[23] Laser desorption mass spectrometry

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ANALYTICALTECHNIQUES

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[23] Laser Desorption Mass Spectrometry By WILLIAM T. MOORE Introduction A full characterization of a synthetic peptide product requires interrogation of structure at several levels using an array of orthogonal techniques. The more complex the technique array, the more stringent the analysis and the most complete the picture of and confidence in the structure. Synthetic peptide products that have interesting biological properties and that will serve as templates for pharmacological design of mimetics will undergo the most extensive characterizations. An extensive analysis includes amino acid analysis, reversed-phase high-performance liquid chromatography (HPLC) using different solvent systems and column matrices, possibly capillary zone electrophoresis (CZE) involving different buffer systems, elemental analysis, mass spectrometry (MS) using different ionization methods and mass filters, and, at the most structurally informative end of the analysis array, three-dimensional (3D) structural analyses using twodimensional (2D) nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography. This extensive range of analytical techniques requires a heavy investment in both time and resources, often the cooperation of individuals in geographically separated groups, and is reserved and justified for only the most interesting model structures that lead to fundamental understanding of biomedically important structure-function relationship problems. However, synthetic peptides not characterized to this degree are often employed in the early phase of a scientific project and are considered by most biomedical investigators to be readily available from either commercial or local core facilities as routine and reliable tools. There is often a mistaken core facility client "vending machine mentality" that assumes so many dollars in, so much peptide out in about the same time that it requires one to acquire their favorite beverage. At the service side, often peptide synthesis is provided by busy and overworked operators who often necessarily have to approach their automated synthesizers with a "black box mentality" with the hope and faith that the instrument reliably performs as promised by the instrument manufacturer. In the recent past, both inside and outside the peptide synthesis core facility, nonpeptide chemists have often taken too much for granted con-

METHODS IN ENZYMOLOGY, VOL. 289

Copyright © 1997 by Academic Press All rights of reproduction in any form reserved. 0076-6879/97 $25.00

[23]

LASER DESORPTION MASS SPECTROMETRY

521

cerning the quality of the peptides that are used to initiate their studies. Peptide synthesis analytical surveys performed in the early 1990s under the auspices of the Association of Biomolecular Resource Facilities (ABRF) have demonstrated the need for a serious reality check. The 1991 A B R F peptide synthesis study 1 of a routine synthetic peptide problem indicated that 17% of the volunteered products had no desired product present and that only 28% of crude products submitted had greater than 75% desired product and only 68% of purified products had desired product in excess of 75%. The next survey2 on a simple synthesis problem showed slight improvement; however, 48% of crude preparations and 21% purified products still contained less than 75% of the desired product. More recent A B R F peptide synthesis surveys3-6 have indicated steady improvement, 6a which has been attributed to instrumental design improvements, more reliable chemistries and reagents provided by commercial sources, and, it is hoped, a heightened awareness of quality control in the minds of those associated with peptide synthesis core laboratories. One positive contributing factor I want to stress is the increased involvement of MS in peptide synthesis core facilities. Either mass spectrometric instrumentation has been acquired by peptide synthesis core facilities or working relationships have been established between peptide synthesis laboratories and MS laboratories. However, although the more recent surveys reveal continuing improvement, the studies indicate the need for constant quality control vigilance. Isolated errors often crop up unexpectedly. Quality control vigilance is aided by MS. 1 A. J. Smith, J. D. Young, S. A. Carr, D. R. Marshak, L. C. Williams, and K. R. Williams, in "Techniques in Protein Chemistry III" (R. H. Angeletti, ed.), p. 219. Academic Press, San Diego, 1992. 2 G. B. Fields, S. A. Carr, D. R. Marshak, A. J. Smith, J. T. Stults, L. C. Williams, K. R. Williams, and J. D. Young, in "Techniques in Protein Chemistry IV" (R. H. Angeletti, ed.), p. 227. Academic Press, San Diego, 1993. 3 G. B. Fields, R. H. Angeletti, S. A. Carr, A. J. Smith, J. T. Stults, L. C. Williams, and J. D. Young, in "Techniques in Protein Chemistry V" (J. W. Crabb, ed.), p. 501. Academic Press, San Diego, 1994. 4 G. B. Fields, R. H. Angeletti, L. F. Bonewald, G. B. Fields, J. S. McMurray, W. T. Moore, J. T. Stults, and L. C. Williams, in "Techniques in Protein Chemistry VI" (J. W. Crabb, ed.), p. 539. Academic Press, San Diego, 1995. s R. H. Angeletti, L. Bibbs, L. F. Bonewald, G. B. Fields, J. S. McMurray, W. T. Moore, and J. T. Stults, in "Techniques in Protein Chemistry VII" (D. R. Marshak, ed.), p. 261. Academic Press, San Diego, 1996. 6 R. H. Angeletti, L. Bibbs, L. F. Bonewald, G. B. Fields, J. W. Kelly, J. S. McMurray, W. T. Moore, and S. T. Weintraub, in "Techniques in Protein Chemistry VIII" (D. R. Marshak, ed.), p. 875. Academic Press, San Diego, 1997. 6a R. H. Angeletti, L. F. Bonewald, and G. B. Fields, Methods Enzymol. 289, [32], this volume (1997).

