A Solid Sample Preparation Method That Reduces Signal

Garden, R. W.; Sweedler, J. V. Anal. Chem. 2000, 72, 30−36. [ACS Full Text ACS Full Text ], [CAS]. (35) . Heterogeneity within MALDI samples as reve...
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Anal. Chem. 2001, 73, 625-631

A Solid Sample Preparation Method That Reduces Signal Suppression Effects in the MALDI Analysis of Peptides Michael Z. Wang and Michael C. Fitzgerald*

Department of Chemistry, Duke University, Durham, North Carolina, 27708

Here we report on the application of a solid-solid (SS) sample preparation protocol for the MALDI analysis of peptides and multicomponent peptide mixtures. Our results with a series of model peptides indicate that a SS MALDI sample preparation protocol is useful for the analysis of peptides in the 1-3 kDa mass range. MALDI mass spectra recorded for peptides in this size range using a SS sample preparation were of a quality comparable to spectra recorded using a conventional drieddroplet (DD) sample preparation. Our results with several model peptide mixtures indicate that one advantage of a SS sample preparation protocol for the MALDI analysis of peptides is that it can significantly reduce signal suppression effects in multicomponent mixtures. MALDI results obtained using a SS sample preparation protocol are also more reproducible than results obtained using a conventional DD sample preparation protocol. MALDI mass spectrometry has rapidly evolved as an effective technique for the mass analysis of a wide variety of biopolymers including peptides, proteins, carbohydrates, and nucleic acids.1,2 By isolating analyte molecules in an appropriate matrix and irradiating the sample with a high-intensity, pulsed laser beam, it is possible to generate intact, gas-phase ions of high molecular weight analytes with the technique. One of the most successful applications of MALDI has been in the area of peptide and protein analysis.3,4,5 The discovery of appropriate matrix compounds6-9 and the refinement of sample preparation procedures10-16 have * Corresponding author. Department of Chemistry, Box 90346, Duke University, Durham, NC 27708-0346. Tel: 919-660-1547. Fax: 919-660-1605. E-mail: [email protected]. (1) Karas, M.; Hillenkamp, F. Anal. Chem. 1988, 60, 2299. (2) Hillenkamp, F.; Karas, M.; Beavis, R. C.; Chait, B. T. Anal. Chem. 1991, 63, 1193-1202. (3) Wang, R.; Chait, B. T. Curr. Opin. Biotechnol. 1994, 5, 77-84. (4) Beavis, R. C.; Chait, B. T. Anal. Chem. 1990, 62, 1836-1840. (5) Stults, J. T. Curr. Opin. Struct. Biol. 1995, 5, 691-698. (6) Strupat, K.; Karas, M.; Hillenkamp, F. Int. J. Mass Spectrom. Ion Processes 1991, 111, 89-102. (7) Beavis, R. C.; Chait, B. T. Rapid Commun. Mass Spectrom. 1989, 3, 432435. (8) Beavis, R. C.; Chaudhury, T.; Chait, B. T. Org. Mass Spectrom. 1992, 27, 156-158. (9) Juhasz, P.; Costello, C. E.; Biemann, K. J. Am. Soc. Mass Spectom. 1993, 4, 399-409. (10) Gobom, J.; Nordhoff, E.; Mirgorodskaya, E.; Ekman, R.; Roepstorff, P. J. Mass Spectrom. 1999, 34, 105-116 (11) Winston, R. L.; Fitzgerald, M. C. Anal. Biochem. 1998, 262, 83-85. 10.1021/ac0009090 CCC: $20.00 Published on Web 12/22/2000

