Disposable Hydrophobic Surface on MALDI Targets for Enhancing MS

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Anal. Chem. 2005, 77, 6609-6617

Disposable Hydrophobic Surface on MALDI Targets for Enhancing MS and MS/MS Data of Peptides Rebekah L. Gundry,† Roy Edward,‡ Thomas P. Kole,§ Chris Sutton,‡ and Robert J. Cotter*,†

Department of Pharmacology and Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, and Shimadzu Biotech, Manchester, U.K.

Automated analyses in MALDI MS are complicated by the uneven distribution of analyte over the sample spot, resulting in areas of analyte localization, or “sweet spots”. Hence, the ability to concentrate and localize samples is advantageous, especially for automated studies involving low concentrations of analyte. A method for rapidly creating a removable and affordable hydrophobic surface that is free from memory effect is presented. The potential for such compounds to serve as a practical coating for MALDI targets is examined. An example compound with a complete methodology is shown to increase sample homogeneity, peak intensity, and resolution when used for peptide mixtures with CHCA and DHB.

with hazardous solvents (THF and hexane) or are difficult to remove via standard plate washing procedures, resulting in a memory effect. An alternative method for rapidly creating a removable and affordable hydrophobic surface that is free from memory effect is presented. The chemical additive used here has been successfully used in “self-cleaning” glass and paint, upon which water will bead and rinse away surface contaminants. The potential for this additive, SILCLEAN 3700, to serve as a practical coating for MALDI targets is examined. The ability for SILCLEAN 3700 to increase sample homogeneity, peak intensity, and resolution when used for peptide mixtures with CHCA (hot matrix) and DHB (cold matrix) is herein described.

A continuing challenge in MALDI MS is the lateral spread of sample and cocrystalline structures required for effective ionization. Typically, in MALDI MS, the analyte is distributed unevenly throughout the sample spot, resulting in areas of analyte localization, or “sweet spots”. The time required to search for these sweet spots can delay high-throughput or automated processes. Consequently, one of the compromises of automation is often an abridged search for sweet spots. This issue becomes increasingly critical as the concentration of available analyte decreases, and the number and quality of the sweet spots is thereby reduced. Hence, the ability to concentrate and localize samples is advantageous, especially for automated studies involving low concentrations of analyte. Several methods for on-target sample concentration have been developed to address this issue. Coatings such as Teflon,1 Scotch Gard,2 dodecylthiol,3 silicone,4 and pentadecafluorooctamido propyltrimethoxysilane5 have been used successfully to reduce sample spot size and increase sensitivity in MALDI analyses. However, these coatings often require complex preparation protocols, lengthy dry times (4-10 h for some), and dilution

EXPERIMENTAL SECTION Materials. All reagents were prepared fresh daily before use. SILCLEAN 3700 (1-methoxy-2-propyl acetate, Byk-chemie) was used as supplied. Matrixes (Sigma, St. Louis, MO) were prepared as saturated solutions in a variety of solvents (see Table 1). CHCA was diluted 1:1 with solvent prior to use. For optimization, reproducibility, and CHCA homogeneity studies, a mixture containing 250 fmol/µL of each of the following peptides (Sigma) in 0.1% aqueous TFA was prepared: bradykinin fragment (m/z 757.39), angiotensin II (m/z 1046.54), and P14R (m/z 1533.85). For the DHB homogeneity study, a mixture of 2.5 pmol/µL each peptide was prepared. The sample was diluted 1:1 with appropriate matrix prior to use. To examine the effects of SILCLEAN 3700 on a complex sample, which is often more challenging than purified peptides, a tryptic digest standard of β-casein (kindly provided by Microm Bioresources, Auburn, CA) was examined. The sample was digested in 25 mM ammonium bicarbonate buffer. A portion of the sample was quantitated by HPLC, and the digest was then accordingly diluted to 500 pmol/tube followed by lyophilization prior to shipment. Upon arrival, the samples were solubilized in 0.1% aqueous TFA to a final concentration of 400 fmol/µL. This sample did not undergo any further cleaning or desalting prior to use. Masking agents (J.T. Baker, Phillipsburg, NJ; Sigma) were standard reagent grade. Sample Preparation. SILCLEAN 3700 was used alone and in combination with a masking agent (Table 1). Masking involved applying 0.3 µL of the masking agent to the sample target immediately prior to spraying SILCLEAN 3700. The purpose of spotting the masking agent was to create a small bead of liquid that would repel the SILCLEAN 3700 such that the SILCLEAN 3700 would dry around the perimeter of the liquid masking agent.