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The remainder of this article concerns the use of only one type of MS, matrix-assisted laser desorption-ionization mass spectrometry (MALDI-MS). The objective of this article is to demonstrate that M A L D I MS is a valuable analytical tool for monitoring peptide synthesis. I hope to show that this form of MS can be applied successfully even at a minimalist level and that it can be a satisfactory approach to evaluate the progress and validate the outcome of most routine synthetic peptide problems that may be encountered in a typical protein laboratory. Examples of M A L D I MS integration into quality control of automated peptide synthesis are presented, pointing out advantages and pitfalls where necessary. Other MSs using other ionization methods and offering enhanced MS/MS capabilities are presented elsewhere in this volume. 6b,c I want to caution that there is no single analytical method for characterization of a synthetic peptide preparation and to advise at the outset that MS should be considered a qualitative method and should only be considered semiquantitative at best. No single analytical method substitutes for an all-encompassing orthogonal approach for the highest analytical stringency. This article illustrates approaches that one can take with a lower cost pioneer instrument lacking costly high-end research instrument refinements to maximize the characterization of a peptide synthesis. Examples of M A L D I - M S characterization of crude preparations and purified preparations derived from analytical and preparative H P L C are presented. Practical issues concerning matrix preparation, sample preparation, matrix selection, mass discrimination, and ion suppression problems as a function of peptide concentration are addressed as well as the use of mass shift assays for clarifying chemistries which would normally require high resolution that might not necessarily be available using external mass spectrometric calibrations. Description of Matrix-Assisted L a s e r D e s o r p t i o n - I o n i z a t i o n Mass S p e c t r o m e t r y Matrix-assisted laser desorption-ionization mass spectrometry (MALDI-MS) is an outgrowth of the direct laser desorption mass spectrometry (LD-MS) of small organic molecules that was initially developed in the 1960s and 1970s. 7'8 Karas and Hillenkamp introduced M A L D I - M S in 6bS. Beranovfi-Giorgianniand D. M. Desiderio, Methods Enzymol. 289, [21], this volume (1997). 6~D. J. Burdick and J. T. Stults, Methods Enzymol. 289, [22], this volume (1997). 7F. J. Vastola, R. O. Mumma, and A. J. Pinone, Org. Mass Spectrom. 3, 101 (1970). 8 M. A. Posthumus, P. G. Kistemaker, H. L. C. Meazelaar, and M. C. ten Neuver de Brauw, Anal Chem. 50, 985 (1978).

[231

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LASER DESORPTION MASS SPECTROMETRY

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19879'1° when they demonstrated that adding a small molecular weight organic acid matrix to an analyte could overcome molecular photodissociation of the sample ions induced by direct laser irradiation of the sample. The Hillenkamp group at Munster and the Chait group at Rockefeller University pioneered the peptide and protein application of this technique through instrument, matrix, and sample handling development, n A major advantage of MALDI-MS is that the mass range extends from the low molecular weight range to the very high molecular weight range up to 200,000 and greater. Time-of-flight (TOF) mass analyzers are suitable for these mass ranges, and several MALDI-MS instruments are commercially available, ranging from the user-friendly benchtop-dimension instruments to stand alone platform research grade instruments with multiple features such as automated sample introduction, multiple lasers, resolution improving reflectron ion mode and "delayed extraction" capabilities, and tandem MS abilities using post source decay analysis to detect fragmentation of a peptide yielding sequence ions permitting primary structure information. A product review of MALDI-MS instruments was published in Analytical Chemistry in 1995.12 The least complex instrument, a typical linear mode MALDI/TOF-MS system, is shown in Fig. 1. Such a system is suitable for monitoring most problems encountered in solid-phase peptide synthesis laboratories. Sample M. Karas, D. Bachmann, U. Bahr, and F. Hillenkamp, Int. J. Mass Spectrom. Ion Proc. 78, 53 (1987). 10 M. Karas and F. Hillenkamp, A n a l Chem. 60, 2299 (1988). 11 F. Hillenkamp, M. Karas, R. C. Beavis, and B. T. Chait, A n a l Chem. 63, 1193A (1991). 12 D. Noble, A n a l Chem. 67, 497A (1995).