© 2001 American Chemical Society

made it possible to routinely acquire high-quality mass spectra of individual peptides and proteins. Moreover, the MALDI technique is generally applicable to a wide variety of peptides and proteins with no apparent limitations imposed by the size or structure (primary, secondary, or tertiary) of the sample. An important characteristic of MALDI is that analytes are ionized with relatively few charges (peptides and small proteins are typically ionized with only one or two charges). This makes the resulting mass spectra relatively simple to interpret. In theory, these unique charging characteristics make MALDI especially well suited to the analysis of multicomponent mixtures. In practice, the MALDI analysis of multicomponent peptide and protein mixtures has been complicated, because the different peptide and protein components of a mixture can experience preferential desorption and/or ionization in the MALDI process.17-20 In some mixtures, signal suppression effects can be so severe that certain peptides and protein analytes are not detected in the presence of others. Such discrimination effects are a severe limitation to MALDI applications that involve the analysis of complex peptide mixtures, such as in peptide ladder sequencing techniques, peptide mapping strategies, and quantitative analyses. Two types of signal suppression effects are generally observed in MALDI: (1) those that are related to contaminants in the sample, and (2) those that appear to be related to the physicochemical properties of the matrix and the sample. The presence of buffer contaminants such as salts, ionic detergents, and involatile solvents can adversely affect signal intensity and the resolving power of MALDI.21 This has led to the development of several strategies to eliminate these contaminants from MALDI samples. They have primarily included the application of micropurification protocols prior to sample deposition and the use on(12) Brockman, A. H.; Dodd, B. S.; Orlando, R. Anal. Chem. 1997, 69, 47164720. (13) Blackledge, J. A.; Alexander, A. J. Anal. Chem. 1995, 67, 843-848. (14) Zhang, H. Y.; Caprioli, R. M. J. Mass Spectrom. 1996, 31, 690-692. (15) Vorm, O.; Roepstorff, P.; Mann, M. Anal. Chem. 1994, 66, 3281-3287. (16) Kussman, M.; Nordhoff, E.; Rahbek-Nielsen, H.; Haebel, S.; Rossel-Larsen, M.; Jakobsen, L.; Gobom, J.; Mirgorodskaya, E.; Kroll-Kristensen, A.; Palm, L.; Roepstorff, P. J. Mass Spectrom. 1997, 32, 593-601. (17) Perkins, J. R.; Smith, B.; Gallagher, R. T.; Jones, D. S.; Davis, S. C.; Hoffman, A. D.; Tomer, K. B. J. Am. Soc. Mass Spectrom. 1993, 4, 670-684. (18) Billeci, T. M.; Stults, J. T. Anal. Chem. 1993, 65, 1709-1716. (19) Amado, F. M. L.; Dominigues, P.; Santa-Marques, M. G.; Ferrer-Correia, A. J.; Tomer, K. B. Rapid Commun. Mass Spectrom. 1997, 11, 1347-1352. (20) Kratzer, R.; Eckerskorn, C.; Karas, M.; Lottspeich F. Electrophoresis. 1998, 19, 1910-1919. (21) Beavis, R. C.; Chait, B. T. Methods Enzymol. 1996, 270, 519-551.