* To whom correspondence should be addressed. Tel: 410-955-3022. Fax: 410-955-3420. E-mail: [email protected]. † Department of Pharmacology, Johns Hopkins University School of Medicine. ‡ Shimadzu Biotech. § Department of Oncology, Johns Hopkins University School of Medicine. (1) Schuerenberg, M.; Luebbert, C.; Eickhoff, H.; Kalkum, M.; Lehrach, H.; Nordhoff, E. Anal. Chem. 2000, 72, 3436-3442. (2) Owen, S. J.; Meier, F. S.; Brombacher, S.; Volmer, D. A. Rapid Commun. Mass Spectrom. 2003, 17, 2439-2449. (3) Konig, S.; Grote, J. Biotechniques 2002, 32, 912, 914-915. (4) Redeby, T.; Roeraade, J.; Emmer, A. Rapid Commun. Mass Spectrom. 2004, 18, 1161-1166. (5) Xiong, S.; Ding, Q.; Zhao, Z.; Chen, W.; Wang, G.; Liu, S. Proteomics 2003, 3, 265-272. 10.1021/ac050500g CCC: $30.25 Published on Web 09/14/2005

© 2005 American Chemical Society

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Table 1. Combinations of SILCLEAN 3700 and Masking Agent Used with Various Matrix and Matrix Solvents in the Optimization Experiments

Upon drying of the SILCLEAN 3700 and masking agent, the small area at the middle of the sample well would be free from SILCLEAN 3700. The volume of 0.3 µL was chosen because it can be reproducibly pipetted manually, which is essential for establishing this work as a practical method for laboratories without robotic spotting capabilities. While the use of SILCLEAN 3700 without masking agent did provide effective concentration 6610

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effects, the masking agent allowed for control over the final location of the sample analytes (Figure 1). As indicated in Table 1, several masking agents were washed with 5 × 2 µL of deionized H2O after drying to eliminate any deleterious effects. SILCLEAN 3700 was diluted 1:1, 1:5, 1:10, 1:25, or 1:50 with acetone and sprayed using 1, 2, or 3 passes over the sample plate with an atomizer (Horiba) onto the MALDI target. The SILCLEAN 3700

Figure 1. Photograph of DHB sample in well overlaid with raster pattern used for quantitation of sample homogeneity across sample spot. Locations (1-20) are labeled in order of acquisition.

and masking agent, if used, were allowed to dry 15 min at room temperature before 1 µL of sample/matrix solution was applied. For optimization experiments, each combination of SILCLEAN 3700, mask, and matrix was spotted in duplicate. As indicated in Table 1, 0.3 µL of 50 mM ammonium persulfate (AP) was added on top of selected samples before drying. Peak intensity, resolution and the presence of salt adducts were examined to determine the best sample preparation combinations. The most favorable conditions as determined by the optimization experiment were used for the homogeneity and reproducibility studies. To examine the limit of volume and organic content that can be retained in a well with SILCLEAN 3700, solutions of 10 mg/ ml CHCA in 50:50, 70:30, 80:20 ACN/ddH2O were prepared and 1-5 µl spotted on SILCLEAN 3700 with water masking and also onto a target with no SILCLEAN 3700. To determine whether the SILCLEAN 3700 would enhance sensitivity, the peptide mixture (250 fmol/µL of each peptide) was diluted 1:1, 1:2, 1:5 and 1:10 prior to mixing 1:1 with matrix. 1 µl of each mixture in either CHCA or DHB was spotted in duplicate onto SILCLEAN 3700 with water or AP masking. To assess the ability to completely remove SILCLEAN 3700 and associated samples on a stainless steel target, the following