524

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is allowed to cocrystallize with matrix on a target prior to introduction into the source of the mass spectrometer. Through a set of fixed optics a pulsed laser beam from a nitrogen laser (337 nm) is directed to the target surface for irradiation of the sample. Light energy absorbed mostly by the matrix induces an explosive ejection of a plume of matrix and analyte ions into the vacuum of the source. Ions are extracted and accelerated by a strong electric field (of the order of 25 to 30 KeV) and the ion packets are focused by a set of high-voltage plates (Einzel lenses) into the field-free region of the flight tube. All ions are given the same kinetic energy, and thereby their respective velocities are mass dependent and reach a multichannel plate electron multiplier ion detector at different times measured in microseconds, with smaller molecular weight ions reaching the detector before larger molecular weight ions. Transient signal recordings representing the spectra are then summed and processed into graphical mass spectra by a computerized data system. The instrument may be calibrated with well-characterized synthetic peptides, known proteins such as cytochrome c, and at the low mass end ions derived from the matrix. The highest mass accuracy is achieved with the use of internal standards; however, the use of this type of calibrant can be problematic owing to ion suppression effects induced by the presence of the calibrant and the time involved to empirically derive proper proportions of calibrant and analyte to minimize these effects and achieve good peak shape for all components, analytes, and calibrants. External calibrations performed at times close to the unknown sample acquisition time in a "pioneer" linear mode instrument such as that depicted in Fig. 1 having resolutions of around 200 yield, in practice, mass accuracy errors of _+0.1 to _+0.5%, which translates to mass error of 1 to 3 amu for most synthetic peptide products. For most synthetic peptide problems where there is foreknowledge of desired product this level of error may be tolerated. Those problems that call for I amu accuracy or less, such as evaluation of deamidation or disulfide formation, require the development of a mass shift assay to clarify the small mass change reactive groups or analysis on a higher resolution instrument. An example of a disulfide formation problem is presented later to illustrate the use of a mass shift assay for analytical clarification when using a low-resolution instrument. Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry Sample Preparation and Matrix Considerations Several matrices are available for MALDI-MS, and various ones are more appropriate for the specific type of analyteJ I Choice of matrix and method of sample deposition remain largely empirical. For synthetic peptide

[23]

LASER DESORPTION MASS SPECTROMETRY

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work it is convenient to work with primarily two matrices, a-cyano-4hydroxycinnamic acid (CHCA) 13 and 2-(4-hydroxyphenylazo)benzoic acid (HABA)) 4 CHCA is usually suitable for smaller peptides (<4000) and is the matrix of choice for most synthetic peptide evaluations. HABA is suitable for larger peptides (>4000) and for some peptides that are not well visualized in CHCA. One has to be cautioned, though, against mass discrimination effects of these matrices. HABA will discriminate for larger peptides and against smaller peptides, and CHCA, vice versa. These matrices are also suitable for peptides that are soluble in the solvent 50% acetonitrile and 0.1% trifluoroacetic acid [50% ACN, 0.1% TFA (v/v)]. The CHCA matrix is prepared by making the solvent 50% acetonitrile, 0.1% TFA saturated in CHCAJ 3 The HABA matrix is prepared by dissolving 10 mg of HABA per 15 ml of 50% acetonitrile as suggested in the original report describing the use of this matrixJ 4 Extremely hydrophobic peptides that are not soluble in 50% acetonitrile and 0.1% TFA will be tested in alternative solvents such as 60% chloroform and 40% methanol, neat methanol, or even tetrahydrofuran or dichloromethane. A matrix prepared in the appropriate solvent chosen for the peptide is used for sample preparation. It should be noted that in 1996 we received from vendors CHCA batches of lower quality than that originally obtained earlier. The more recent material has a darker brownish color when compared to the original lighter yellow preparations and if used directly results in the acquisition of poor spectra with at least a 10-fold loss in sensitivity. The material may be recrystallized, but we have found it convenient to simply extract-wash it one time with 50% acetonitrile, 0.1% TFA. This can be simply done in a 1.5-ml graduated Eppendorf tube by adding matrix powder to the 0.25-ml mark (-80 to 100 mg), vortexing, suspending in 1.4 ml 50% acetonitrile, 0.1% TFA for 30 sec, and discarding the first supernatant after centrifugation. Vortex resuspension of the wash-extracted pellet in a 1.4-ml fresh aliquot of 50% acetronitrile, 0.1% TFA saturates the fresh solvent aliquot in the remaining wash-extracted CHCA, and, after centrifugation, the supernatant serves an appropriate matrix with a 10- to 30-fold improvement in sensitivity. Figure 2A shows the differences in the CHCA-related matrix ions for the unextracted and wash-extracted CHCA. Figure 2A (top) shows dominant ion signals at masses 165 and 102. Figure 2A (bottom) indicates a marked reduction of the 165 and 102 ion signals and the dominance of the ion signals usually observed with higher quality CHCA, especially the 190 13 R. C. Beavis, T. Chaudhary, and B. T. Chait, Org. Mass Spectrom. 27, 156 (1992). 14 p. Juhasz, C. E. Costello, and K. Bieman, J. Am. Soc. Mass Spectrom. 4, 399 (1993).

526

[23]

ANALYTICALTECHNIQUES 100%

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FIG. 2. R e s t o r a t i o n o f m a t r i x e f f i c i e n c y b y simple w a s h - e x t r a c t i o n o f the l o w e r q u a l i t y d a r k e r y e l l o w batches o f c y a n o - 4 - h y d r o x y c i n n a m i c acid ( C H C A ) that are p r e s e n t l y c o m m e r c i a l l y available. ( A ) Top: M a t r i x b a c k g r o u n d f o r the l o w e r q u a l i t y C H C A ; bottom: m a t r i x b a c k g r o u n d o b s e r v e d a f t e r the w a s h - e x t r a c t i o n step. ( B ) Top: M A L D I - M S analysis o f 2 p m o l o f a test p e p t i d e in the m a t r i x p r i o r to w a s h - e x t r a c t i o n ; bottom: result o b t a i n e d f o r 2 p m o ] o f test p e p t i d e f o l l o w i n g w a s h - e x t r a c t i o n .