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target washing procedures after sample deposition.10-14,19 These protocols and procedures can significantly enhance the intensity and resolution of peptide and protein signals in MALDI. However, even when very pure samples are analyzed, the relative ion yields of different peptide and protein analytes can vary significantly. Studies have shown that the relative desorption and/or ionization peptides and proteins can be affected by predesorption factors, including the choice of matrix, the sample matrix composition, and pH, as well as the rates at which sample-matrix cocrystals are grown.22-24 The physicochemical properties of peptide and protein analytes (e.g., their charge, hydrophobicity, and solubility) can also influence their MALDI behavior when using conventional sample preparation methods.25-29 Here we report on the application of a solid-solid (SS) sample preparation protocol for the MALDI analysis of peptides, proteins, and multicomponent peptide mixtures. The protocol employed in this work is very similar to the SS sample preparation protocol recently reported by Skelton et al. for the MALDI analysis of insoluble polymers.30 Solid samples of matrix and analyte are finely ground with a mortar and pestle, and the resulting powder is affixed to a stainless steel sample stage using double-sided adhesive tape. Our results indicate that a SS sample preparation protocol is applicable to the MALDI analysis of peptides in the 1-3 kDa mass range. Moreover, our results with several model peptide mixtures demonstrate that one advantage of a SS sample preparation protocol for the MALDI analysis of peptides is that it can significantly reduce signal suppression effects in multicomponent peptide mixtures. EXPERIMENTAL SECTION Peptide Synthesis, Purification, and Quantitation. The model peptides I, II, and III of sequence GHFGIGGELASRARAGHFGIGGELASK, GHFGIGGELASK, and IAQIHILEGRSDEQK (respectively) were manually synthesized from protected L-amino acids in stepwise fashion using established, solid-phase peptide synthesis (SPPS) protocols for tert-butoxycarbonyl (Boc) chemistry.31 Appropriately protected Boc-amino acid derivatives were purchased from either Novabiochem (San Diego, CA) or Peptides International (Osaka, Japan). Side chain protection was Arg(Tos), Asp(OcHxl), Glu(OcHxl), Lys(2ClZ), Ser(Bzl), and His(Bom); where OcHxl is cyclohexyl, 2ClZ is 2-chlorobenzyloxycarbonyl, Bom is benzyloxymethyl, and Bzl is benzyl. All syntheses were initiated on pre-loaded Boc-L-Lys(2-Cl-Z)-Obzl-4-(carboxamidomethyl)-resin lysine resin from PE Biosystems (Foster City, CA). After chain assembly was complete, removal of side chain protecting groups and cleavage of peptide from the resin was achieved by treatment with anhydrous hydrofluoric acid (HF). (22) Xiang, F.; Beavis, R. C. Org. Mass Spectrom. 1993, 28, 1424-1429. (23) Cohen, S. L.; Chait, B. T. Anal. Chem. 1995, 68, 31-37. (24) Chou, J. Z.; Kreek, M. J.; Chait, B. T. J. Am. Soc. Mass. Spectrom. 1994, 5, 10-15. (25) Olumee, Z.; Sadeghi, M.; Tang, X.; Vertes, A. Rapid Commun. Mass Spectrom. 1995, 9, 744-752. (26) Olumee, Z.; Vertes, A. J. Phys. Chem. B 1998, 102, 6118-6122. (27) Zhu, Y. F.; Lee, K. L.; Tang, K.; Allman, S. L.; Taranenko, N. I.; Chen, C. H. Rapid Commun. Mass Spectrom. 1995, 9, 744-752. (28) Krause, E.; Wenschuh, H.; Jungbult, P. R. Anal. Chem. 1999, 71, 41604165. (29) Valero, M. L.; Giralt, E.; Andreu, D. Lett. Pept. Sci. 1999, 6, 109-115. (30) Skelton, R.; Dubois, F.; Zenobi, R. Anal. Chem. 2000, 72, 1707-1710. (31) Schnolzer, M.; Alewood, P.; Jones, A.; Alewood, D.; Kent, S. B. H. Int. J. Pept. Protein Res. 1992, 40, 180-193.

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After removal of HF under reduced pressure, the crude peptide products were precipitated and washed with anhydrous ether, dissolved in a 70% acetonitrile water solution containing 0.1% TFA diluted with water, and lyophilized. Peptides I, II, and III were purified by reversed-phase HPLC using a Rainin Dynamax system. Semipreparative RP HPLC was performed on a C18 Vydac column (1.0 × 25.0 cm, 300 Å) using a flow rate of 3 mL/min, 230-nm detection, and linear mobile phase gradients of buffer B in A (A ) 0.1% trifluoroacetic acid (TFA) in water; B ) 90% acetonitrile and 0.09% TFA in water). Linear mobile phase gradients of 10-50% buffer B in A were utilized for the purification of peptides I, II, and III (respectively). The BrAc-III peptide construct was prepared by treating side chain protected peptide-resin of III (approximately 355 mg of 0.63 mmol/g peptide-resin) with 239 mg of bromoacetic acid and 130 µL of diisopropylcarbodiimide in 1.5 mL of dichloromethane. This N-terminal bromoacetlyation reaction was allowed to proceed for 40 min. Ultimately, the peptide was deprotected and cleaved from the resin by treatment with HF. The lyophilized, crude synthetic product obtained from this solid-phase synthesis of BrAc-III was used directly in these studies without any further purification. The relative concentrations of the peptides in our multicomponent mixtures were established by analytical RP HPLC and peak area analyses. Analytical RP HPLC was performed on a C18 Vydac column (0.46 × 15.0 cm, 300 Å) using flow rates of 1 mL/min, 214-nm detection, and a linear mobile phase gradient of 0-67% buffer B in A. MALDI Sample Preparation. Peptide samples were prepared for MALDI analysis using either a conventional dried-droplet (DD) protocol or a SS sample preparation technique that we adapted from the solid-sample preparation method recently reported by Skelton et al. for the MALDI analysis of insoluble polymers.1,30 In both protocols, R-cyano-4-hydroxycinnamic acid (4HCCA) was employed as the matrix. 4HCCA was obtained in crystalline form from Aldrich Chemical Co. (Milwaukee, WI) and was used without further purification. For the DD protocol, the 4HCCA matrix was prepared as a saturated, aqueous solution that contained 50% acetonitrile and 0.1% TFA, and peptide samples were prepared in an aqueous solution that also contained 50% acetonitrile and 0.1% TFA. In the DD protocol, equal volumes of the peptide solution (approximately 2-10 µM in peptide) and the saturated 4HCCA matrix solution were mixed prior to depositing 2 µL of the matrix-peptide mixture on the MALDI sample stage where the solvent was evaporated under ambient conditions. In our SS preparation protocol, we combined 100-200 mg of solid 4HCCA matrix with approximately 0.1-2 mg of solid, lyophylized peptide. The solid, lyophylized peptide material that we used in SS preparations of the binary mixtures in this work was from the lyophilized powder from an equimolar solution of the appropriate peptides. Ultimately, solid matrix and peptide mixtures were finely ground with a mortar and pestle, and the resulting powder was affixed to the MALDI sample stage using double-sided adhesive tape. Any loose powder was removed under a gentle stream of nitrogen gas prior to inserting the sample stage into the mass spectrometer.