standard washing procedure was followed (each step was performed in a ultrasonic shaking bath, 15-min duration): (I) the target was first wiped with a methanol-soaked tissue to remove loose material and then bathed sequentially in (II) 1% formic acid, (III) acetone, (IV) methanol, and (V) water and finally flooded with methanol, drained, and placed in a drier box /oven for 6090 min. Following this treatment, the target was tested for memory of coating or residual peptides. No evidence of this was seen (data not shown.) Targets were thereafter used for recoating with SILCLEAN 3700. Acquisition Methods. In all cases, a circular raster was used to mimic automated operation and to reveal any useful concentration effect. The diameter of raster was set to 1000 µm for all CHCA samples and 1500 µm for all DHB samples, due to the pronounced crystallization of matrix in the outer ring of the dried spot for those samples spotted without SILCLEAN 3700. The center of the raster pattern was set to the center of the dried sample spot. All analyses were performed on an AXIMA-CFR MALDI-TOF (Shimadzu Biotech, Manchester U.K.) mass spectrometer equipped with 50µm-diameter nitrogen laser. To determine the effect of SILCLEAN 3700 on sample homogeneity across a spot, three sample spots of each combination (Table 2) were plated. Spectra (20 laser shots) were acquired at each of the 20 different locations across the spot (Figure 1). For each of the 20 locations, intensity (mV), S/N, and resolution were recorded for each peptide. To determine the reproducibility of the benefits of SILCLEAN 3700 for peak intensity, 20 sample spots of each combination (Table 2) were plated and 50 profiles (containing the accumulated spectra from 5 laser shots per profile) each were averaged across the spot. For each spot, intensity (mV) and resolution were recorded for each peptide. For MS/MS analyses, 300 profiles containing 10 shots each were acquired. For all other analyses, 100 profiles containing 5 shots each were acquired. Statistical Analyses. Statistical parameters were evaluated using custom analysis programs written in Matlab. The peak intensity and resolution values for each condition were compared using the method of multiple comparisons to determine the reproducibility of each experimental treatment. Differences between SILCLEAN 3700 and no SILCLEAN 3700 conditions were

Table 2. Combinations of SILCLEAN 3700, Mask, and Matrix Preparation Used for Homogeneity and Reproducibility Studies study homogeneity

reproducibility

SILCLEAN 3700

masking agent

matrix

no no yes yes yes

no 50 mM AP no ddH2O 50 mM AP

CHCA CHCA CHCA CHCA CHCA

50:50 EtOH/ddH2O 50:50 EtOH/ddH2O 50:50 EtOH/ddH2O 50:50 EtOH/ddH2O 50:50 EtOH/ddH2O

HC-1 HC-2 HC-3 HC-4 HC-5

no no yes yes yes no yes yes no yes yes

no 50 mM AP no ddH2O 50 mM AP no ddH2O 50 mM AP no ddH2O 50 mM AP

DHB DHB DHB DHB DHB CHCA CHCA CHCA DHB DHB DHB

30:70 ACN/0.1% TFA 30:70 ACN/0.1% TFA 30:70 ACN/0.1% TFA 30:70 ACN/0.1% TFA 30:70 ACN/0.1% TFA 50:50 EtOH/ddH2O 50:50 EtOH/ddH2O 50:50 EtOH/ddH2O 30:70 ACN/0.1% TFA 30:70 ACN/0.1% TFA 30:70 ACN/0.1% TFA

HD-1 HD-2 HD-3 HD-4 HD-5 RC-1 RC-2 RC-3 RD-1 RD-2 RD-3

matrix solvent

combination

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Figure 2. Comparison of various SILCLEAN 3700 and masking agent combinations for a peptide mixture. Panel A shows peptide mixture in DHB. S/N for each peptide averaged over 3 spots per combination is indicated in italics. Panel B shows magnification of a peptide (m/z 1533.85) from the spectra in panel A. Panel C shows magnification of the same peptide (m/z 1533.85) as seen in various combinations with CHCA. Sodium and potassium adducts are indicated. In each panel, presence/absence of SILCLEAN 3700 ((), and masking agent are labeled.