[23]

L A S E R D E S O R P T I O N MASS S P E C T R O M E T R Y

527

and 379 ion signals representing the (M + H) + for the monomer and dimer of CHCA, respectively. To demonstrate the effect of the CHCA wash-extraction on the restoration of the sensitivity of detection, we compared analyses of a synthetic peptide. Figure 2B shows the MALDI-MS analysis of 2 pmol of the 45-residue synthetic peptide Cys-Cys-His-HisGly-Gly-Arg-Arg-Gly-Gly-Thr-Thr-Cys-Cys-Asn-Asn-Tyr-Tyr-Tyr-TyrSer-Asn-Ser-Ser-Tyr-Tyr-Ser-Ser-Phe-Phe-Trp-Leu-Ala-Ser-Leu-AsnPro-Glu-Arg-Met-Phe-Arg-Lys-Pro-Pro. Figure 2B (top) shows MALDI-MS analysis of the 2 pmol of peptide spotted in the poorer quality CHCA matrix directly. Figure 2B (bottom) shows the restoration of sensitivity after the simple wash-extraction step. At least a 10- to 30-fold increase in sensitivity is achieved. A drawback to MALDI-MS is that the quantitation or the relative signal height intensities of the peptide components revealed in a MALDI-MS profile can be affected by peptide concentration. 4 To address this problem in our laboratory and to carefully assess the complexity and quantity of components that we may find in a peptide product, we examine the peptide product at different concentrations. For a typical analysis of a synthetic peptide preparation we prepare synthetic peptide solutions at 1 mg/ml and perform serial dilutions at 1/10, 1/100, and 1/1000 in the matrix and interrogate the peptide at the three dilutions. These dilutions represent peptide amounts ranging from a few picomoles to hundreds of picomoles of product. If the peptide is relatively pure, similar spectra are observed for all three dilutions. Figure 3A shows a MALDI-MS analysis of an HPLCpurified preparation of the 45-residue peptide described above at the 2, 20, and 200 pmol level (top to bottom, respectively). Essentially the identical ion profile is observed in each spectrum. The only differences in the spectra are the emergences of the doubly charged ions for the peptide at the more dilute concentrations. If the synthetic peptide product arising from a problematic synthesis turns out to be a complex mixture, it is essential to interrogate at serial dilution to evaluate the quantitative relationships of the components by permitting the release of ion suppression phenomena that are dependent on peptide concentration. Different ion signal intensities are often observed for the same component in the spectra for the diluted samples owing to ion suppression effects. Figure 3B shows the MALDI-MS interrogation at dilution of an attempt to synthesize a 22-residue peptide having the sequence Ala-Pro-Val-Gly-Leu-Val-Ala-Arg-Leu-Ala-Asp-GluSer-Gly-His-Val-Val-Leu-Arg-Trp-Leu-Pro [theoretical (M + H) ÷ = 2357]. Figure 3B (top) represents approximately 500 pmol of the mixture, and Fig. 3B (middle and bottom) represents 50 and 5 pmol, respectively.

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123]

LASER DESORPTION MASS SPECTROMETRY

529

The MALDI-MS analysis (Fig. 3B) indicated that the particular synthesis was extremely problematic. The multitude of ion signals are consistent with a very complex mixture of various amounts of deletion products lacking in various combinations of Pro, Leu, Val, Trp, Arg, His, and Asp residues. The varying ion signal intensities for the respective components depending on the concentration of the peptide mixture also provide a good example for demonstrating the limitations of quantitative estimation from such data. Misleading ion suppression and mass discrimination effects are observed in this set of comparative data. Note in Fig. 3B (bottom) showing the MALDI-MS analysis of a 5 pmol amount of the mixture that the 1355 ion signal representing an extensively deleted peptide in 8 to 10 residues appears to be the dominant product. The spectra representing 50 and 500 pmol of product (Fig. 3B, middle and top, respectively) show a different distribution and are for the most part in agreement with one another. In these two spectra examination of the quantitative relationships suggests that the ion signal having a 2148 mass assignment is the major product. This example is explored further to show the necessity of coupling MALDI-MS with an HPLC analysis to achieve a more realistic clarification of the quantitative relationhips of the various components.

Characterization of a Peptide Synthesis by Coupling Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry and High-Performance Liquid Chromatography Analysis The example of the problematic synthesis of the 22-residue peptide having the sequence Ala-Pro-Val-Gly-Leu-Val-Ala-Arg-Leu-Ala-AspGlu-Ser-Gly-His-Val-Val-Leu-Arg-Trp-Leu-Pro [theoretical (M + H) + = 2357] introduced above has been exploited to show the definite advantage of coupling MALDI-MS with analytical and preparative reversed-phase (RP) HPLC analysis. In this particular experiment, even though the overall synthesis could be considered a problematic or even a "failed" synthesis, the coupling of MALDI-MS analysis to the HPLC analyses permitted the harvesting of several milligrams of the desired product. Figure 4A shows both the analytical and preparative (inset) HPLC profiles for the complex crude peptide mixture derived from the synthesis having the MALDI-MS analysis presented in Fig. 3B. The conditions for the respective RP-HPLC runs are presented in the legend. Analytical HPLC analysis on a 40-/~g load of the mixture indicated three major components, labeled 1, 2, and 3 in the analytical HPLC profile shown in Fig. 4A. As shown in Fig. 4B, MALDI-MS analysis of 2-~1 aliquots of peaks, 1, 2, and 3 permitted immediate mass identification of the major products. Peak fractions 1 and 2 were