In some cases, DD sample preparations were prepared directly from SS sample preparations. In these cases, 2 mg of the finely ground SS sample containing matrix and peptide was dissolved in 0.5 mL of 50% buffer B in A. A 2-µL aliquot of the resulting solution was deposited on the MALDI sample stage, and the solvent was evaporated under ambient conditions. MALDI Instrumentation. All MALDI/MS mass spectra were acquired on a Voyager DE Biospectrometry Workstation (PerSeptive Biosystems, Inc., Framingham, MA) in the linear mode using a nitrogen laser (337 nm). Mass spectra were collected in the positive-ion mode using an acceleration voltage of 20 kV and a delay of 200 ns. Each spectrum obtained was the sum of approximately 50 laser shots. RESULTS AND DISCUSSION Peptide and Protein Analysis. We initially explored the utility of a SS sample preparation method for the MALDI analysis of polypeptides using a series of model peptides and proteins. The peptide and protein samples that we studied included: several synthetic peptides in the 1-7 kDa molecular mass range; lysozyme (mol. mass ) 14 327 Da); and bovine serum albumin (mol. mass ) 66 431 Da). No protein ion signals were detected for the bovine serum albumin sample using the SS preparation method, and the protein ion signals that we detected for polypeptide analytes in the 5-15 kDa molecular mass range were of relatively poor quality (low signal-to-noise ratio, S/N, and low resolution), as compared to spectra that we obtained on the same samples using a conventional DD sample preparation. In contrast to the poor results that we obtained using a SS sample preparation method for the MALDI analysis of these large peptides and proteins (mol. mass > 5 kDa), we were able to acquire good quality MALDI spectra of small peptides (mol. mass < 5 kDa) using a SS sample preparation protocol. The MALDI mass spectrum that we obtained for peptide I (mol. mass ) 2653.0 Da) using a SS sample preparation is shown in Figure 1A. For comparison, the MALDI mass spectrum that we recorded for peptide I using a conventional DD sample preparation protocol is shown in Figure 1B. Intense, highresolution ion signals corresponding to singly protonated peptide I molecules were detected in both of the spectra shown in Figure 1. The S/N ratio and the resolution (full width half-maximum, fwhm) of the major peak in Figure 1A were 62 and 450, respectively. The S/N and the resolution (fwhm) of the peptide ion signal in Figure 1B were 143 and 600, respectively. It is noteworthy that the S/N ratio and the resolution of the peptide ion signal in the MALDI mass spectrum generated from the SS sample (Figure 1A) were slightly lower than in the MALDI mass spectrum generated from the DD sample (Figure 1B). Although small, these differences in spectral quality were consistently observed between SS and DD samples. These observations suggest that peptide ionization by MALDI when a SS sample preparation is employed is less efficient than when a DD sample preparation is utilized. However, it is clear from our results above with peptide I and from our results with several other synthetic peptides in the 1-3 kDa size range (data not shown) that good quality MALDI spectra of small peptides can be acquired using a SS sample preparation protocol. Our success in analyzing small peptides using a SS sample preparation protocol is not surprising, considering the recent work of Horneffer et al. in which it was

Figure 1. MALDI mass spectra of peptide I (mol. mass ) 2653.0 Da) that was obtained when using (A) a SS sample preparation protocol and (B) a conventional DD sample preparation method.

