the problems of limited exposed surface area and binding times. The ability of this approach to desalt samples is shown by the analysis of solutions containing 1 µg/mL and 1 ng/mL renin substrate dissolved in 2 M sodium acetate (Figure 4A and B, respectively). Both of these spectra are free of the sodium interference that prevented analysis of these solutions by conventional MALDI. Although, the extended incubation approach is not as fast as our single-droplet extraction approach and requires larger sample volumes (>100 vs 1 µL), the spectra are significantly easier to obtain with the extended incubation period, since the analyte appears to be fairly evenly deposited over the entire probe surface. The surface characteristics of C18-modified probes also require a judicious choice of solvents for the matrix. For conventional MALDI-MS experiments, we typically dissolve the matrix in 90% methanol or ethanol. However, these solutions form very unstable droplets on the C18-derivatized probe surface and quickly spread over the entire probe surface. An ACN/H2O/matrix droplet, on the other hand, forms a bead on the C18-derivatized probe. This geometry helps to maximize sample uniformity and homogeneity and can be a critical factor when surface separations in which a limited amount of total analyte has been isolated are analyzed. We implemented various controls to ensure that the C18 assembly was the cause of our successful isolation of the renin substrate. We were unable to acquire a spectrum after a methanol wash of a C18 surface that had been incubated for 8 h in a 1 µg/ mL solution of renin substrate. This experiment demonstrates that the binding is reversed by washing with a hydrophobic solvent and is highly suggestive that the peptides are held to the surface via hydrophobic interactions. Spectra could also not be acquired from an underivatized surface that had been incubated for 8 h in a 1 µg/mL solution of renin substrate with 2 M sodium acetate and washed with deionized water. These results show that an underivatized surface does not share the binding characteristics of the C18 surface. Finally, we were unable to acquire a spectrum by taking a 1 µL sample from the 1 µg/mL renin substrate/2 M sodium acetate solution and homogenizing it with 1 µL of matrix (as would be done in a conventional MALDI experiment). The absence of signal confirmed that conventional preparatory methods are not suitable for this sample. Analytical Chemistry, Vol. 69, No. 22, November 15, 1997

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Figure 5. MALDI spectrum of C18-isolated renin substrate from a 1 µg/mL solution of the peptide in 6 M urea.

Figure 6. (A) Conventional MALDI spectrum of an unknown peptide from T. cruzi-infected heart extract obtained from the nonvolatile HPLC eluent. (B) MALDI spectrum of an unknown peptide from T. cruzi-infected heart extract obtained from the nonvolatile HPLC eluent using the C18 methodology.

Samples dissolved in solutions containing high concentrations of chaotropic agents offer an attractive application of C18 MALDI, since many transmembrane and hydrophobic proteins are kept in solution using this class of compounds. Figure 5 shows a MALDI-MS spectrum of renin substrate isolated from a 1 µg/mL solution in 6 M urea using a C18-modified probe with an 8 h incubation period. Despite the high concentration of the nonvolatile chaotropic agent in this sample, the protonated molecular ion is the most abundant ion in the spectrum. This spectrum contains a variety of unidentifiable peaks perhaps due to contaminants in the urea, but the peak from protonated renin substrate is the most abundant and is easily recognizable. The molecular ion from renin substrate could not be detected from 6 M urea solutions when we lowered the peptide concentration to 1 ng/ mL. This result can be explained, as strong chaotropes (such as urea or guanidine hydrochloride) work by limiting the hydrogen bonding critical to the success of the hydrophobic interactions necessary for C18 isolation.21 Another application of C18-modified probes involves desalting HPLC fractions collected by using separation protocols employing nonvolatile buffers. Phosphate-containing buffers used with RPHPLC is an excellent example of a good separation strategy that is ordinarily avoided by bioanalytical chemists because of the problems associated with the nonvolatile mobile phase. The use of phosphate as an ion-pairing agent alters the selectivity and increases the resolution and recovery of RP-HPLC separations.22

Hence, we decided to incorporate a phosphate-containing buffer in our RP-HPLC separation protocol of MHC class I peptides obtained from T. cruzi-infected heart extracts because of the benefits of this buffer and the ability to analyze these fractions with the C18-modified MALDI probes. The conventional MALDI spectrum from one of these fractions is shown in Figure 6A. Unfortunately, the nonvolatile buffer prevented all of the peptides from being detected. The spectrum obtained from a C18-modified probe (Figure 6B), however, demonstrates that numerous peptides are in fact present in this fraction. These results indicate that the C18 MALDI method is superior to conventional MALDI for fractions collected from separation strategies employing nonvolatile buffers.

(21) Hermanson, G. T.; Mallia, A. K.; Smith, P. K. Immobilized Affinity Ligand Techniques; Academic Press, Inc.: San Diego, 1992; Chapter 4. (22) Guo, D.; Mant, C. T; Hodges, R. S. J. Chromatogr. 1987, 386, 205.

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ACKNOWLEDGMENT The authors thank Dr. Rick L. Tarleton of the University of Georgia Department of Cellular Biology for access to the Rainin HPLC, parasites, mice, and useful discussions. We also thank Dr. Mark Farmer of the University of Georgia Department of Cellular Biology for continuing access to his gold-sputtering apparatus. Finally, we greatly appreciate financial support from NIH Grant T32 AI07322 and NSF Grant 9626835, which made this project possible. Received for review June 23, 1997. Accepted September 11, 1997.X

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Abstract published in Advance ACS Abstracts, October 15, 1997.