530

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found to be deletion peptides, and peak 3 was found to have a mass consistent with that of the desired product. For purification of the desired product, 135 mg of the synthetic peptide mixture was subjected to preparative RP-HPLC. The inset of Fig. 4A presents the preparative RP-HPLC profile. Further analytical HPLC and MALDI-MS analyses of the center cuts of the preparative peak fractions 2 and 3 (inset of Fig. 4A) are presented in Figs. 5 and 6, respectively. The analytical HPLC profile shown in Fig. 5A and the MALDI-MS analysis shown in Fig. 5B indicate that the major peak (fraction 2) in the preparative HPLC analysis (inset of Fig. 4A) corresponded to fraction 2 in the analytical HPLC profile of the complex mixture (Fig. 4A). The analytical HPLC profile shown in Fig. 6A and the MALDI-MS analysis shown in Fig. 6B indicate that the fractions labeled 3 in both the analytical and preparative HPLC profiles (Fig. 4A) corresponded to the desired product. Figure 5A is the analytical HPLC profile derived from an aliquot of the eluant from the center cut of the preparative HPLC peak labeled 2 in the Fig. 4A inset. Figure 5B is the MALDI-MS interrogation by serial dilution of this material. Once again notice the marked and misleading ion discrimination effects observed for the set of data presented in Fig. 5B. The MALDI-MS analysis at high concentration (1/10 dilution, Fig. 5B, top) suggests that the preparative HPLC fraction 2 is pure, containing a deletion product at mass 2146. However, on further dilution the additional presence of a more extensively deleted product at mass 1805 becomes evident. At the lowest concentration (1/1000 dilution, Fig. 5B, bottom) this more extensively deleted product is the major ion signal in the spectrum. It should be noted that the MALDI-MS analyses of the 1/100 dilutions in both Figs. 3B and 5B appear to be the only spectra that depict realistically semiquantitative information and that are in agreement with the quantitative relationships observed in the respective analytical RP-HPLC profiles (Figs. 4A and 5A). Analytical HPLC and MALDI-MS analyses of the desired product derived from the center cut of fraction 3 of the preparative RP-HPLC run (inset of Fig. 4B) are shown in Fig. 6A,B, respectively. This product is shown to be highly pure by both MALDI-MS interrogation at

FIG. 4. Characterization of a highly impure crude synthetic peptide product by analytical and preparative reversed-phase HPLC and identification of the peaks by MALDI-MS analysis. (A) Analytical HPLC performed on a 4.6 X 200 mm Vydac C18 column at a flow rate of 1.5 ml/min. Solvent A, 0.1% TFA, solvent B, 0.1% TFA in acetonitrile; elution with a linear gradient of 5 to 90% B/30 min. Inset: Preparative HPLC performed on a 20 x 200 mm Vydac C18 column at a flow rate of 10 ml/min. Elution with a linear gradient of 5 to 90% B/40 min. (B) MALDI-MS analysis of fractions derived from the analytical HPLC fractions labeled 1, 2, and 3.

532

[23]

ANALYTICAL TECHNIQUES

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FIG. 5. Characterization of the preparative HPLC fraction 2 (Fig. 4A) by analytical HPLC and MALDI-MS. (A) Analytical HPLC profile of tile center cut of fraction 2 derived from the preparative HPLC run presented in the inset of Fig. 4A. Conditions are those described in the legend to Fig. 4. (B) MALDI-MS interrogation by dilution of the preparative HPLC fraction 2.

[23]

LASER DESORPTION

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FIG. 6. Characterization of the preparative HPLC fraction 3 (Fig. 4A) by analytical HPLC and MALDI-MS. (A) Analytical HPLC profile of the material obtained from the center cut of preparative fraction 3. (B) MALDI-MS interrogation by dilution of the preparative HPLC fraction 3.