determined to be significant if their p-value was less than 0.05. Sample heterogeneity was assessed using the normalized variance (σ2/µ2) to compare experimental treatments. RESULTS AND DISCUSSION Initial studies were directed toward optimizing the amount of SILCLEAN 3700 necessary to provide the most even coating with desired concentration effect yet minimal interference or background. Dilutions of 1:1, 1:10, 1:25, and 1:50 SILCLEAN 3700 in acetone were examined, and the 1:10 dilution with two passes of the atomizer provided the optimum conditions (data not shown). Not unlike most MALDI experiments, some unwanted background was observed when the sample was applied directly on top of the SILCLEAN 3700 (Figure 2A: SILCLEAN 3700, no mask). However, this background was not significantly more than that observed for sample alone, without SILCLEAN 3700 (Figure 2A: no SILCLEAN 3700, no mask). Similar results were obtained 6612 Analytical Chemistry, Vol. 77, No. 20, October 15, 2005

when sample was spotted directly to a well in which SILCLEAN 3700 had been sprayed with no masking agent but was rinsed with water before sample deposition (data not shown). Therefore, the potential for using a masking agent to eliminate any unwanted background was investigated in order to even further enhance the signal-to-noise ratio. Masking agents with varying degrees of viscosity and surface tension were examined for their ability to effectively mask the SILCLEAN 3700 and either enhance or at least not impair the spectra. While many of the masking agents successfully masked the SILCLEAN 3700, they also proved deleterious to the sample unless rinsed off the plate with water (glycerol, triethanolamine, poly(acrylic acid), glycols). With highthroughput analysis in mind, it was preferable to find a masking agent that did not require washing, preferably one that could also enhance the spectra. Use of octyl β-D-glucopyranoside, trifluroacetic acid, and the ammonium salts proved to be effective at

Figure 3. Photographs of dried 1-µL sample spots of DHB with various combinations of SILCLEAN 3700 and masking agent.

Figure 4. Average normalized peak intensity variance for each peptide under five conditions as listed in Table 2 for CHCA and DHB.

masking the SILCLEAN 3700 while also enhancing the quality of the spectra, with the ammonium salts providing the best enhancement. More specifically, while results with both ammonium bisulfate and ammonium persulfate gave substantially higher peak

intensities than the other masking agents, the ammonium persulfate was ultimately chosen as the ideal masking agent due to its added ability to eliminate salt adducts (Figure 2C), though water was also used in the remainder of the study for comparison Analytical Chemistry, Vol. 77, No. 20, October 15, 2005

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Table 3. Normalized Variance of Peak Intensity and Resolution for Each Peptide Across a Sample Spot for Various SILCLEAN 3700 and Masking Agent Combinations As Listed in Table 2a normalized variance of peak intensity

normalized variance of resolution

combo

peptide 1

peptide 2

peptide 3

average

peptide 1

peptide 2

peptide 3

average

HC-1 HC-2 HC-3 HC-4 HC-5 HD-1 HD-2 HD-3 HD-4 HD-5

0.56683 1.1379 0.41788 0.21666 0.89426 4.4709 6.2684 2.1609 4.4458 3.3026

0.7101 0.69792 0.18425 0.12454 0.20255 5.0552 5.432 1.6403 4.0251 4.1608

0.6775 0.8463 0.2688 0.22399 0.34284 6.1346 4.6472 3.7785 3.6575 4.8806

0.6515 0.8940 0.2903 0.1884 0.4799 5.2202 5.4492 2.5266 4.0428 4.1147

0.03287 0.16751 0.038787 0.01789 0.036802 1.335 0.8902 0.27279 0.69544 0.88798

0.044157 0.13065 0.074352 0.067218 0.031787 1.125 1.224 0.36156 0.88146 0.91299

0.15191 0.17824 0.11936 0.10723 0.049204 1.2043 1.3939 1.0656 0.81821 1.2953

0.0763 0.1588 0.0775 0.0641 0.0393 1.2214 1.1693 0.5666 0.7983 1.0320

a

Average variance for all three peptides is also listed.