shown that protein incorporation into matrix crystals of solid MALDI matrixes is helpful, but not a prerequisite, for MALDI.32 Salts such as sodium chloride (NaCl) are commonly associated with peptide samples. Therefore, we investigated how the presence of NaCl might affect spectral quality using our SS sample preparation technique. We prepared a series of SS MALDI samples of peptide I that contained increasing amounts of solid NaCl. In these experiments, 100-mg samples of solid matrix-peptide mixtures containing an approximate 1000-fold molar excess of matrix over peptide were combined with 0, 7, and 20 mg of solid NaCl. The resulting mixtures of solid matrix, solid peptide, and solid NaCl were each finely ground with a mortar and pestle. The resulting powders were fixed to a MALDI sample stage using double-sided adhesive tape. The MALDI mass spectra that we acquired on these SS sample preparations containing increasing amounts of NaCl are shown in Figure 2. The major peak in each mass spectrum shown in Figure 2 corresponds to singly protonated molecules of peptide I. The peaks in each spectra that are labeled with an asterisk (*) correspond to sodium adducts of the peptide. The number and intensity of these sodium adducts increased with increasing amounts of solid NaCl in the SS sample. When the amount of solid NaCl in the SS sample was less than 10% (w/w) the number and intensity of peaks due to salt formation was relatively low and comparable to the number and intensity of peaks due to salt formation in the MALDI analysis of DD samples (see Figure 2). At higher salt concentrations (>10% w/w), the number and intensity of peaks due to salt formation observed with SS MALDI samples was significantly higher than with DD samples containing comparable amounts of salt (see Figure 2). In a series of control experiments using our SS MALDI sample preparation method, we confirmed that the matrix was required for peptide ionization. No peptide ion signals were detected in the MALDI mass spectrum of peptide I when only solid NaCl and (32) Horneffer, V.; Dreisewerd, K.; Ludemann, H. C.; Hillenkamp, F.; Lage, M.; Strupat, K. Int. J. Mass Spectrom. 1999, 185/186/187, 859-870.

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Figure 2. MALDI mass spectra of peptide I (mol. mass ) 2653.0 Da) that was obtained when using SS DD sample preparation protocols. The spectra in a-c were acquired from SS sample preparations that contained 0, 7, and 20% NaCl (w/w), respectively. The spectra in a′-c′ were acquired from DD sample preparations that contained 0, 7, and 20% NaCl (w/w), respectively. The peaks labeled with an asterisk (*) correspond to sodium adducts of the peptide.

no matrix were used in our SS sample protocol. Likewise, we also did not detect MALDI ion signals of peptide I when the lyophilyzed peptide, finely ground with a mortar and pestle, was affixed directly to the adhesive tape on the sample stage. The MALDI matrix clearly plays an essential role in the desorption/ionization process. Mixture Analysis. One goal of this work was to determine if a SS sample preparation method would reduce signal suppression effects in the MALDI analysis of peptide mixtures. Studies have shown that the ionization efficiency of peptides and proteins in multicomponent mixtures can vary dramatically in MALDI when a conventional DD sample preparation method is used.18-20,23,24 It has been suggested that different peptide and protein samples may have a different propensity to incorporate into and/or associate with the matrix in a DD sample preparation due to their different physicochemical properties in the liquid phase.23 Such matrix exclusion events have been observed in several studies.33-35 We reasoned that the physicochemical properties of different peptides in the solid phase would be similar, and we hypothesized that a SS sample might reduce signal suppression effects in the MALDI analysis of multicomponent peptide mixtures. To determine if a SS sample preparation method would reduce signal suppression effects in the MALDI analysis of multicomponent peptide mixtures, we studied two model peptide mixtures. One mixture contained peptides I and II of sequence GHFGIGGELASRARAGHFGIGGELASK and GHFGIGGELASK, respectively. A second mixture contained peptides II and III in which the sequence of peptide III was IAQIHILEGRSDEQK. The amino acid sequences of peptides I, II, and III are derived from the primary amino acid sequence of 4-oxalocrotonate (4OT), a small bacterial enzyme that is currently under study in our laboratory. We first observed signal suppression effects in the MALDI analysis of peptides I, II, and III during the course of MALDI-based peptide mapping experiments with the 4OT enzyme molecule. (33) Yuqin D.; Whitttal, R. M.; Li, L. Anal. Chem. 1996, 68, 2494-2500. (34) Strupat, K.; Kampmeier, J.; Horneffer, V. Int. J. Mass Spectrom. Ion Processes 1997, 169, 43-50. (35) Garden, R. W.; Sweedler, J. V. Anal. Chem. 2000, 72, 30-36.