534

ANALYTICALTECHNIQUES

[231

dilution and high resolution analytical RP-HPLC criteria. Note the lack of any emerging ion signals on MALDI-MS interrogation by serial dilution, indicating the high degree of purity. Monitoring Automated Peptide Synthesis Stepwise by Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry In the case of troubleshooting problematic syntheses it is advantageous to be able to generate a cycle-by-cycle stepwise historical record of a particular synthesis by coupling microcleavage and deprotection chemistries and mass spectrometric analysis of the generated products. This information would be useful in locating trouble spots and identifying any specific sites of deletion or insertion. This capability was first demonstrated using fast atom bombardment (FAB)-MS and exploiting microcleavage chemistries on peptide-resin aliquots that were automatically removed by a resin sampiing feature that is available on some automated peptide synthesizers [Perkin-Elmer Applied Biosystems (PE-ABI, Foster City, CA) Model 430 and 431A peptide synthesizers]. 15 Subsequently, the potential of MALDI-MS analysis for this purpose was also demonstrated. 16 Figure 7 shows the utility of this approach for the evaluation of a successful synthesis of a 22-residue peptide having the sequence Asp-Val-Arg-Val-Gln-ValLeu-Pro-Glu-Val-Arg-Gly-Gln-Leu-Gly-Gly-Thr-Val-Glu-Leu-Pro-Cys. From four TFA microcleavage and deprotection chemistries performed on four resin pools, and MALDI-MS analysis of the product mixtures derived from the respective pools, the entire stepwise assembly record of this particular synthesis was obtained. Individual peptide-resin sample aliquots were sorted into four pools to minimize any ion suppression effects that may arise if all the peptide-resin samples had been pooled and cleaved in one reaction to generate the historical record. Four pools were chosen to limit the number of microcleavage reactions and thus to minimize effort and time. The first pool consisted of peptide-resins obtained from synthetic cycles 1 through 7, covering assembly of the peptide from the C-terminal Cys to the next six residues (Cys-Pro-Leu-Glu-Val-Thr-Gly) toward the N terminus, the direction of chemical synthesis. The second pool consists of peptide-resins removed after cycles 8 through 12 comprising the next region of extension (Gly-Leu-Gln-Gly-Arg). The third pool of peptide-resin products, from cycles 13 through 17, covers the region Val-Glu-Pro-Leu-Val, and the fourth 15W. T. Moore and R. M. Caprioli,in "Techniquesin Protein ChemistryII" (J. Villifranca, ed.), p. 511. AcademicPress, San Diego, 1991. 16B. T. Chait, R. Wang, R. C. Beavis, and S. B. H. Kent, Science 262, 89 (1993).

[231

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m/z FIG. 7. Stepwise assessment of the automated solid-phase synthesis of a 22-residue peptide by microcleavage/MALDI-MS analysis. Peptide synthesis was performed on a PE-ABI Model 431A automated peptide synthesizer set up and programmed for an Fmoc/tert-butyl synthesis strategy. After each addition cycle the automated peptide-resin sampling feature obtained a peptide-resin aliquot. Peptide-resins were pooled into four groups, subjected to microcleavage chemistry, and analyzed by MALDI-MS. The four spectra obtained from each pool are presented as overlapped spectra.

pool covers cycles 18 through 22 comprising the extension of the peptide to the N terminus (Gln-Val-Arg-Val-Asp). The four resin pools were subjected to a microcleavage and deprotection procedure similar to that previously described 15but adapted for a 9-fluorenylmethyloxycarbonyl (Fmoc) synthesis strategy (a microtization of the Fmoc cleavage reagent K17). Cleaved and deprotected peptide products were then interrogated by MALDI-MS analysis on 10-fold serial dilutions in CHCA matrix. The four spectra derived from those dilutions that yielded maximal information are presented as overlapping spectra in Fig. 7. Note the ion 17 D. King, C. G. Fields, and G. B. Fields, Int. J. Pept. Protein Res. 36, 255 (1990).

536

ANALYTICAL TECHNIQUES

[23]

suppression effect that is observed on MALDI-MS analysis for the peptides derived from the microcleavage of the second peptide-resin pool (ion signals for peptides having the assembly sequence Gly-Leu-Gln-Gly-Arg having masses 774 through 1229). The peptide product resulting from the incorporation of the extremely basic Arg residue designated at mass 1229 suppresses the ion signal intensities of the prior assembly products present in this pool. Note the low signal heights for the products labeled in the ion signal sequence G, L, Q, and G falling between masses 774 through 1073 (Fig. 7). It should also be noted that a microcleavage-derived pyroglutamic acid formation was detected for the product labeled Q at mass 1895, as indicated by the - 1 7 amu satellite peak observed for this product. The lack of this - 1 7 amu component in the final crude product (data not shown) indicates that pyroglutamic acid formation occurred as a consequence of the microcleavage chemistry and did not occur during the automated synthesis. Because the analysis of the peptide-resin pool products is stepwise and "time-based," the position of a deletion, insertion, or any modification, if either had occurred, could have been unambiguously and quickly determined. The premise is that any change would be detected as a mass shift in descendants derived only from that cycle where the event occurred. An interesting variation on this theme has been described exploiting either very acid-labile 18-2° or photolabile linkages 21 to the resin beads. With this approach peptide-resin beads may be exposed to matrix and directly irradiated, with detection of released resin-bound peptides. Either the acidity in the matrix or the laser light promotes a dissociation of a subset of the peptide products from the bead. The postsynthesis cleavage and deprotection steps are thereby eliminated. These modifications lead to the potential for a more direct analysis approaching an on-line peptide synthesis monitoring strategy involving MALDI-MS. Monitoring Disulfide Bond F o r m a t i o n by Matrix-Assisted Laser Desorption-lonization Mass Spectrometry-Based Mass Shift Assay This section covers a MALDI-mass spectrometric assay devised to evaluate a disulfide formation problem. MALDI-MS was performed on a low resolution instrument using external calibrations that resulted in a mass accuracy error that would not permit reliable discernment of the 2 amu 18B. J. Egner, G. J. Langley,and M. Bradley,I. Org. Chem. 60, 2652 (1995). 19B. J. Egner, M. Cardno, and M. Bradley, J. Chem. Soc., Chem. Commun., 2163 (1995). 2oG. Talbo, J. D. Wade, N. Dawson, M. Manoussios, and G. W. Tregear, Lett. Pept. Sci. 4, 121 (1997). 21M. C. Fitzgerald, K. Harris, C. G. Shevlin, and G. Siuzdak, Bioorg. Med. Chem. Lett. 6, 979 (1995).