Figure 5. Average intensity (column 1) and variance in peak intensity (column 2) across a sample spot in DHB for conditions HD-1-HD-5 as listed in Table 2. The 20 raster locations were grouped according to their position on the spot, edge, or center.

purposes. Optimum matrix solvents for use with SILCLEAN 3700 were determined to be 50:50 EtOH/ddH2O for CHCA and 30:70 ACN/0.1% TFA for DHB. After optimizing the plate preparation conditions, the effects of SILCLEAN 3700 on sample morphology, sample heterogeneity, peak intensities, and resolution were investigated. Sample morphology was examined under various combinations of SILCLEAN 3700 and masking agent for both matrixes. As can be seen for DHB (Figure 3), the diameter of the dried sample spot with SILCLEAN 3700 is visibly smaller than that without SILCLEAN 6614

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3700. Furthermore, the choice of masking agent does not appear to affect spot diameter. A constant challenge in MALDI MS is the heterogeneity present across a sample spot. The ability of SILCLEAN 3700 to decrease heterogeneity across a sample spot, or to localize the sample, was evaluated by calculating the normalized variance of peak intensity and resolution for each of the three peptides (Table 3). The overall variance for CHCA is much lower than that for DHB (Table 3 and Figure 4). This observation is not unexpected due to the fact that when DHB is used as the matrix the sample

Figure 6. Reproducible effect of SILCLEAN 3700 and masking agent on peak intensity of mixture of peptide standards. Average intensity (mV) for each peptide is shown. * indicates difference between that condition and no SILCLEAN 3700 is significant (p < 0.05); (() indicates presence or absence of SILCLEAN 3700.

tends to be localized to the outer ring of the dried spot. The normalized variance of peak intensity and resolution using

SILCLEAN 3700 alone (HC-3, HD-3) or with water mask (HC-4, HD-4) and AP mask (HC-5, HD-5) are lower than that for no SILCLEAN 3700 (HC-1, HD-1) for the same matrix preparation. The lowest variance for CHCA was observed for SILCLEAN 3700 with water mask. The lowest variance for DHB was observed for SILCLEAN 3700 with no mask. It is noted that the AP mask alone did not improve homogeneity for either matrix. In addition to the overall heterogeneity across the spot, the localization of sample to the center versus the outer edge was calculated for DHB (Figure 5). The 20 raster locations were grouped according to their position toward the center or the outer edge of the spot (Figure 5). The average intensity across the center locations was higher with SILCLEAN 3700 than without. Additionally, the average intensity with SILCLEAN 3700 was higher in the center than across the entire spot or the outer edge. Furthermore, the variance across the center locations was lower with SILCLEAN 3700 than without and similarly lower in the center than it was for the entire spot or the outer edge. Therefore, the SILCLEAN 3700 effectively concentrates the sample to the center of the well. In addition to heterogeneity, the effect of SILCLEAN 3700 on peak intensities was also examined. SILCLEAN 3700 with water and AP masking reproducibly and significantly increased the peak intensities for peptides in both matrixes (Figure 6). SILCLEAN 3700 with water mask yielded best results for CHCA, and SILCLEAN 3700 with AP mask yielded best results for DHB. It is noted that the AP mask alone, while it did increase peak intensity, did not yield intensities as high as those for AP mask combined with SILCLEAN 3700 (Figure 5, HD-2). Furthermore, the AP mask alone, without SILCLEAN 3700, did increase signal-to-noise ratio,

Figure 7. Spectra illustrating effect of SILCLEAN 3700 on peak intensity for low concentrations of three peptide mixture (1, 757.39 Da; 2, 1046.54 Da; 3, 1533.85 Da) in CHCA (A) and DHB (B). Peptides are labeled 1-3. Total peak intensities (mV) are provided for each condition. Presence or absence of SILCLEAN 3700 (() and masking agent are indicated.