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Figure 3. MALDI mass spectra recorded for (A) an equimolar mixture of peptides I (mol. mass ) 2653.0 Da) and II (mol. mass ) 1171.3 Da) and (B) an equimolar mixture of peptides II (mol. mass ) 1171.3 Da) and III (mol. mass ) 1734.9 Da). Both spectra were acquired when using a conventional DD sample preparation method. The peak labeled ∆ in A is the [M + 2H]+2 ion of peptide I.

Shown in Figure 3 are the mass spectra that we obtained using a DD sample preparation for the MALDI analysis of an equimolar mixture of peptides I and II (Figure 3A) and for the MALDI analysis of an equimolar mixture of peptides II and III (Figure 3B). Although high quality (good S/N ratio and high resolution) MALDI mass spectra of peptides I, II, and III are easily acquired when each peptide is analyzed alone (data not shown), our MALDI results in Figure 3 show that the ion signal of peptide II is significantly suppressed when this peptide is analyzed in the presence of either peptide I or peptide III when using a DD sample preparation protocol. In Figure 3A, the signal intensity of the singly protonated peptide II ion is approximately 10% of the singly protonated peptide I ion. In 25 different MALDI spectra that we recorded for this sample, including 5 different spectra acquired at 5 different laser positions in 5 different DD sample preparations, the relative signal intensity ratio II/I (based on the relative signal intensities of the singly protonated molecules) varied from 0.01 to 0.25, with an average value of 0.08 ( 0.05. This was despite the equimolar concentrations of peptides I and II (2 pmol/µL/ peptide) in the sample. The relative concentration of the peptides in the sample was confirmed by RP HPLC and peak area analysis of a concentrated stock solution of the peptide mixture (data not shown). In Figure 3B, the signal intensity of the singly protonated peptide II molecules was also approximately 10% of the signal of the singly protonated peptide III molecules. In 25 different MALDI spectra that we recorded for this sample, including 5 different spectra acquired at 5 different laser positions in 5 different DD sample preparations, the relative signal intensity ratio II/III (based on the signal intensity of the singly protonated molecules) varied from 0.07 to 0.20 with an average value of 0.12 ( 0.03. This was, again, despite the equimolar concentrations of peptides II and III in the sample. The relative concentration of the peptide in this sample was also confirmed by RP HPLC analysis of a concentrated stock solution of the peptide mixture. It is noteworthy that the

Figure 4. MALDI mass spectra recorded for an equimolar mixture of peptides I and II when using (A) a SS sample preparation and (B) a DD sample preparation prepared directly from the SS sample in A.

peptide mixtures described above displayed the same signal suppression effects when different variations of the DD sample preparation protocol such as the fast evaporation method of Vorm et al. were used for sample preparation.15 Remarkably, the dramatic signal suppression effects that we observed with peptide II when it was analyzed in the presence of peptide I or III using a DD MALDI sample preparation were not observed when a SS sample preparation protocol was utilized. Typical MALDI mass spectra that we acquired for an equimolar, binary mixture of peptides I and II and for an equimolar, binary mixture of peptides II and III when using a SS sample preparation are shown in Figures 4A and 5A. The peptide material used in the SS sample preparations of these binary mixtures was the lyophylized powder from an equimolar solution of the appropriate peptides. Shown in Figures 4B and 5B are the results from the MALDI analysis of DD sample preparations that were prepared directly from the SS samples that were analyzed in Figures 4A and 5A. Each of these DD samples was prepared by dissolving 2 mg of the finely ground matrix-peptide powders from each SS sample in 0.5 mL of an aqueous solution containing 50% acetonitrile and 0.1% TFA. The resulting solutions were approximately 10 mM in matrix and 10 µM in total peptide. Ultimately, 2 µL of each matrix-peptide solution was deposited on the MALDI sample stage, where it was allowed to air-dry. Our results in Figures 4 and 5 show that the relative MALDI ion signal of peptide II in the presence of peptide I and in the presence of peptide III is significantly enhanced when a SS sample preparation method is used. The relative MALDI signal ratio of peptide II/I was approximately 1.6 ( 0.18 when using a SS sample preparation (Figure 4A), as compared to only 0.07 (0.03 when using a DD sample preparation protocol (Figure 4B). Similarly, the relative MALDI signal ratio of peptide II/III was approximately 0.77 ( 0.06 when a SS sample preparation was used (Figure 5A), as compared to only 0.13 ( 0.04 when a DD sample preparation protocol was utilized (Figure 5B). A SS sample preparation protocol did not completely eliminate signal suppression effects in the MALDI analysis of our model