[23]

LASERDESORPTIONMASSSPECTROMETRY

537

mass differences that would be required for distinguishing between the oxidized and reduced forms of the peptide. This assay was developed in response to the challenge presented by the ABRF-1995 Peptide Synthesis study by the Peptide Synthesis Research Committee (PSRC) of the Association of Biomolecular Resource Facilities (ABRF)P This study was designed to evaluate four peptide thiol oxidation procedures for the generation of cyclic peptides. The target peptide for this study was cyclo[Cys-Phe-TrpLys-Thr-Cys]-Thr-NH2 [(M + H) + -- 1033.2]. The MALDI-MS mass shift assay for free thiols was based on the previous work of Zaluzec e t al. 22 demonstrating the use of the organomercurial compound p-hydroxymercuribenzoate (PHMB) to probe free thiols in peptides and proteins by MALDI-MS analysis. The synthetic product was subjected to four different cyclization methods. Methods I and II were postcleavage procedures, and methods III and IV were on-resin procedures. 5 Cyclized product was harvested by lyophilization and dissolved in 50% acetonitrile, 0.1% TFA at 1 mg/ml concentration. This material was diluted 1/10 into a 1.9 mM solution of PHMB dissolved in 0.1% ammonium hydroxide. The reaction was allowed to proceed for 15 min at room temperature. The PHMB peptide reaction mixtures were diluted 1/10 in CHCA matrix and spotted for subsequent MALDI-MS analysis. Figure 8 is the set of data that we obtained for the peptide product that we submitted for evaluation by the ABRF PSRC. Figure 8A is the PHMB/ MALDI-MS analysis result for the linear form of the peptide. The ion signals having observed masses at 1358 and 1680 represent the addition of one (+321) and two molecules of PHMB (+642), respectively, to the available thiols in the linear peptide. Figure 8B is the result for the peptide cyclized by method I involving treatment of the linear peptide in 7 g/1 ammonium acetate for 3 days prior to harvest by lyophilization. The MALDI-MS profile in Fig. 8B indicates that some material was oxidized (note the ion signal at mass 1032) and that some material was still in the linear form as demonstrated by the PHMB adducted material (note 1356 and 1676 species). Spectrophotometric evaluation of the free sulfhydryl groups by Ellman's reagent, 5,5'-dithiobis(nitrobenzoic acid), 23 indicated that this product was only 24% cyclized, which correlates well with the PHMB/MALDI-MS analysis. Figure 8C shows the profile derived from the PHMB/MALDI-MS analysis of the method II cyclization procedure involving exposure of the linear form of the peptide to direct oxygenation in z2E. J. Zaluzec,D. A. Gage, and J. T. Watson,J. A m . Soc. Mass Spectrom. 5, 359 (1994). 23j. M. Stewart and J. D. Young, "Solid Phase Peptide Synthesis,"2nd ed., p. 116. Pierce, Rockford, Illinois,1984.

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FIG. 8. Assessment of four different peptide eyclization methods through disulfide formation by a M A L D I - M S mass shift assay using the thiol adducting reagent PHMB. (A) PHMB! M A L D I - M S analysis of the linear thiol-containing form of the peptide. (B) PHMB! M A L D I - M S analysis of a peptide subjected to oxidation method I. (C) P H M B / M A L D I - M S analysis of a peptide subjected to oxidation method II. (D) P H M B / M A L D I - M S analysis of a peptide subjected to oxidation method III. (E) P H M B / M A L D I - M S analysis of a peptide subjected to oxidation method IV. PHMB adds a mass of 32l for one thiol and 642 for two tfiiols.

[23]

L A S E R D E S O R P T I O N MASS S P E C T R O M E T R Y

539

0.1 M ammonium bicarbonate buffer for 24 hr. Very little PHMB adducted material was observed, indicating an extensive cyclization. Spectrophotometric evaluation using Ellman's reagent indicated 100% cyclization. Figure 8D,E shows the PHMB/MALDI-MS evaluations for the onresin cyclizations methods III and IV, respectively. Method III required that the peptide-bound resin be treated with a 1.5 molar excess of 0.4 M thallium trifluoroacetate in dimethylformamide for 1 hr before washing and cleaving. The PHMB/MALDI-MS analysis profile in Fig. 8D indicates an extensively cyclized product that was confirmed by spectrophotometric assay using Ellman's reagent, suggesting 100% cyclization by this method also. The PHMB/MALDI-MS analysis of the product generated by the method IV cyclization procedure clearly indicates a problem with this method. The ion signal at mass 1234 is a Hg (+200) adduct of the peptide. Method IV required treating the peptide-resin with a 4-fold molar excess of 0.1 M mercuric acetate in dimethylformamide for 1 hr followed by treatment with a 10-fold molar excess of 2-mercaptoethanol prior to washing and cleavage. The problem of mercury adduction has been previously noted with this procedure. It is of interest to note that spectrophotometric assay using Ellman's reagent suggested 100% cyclization. The product is cyclized but via a mercuric ion insertion. Divalent mercuric ions (Hg 2+) are known to have the potential to bridge two thiol groups? 4