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Figure 8. Spectra of 400 fmol of β-casein digest with and without SILCLEAN 3700 in CHCA (A) and DHB (B). Peak intensity (mV) of highest peak is provided. Phosphopeptide is marked (*). Presence or absence of SILCLEAN 3700 (() and masking agent are indicated.

Figure 9. MS/MS of phosphorylated peptide (m/z 2061.8) from 400 fmol of β-casein digest in DHB. Presence or absence of SILCLEAN 3700 (() and masking agent are indicated.

but not as highly as when coupled with SILCLEAN 3700 (Figure 2A, no SILCLEAN 3700, AP mask vs SILCLEAN 3700, AP mask). A practical limit of stainless steel is the volume of solution that can be retained in a well. This is exacerbated as the organic or nonpolar component of the sample increases, e.g., derived from eluent fractions of a HPLC gradient reach 70:30 organic/aqueous 6616 Analytical Chemistry, Vol. 77, No. 20, October 15, 2005

solution. As shown in Figure 1S (Supporting Information), the maximum volume of organic/aqueous solution that can be retained in a well sprayed with SILCLEAN 3700 on a water mask is 4 µL for 50:50, 3 µL for 70:30, and 2 µL for 80:20. On a stainless steel target with no SILCLEAN 3700 applied, 2 µL was the maximum for all organic concentrations that could be retained in

a well (data not shown). In addition to increasing the volume of sample that can be retained in a well, the SILCLEAN 3700 was also effective at increasing the peak intensities for low levels of peptide (63 and 11 fmol) in both CHCA and DHB (Figure 7). However, at 11 fmol, a higher background for SILCLEAN 3700 with AP mask is observed. Finally, the effects of SILCLEAN 3700 were tested on a tryptic digest of β-casein to examine its effects on more complex peptide mixtures and MS/MS spectra. Suppression effects and salt adducts are common unwanted consequences of uncleaned digest samples. For these studies, the sample was not cleaned/desalted prior to use in order to determine whether SILCLEAN 3700 could eliminate the need for this step that is typically required prior to MALDI MS analyses. Figure 8 shows a significant decrease in background and increase in peptide intensity for SILCLEAN 3700 with water and AP masking for both matrixes. Furthermore, in addition to improving peak intensity and resolution, SILCLEAN 3700 also significantly improves MS/MS spectra of low abundance and phosphorylated peptides (Figure 9).

does improve the quality of peptide spectra, some unwanted background is observed. When coupled with a water or AP mask, the spectra with SILCLEAN 3700 are markedly improved when compared to samples deposited on a standard stainless steel target. Additionally, use of an AP mask yields spectra with fewer salt adducts, resulting in less complicated spectra. Further to benefiting MALDI MS spectra of peptide mixtures, the quality of MS/MS spectra are also significantly improved with the use of SILCLEAN 3700 and masking agent. The on-target concentration of MALDI MS samples, as achieved with the use of SILCLEAN 3700, is clearly beneficial for automated analyses of samples containing low concentrations of peptides, akin to those samples routinely encountered in proteomics analyses.

CONCLUSIONS This rapid, uncomplicated protocol for creating a removable hydrophobic surface is an attractive approach for on-target concentration of MALDI MS samples for routine and automated analyses. SILCLEAN 3700 allows for a larger volume and higher organic content sample to be contained in a well while improving homogeneity, peak intensity, and peak resolution for peptide mixtures in MALDI MS. While SILCLEAN 3700 without masking

SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

ACKNOWLEDGMENT Michrom Bioresources (Auburn, CA) kindly donated the tryptic digest of β-casein. Funding for R.J.C. and R.L.G. provided by NIH grants N01HV28180 and R01GM64402.

Received for review March 24, 2005. Accepted August 8, 2005. AC050500G

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