Figure 5. MALDI mass spectra recorded for an equimolar mixture of peptides II and III when using (A) a SS sample preparation and (B) a DD sample preparation prepared directly from the SS sample in A.

binary peptide mixtures. However, such effects were significantly reduced using a SS sample preparation, as compared to a DD sample preparation. These results suggest that the differential associations of peptides with matrix crystals in DD MALDI sample preparations can be a significant factor in defining the relative desorption/ionization efficiency of peptides in multicomponent mixtures. However, it is clear that they are not the only factor. Other factors such as proton affinity are likely to play a role as well. This, for example, may explain why, even when using the SS sample preparation for the analysis of an equimolar peptide III and II mixture, the MALDI ion signal of peptide II was still suppressed, as compared to the MALDI ion signal of peptide III. Unlike peptide III, peptide II does not have an arginine residue in its amino acid sequence. Arginine has the highest proton affinity of any of the 20 naturally occurring amino acids.36 The relative MALDI ion yields that we observed for the peptides in our model mixtures were very reproducible using a SS sample preparation protocol. The relative standard deviations that we recorded for the MALDI signal ratio of peptide II/I and for the MALDI signal ratio of peptides II/III were 11.3% and 7.8%, respectively, when our SS sample preparation method was used. These relative standard deviations are significantly less than the values of 42.9% and 30.7% that we observed when the DD method was used for sample preparation. These results are consistent with the SS sample preparation’s being more homogeneous in peptide and matrix. Peptide analytes appear to be more evenly dispersed in the finely ground powder of SS MALDI samples than they are in the peptide-matrix crystals formed in DD MALDI samples. Peptide-matrix inhomogeneities in DD sample preparations can result from several factors, including the differential rates of solvent evaporation (i.e., evaporation rates are slower in the center of the droplet, as compared to the edges) and the differential association/incorporation rates of peptide analytes with the matrix (36) Bojesen, G.; Breindahl, T. B. J. Chem. Soc., Perkin Trans. 1994, 2, 10291037.

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during the crystallization process. Both of these effects would be minimized with a SS sample preparation procedure. This likely explains why the SS sample preparation method yielded more reproducible MALDI results. Applications. Our results with the model peptide mixtures in this work demonstrate that there is some advantage to be gained by using a SS sample preparation for the MALDI analysis of multicomponent peptide mixtures. When compared to conventional DD sample preparation methods, a SS sample preparation protocol can provide a more accurate representation of the peptide components that are present in a given mixture by increasing shotto-shot sample reproducibility and by reducing signal suppression effects. We have observed these advantages in the two model peptide mixtures shown above, as well as with several other welldefined peptide mixtures and other synthetic product mixtures. Our results with the model peptide mixtures described above and those with several additional samples suggest that a SS preparation protocol is generally a useful approach for increasing shot-to-shot sample reproducibility and for reducing signal suppression effects in the MALDI analysis of peptides. However, one drawback to using a SS preparation protocol for the MALDI analysis of peptides is that it requires relatively large amounts of sample. We typically used 0.1 to 2 mg quantities of lyophylized, solid peptide in our SS sample preparations. The use of smaller quantities of peptide in a SS sample is not feasible, due to difficulties associated with the manipulation of 0.1 mg) are available for analysis. One important application in which a SS sample preparation protocol is potentially useful is in the MALDI analysis of synthetic peptides. Peptides are generally synthesized by stepwise solidphase peptide synthesis (SPPS) methods in relatively large quantities (mg to g quantities are typical). Therefore, it is straightforward to analyze the lyophilized crude synthetic products from a particular synthesis using a SS sample preparation protocol. The MALDI analysis of crude synthetic products from SPPS can provide important information about the overall quality of a synthetic peptide product. However, in order to accurately assess the quality of a synthetic peptide by MALDI, it is important to have all the products and coproducts of a particular synthesis accurately represented in a MALDI mass spectrum. Signal suppression effects can potentially lead to an inaccurate representation of the relative product yield if such a measurement is based solely on the relative ion signals detected in a MALDI mass spectrum. Our results with the model peptide mixtures described above indicate that a SS sample preparation could be useful for the MALDI analysis of synthetic peptide products to give an accurate representation of the overall product yield and to define specific problems association with a particular synthesis (i.e., incomplete coupling reactions or incomplete deprotection). Shown in Figure 6 are the MALDI mass spectra that we obtained with a crude synthetic peptide sample when using a SS sample preparation protocol (Figure 6A) and a DD sample preparation protocol (Figure 6B,C). The synthetic peptide product, BrAc-III, that we analyzed in Figure 6 was a chemically modified version of peptide III in which the N-terminus of peptide III was bromoacetylated. The BrAc-III peptide construct was synthesized 630 Analytical Chemistry, Vol. 73, No. 3, February 1, 2001

Figure 6. MALDI mass spectra recorded for a crude synthetic peptide product mixture when using (A) a SS sample preparation protocol, (B) a DD sample preparation method utilizing ∼40 pmol of total peptide product, and (C) a DD sample preparation method utilizing ∼4 pmol of total peptide product. The major products from the synthesis were peptide III and BrAc-III. Other more minor peptide products from the synthesis are identified in the spectra according to their m/z.

on the solid phase from appropriately protected L-amino acids using standard SPPS protocol for Boc-chemistry. The N-terminal bromoacetylation reaction that was used to prepare BrAc-III was purposely not allowed to proceed to completion so that we could generate a mixture of peptide III and the BrAc-III construct. As expected, our MALDI analysis of the crude synthetic product obtained from the BrAc-III synthesis that was described above indicated that the N-terminal bromoacetylation reaction did not go to completion. The two most intense ion signals in each of the spectra shown in Figure 6 correspond to singly protonated molecules of peptide III and BrAc-III. A series of other, more minor peptide products are also detected in each spectrum. The relative peak intensities of the different peptide products varied significantly, depending on how the MALDI sample was prepared. This variability in peak intensities was most dramatic for the two major synthetic products, peptide III and BrAc-III. In 25 different spectra that we recorded for this synthetic peptide sample when using a SS sample preparation protocol (see Figure 6A for a typical spectrum), the relative signal intensity ratio BrAc-III/III (based on the signal intensity of the singly protonated molecules) was 0.43 ( 0.04. In 25 different spectra that we recorded for this sample when using a concentrated sample solution (approximately 20 µM in total peptide) and a DD sample preparation method (see Figure

6B for a typical spectrum), the relative signal intensity ratio BrAcIII/III (based on the signal intensity of the singly protonated molecules) was 1.37 ( 0.23. In 25 different spectra that we recorded for this sample when using a dilute sample solution (approximately 2 µM in total peptide) and a DD sample preparation method (see Figure 6C for a typical spectrum), the relative signal intensity ratio BrAc-III/III (based on the signal intensity of the singly protonated molecules) was 0.67 ( 0.11. Concentration-dependent MALDI results have been reported for other peptide mixtures when a DD sample preparation was used.18,23 Clearly, this phenomenon can be problematic for quantitative analyses. It is noteworthy that our SS sample preparation method was not highly sensitive to the total protein content of the sample. MALDI spectra that were comparable to the spectrum shown in Figure 6A were obtained using as little as 0.1 mg or as much as 10 mg of the crude synthetic peptide in the SS sample preparation. The SS MALDI sample preparation of the crude synthetic peptide also gave the most accurate and precise representation of the two major peptide products in the sample, peptide III and the BrA-III peptide construct. RP HPLC and peak area analysis of the crude synthetic peptide product mixture revealed that the relative ratio of BrAc-III to III in the sample was 0.55. This is in relatively good agreement with the relative ratio value of 0.43 calculated from the MALDI mass spectrum that was acquired from the SS sample preparation. A relative ratio value

of 0.67 was calculated from the MALDI mass spectrum acquired using the DD sample preparation method on the dilute sample (Figure 6C). This value is also relatively close to the value calculated by RP HPLC analysis; however, the relative standard deviation of the measurement was greater than that observed when a SS sample was analyzed. These results are consistent with those that we obtained with the model binary peptide mixtures in this study. CONCLUSIONS We have demonstrated that a SS sample preparation protocol is applicable to the MALDI analysis of small (