Probing Synthetic Peptide Racemization Problems by Coupling Enzymatic Treatment with Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry Enantioselective exopeptidase hydrolysis has been employed to address racemization problems in peptide synthesis.25 In this type of study, the release of terminal amino acids are quantitatively measured by amino acid analysis. The present example, inspired by the ABRF-1996 PSRC study,6 employs a similar approach with a test peptide designed to address the racemization that is possible with activated His derivatives,a6 However, for the ABRF-1996 PSRC study,6 enantioselective enzymatic hydrolysis was coupled with MALDI-MS to investigate any problems encountered in the synthesis of the test peptide Arg-Glu-Arg-His-Ala-Tyr [(M + H) + =

8321. 24T. M. Jovin, P. T. England,and A. Kornberg,J. Biol. Chem. 244, 3009 (1969). 2sM. Bodanskyand A. Bodansky,"The Practice of Peptide Synthesis,"p. 237. SpringerVerlag, New York, 1984. 26G. B. Fields, Z. Tian, and G. Barany, in "SyntheticPeptides: A User's Guide" (G. A. Grant, ed.) p. 77. Freeman, New York, 1992.

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[23]

LASER DESORPTION MASS SPECTROMETRY

541

To demonstrate the power of MALDI-MS in this type of racemization study, results obtained on two reference peptides are presented. One reference peptide was made with all-L amino acids (Arg-Glu-Arg-His-Ala-Tyr). The other reference peptide was made with all-L amino acids except for His, which was incorporated as the D-form of the amino acid (Arg-GluArg-D-His-Ala-Tyr). Figure 9A shows the results obtained by applying a coupled carboxypeptidase A/MALDI-MS analysis of the reference peptides. The top two spectra of Fig. 9A are the results obtained from nonenzyme-treated control all-L peptide (top) and the carboxypeptidase A-treated all-L peptide (secondfrom top). The ion signal at mass 461 seen in the spectrum in Fig. 9A (second from top) is for the tripeptide product Arg-Glu-Arg. Carboxypeptidase A removed the three C-terminal residues, Tyr, Ala, and His. The third spectrum from the top in Fig. 9A represents the product containing the D-His residue (non-enzyme-treated control). Treatment of this reference peptide with carboxypeptidase A resulted in removal of solely the C-terminal Tyr as indicated by the observed ion signal at mass 670 (Fig. 9A, bottom). The presence of a D-His at the P2 subsite on the substrate (Schecter and Berger nomenclature for protease subsites 27) was enough to prevent further processing by the carboxypeptidase A. The theoretical (M + H) ÷ for the sequence Arg-Glu-Arg-D-His-Ala is 669, and the presence of this ion is taken to be a signature for racemization at the His position in this particular peptide. Note that the slight amount of 670 ion signal in the second spectrum from the the top in Fig. 9A suggests that a slight amount of racemized product was obtained from the synthesis of the all-L peptide. Interestingly, as shown in Fig. 9B, treatment of the reference peptides with trypsin did not show any enantiomeric selectivity of trypsin for these synthetic peptide substrates. In both preparations the identical tryptic products were observed: Arg-Glu-Arg [(M + H) ÷ = 461] and His27 I. Schecter and A. Berger, Biochem. Biophys. Res. Commun. 27, 157 (1967).

Fie. 9. Racemization analysis by coupling enantioselective enzymatic hydrolysis with MALDI-MS. (A) Carboxypeptidase A treatment of reference peptides. The top two spectra represent the all-L form of the peptide, Arg-Glu-Arg-His-Ala-Tyr. The top most spectrum is the untreated control, and the second spectrum from the top is carboxypeptidase A treated. The bottom two spectra are the fully "racemized" reference peptide Arg-Glu-Arg-D-His-Tyr. The third spectrum from the top is the untreated control, and the bottom most panel is carboxypeptidase A treated. (B) Trypsin treatment of the same reference peptides. The top two spectra represent the all-L form peptide Arg-Glu-His-Ala-Tyr. The top most spectrum is the untreated control, and the second spectrum from the top is trypsin treated. The bottom two spectra represent the fully "racemized" reference peptide Arg-Glu-Arg-D-His-Tyr. The third spectrum from the top is the untreated control, and the bottom most spectrum is trypsin treated.

542

ANALYTICAL TECHNIQUES

[23]

Ala-Tyr [(M + H) + = 391] whether or not D-His was present in the P~ position (Schecter and Berger nomenclature for protease substrate subsites27).

Summary The examples presented indicate that MALDI-MS is a useful tool for evaluating the progress of peptide synthesis at all the necessary levels: automated assembly, cleavage and deprotection chemistries, RP-HPLC analyses and purifications, and structural validation of the final product. The technique, if judiciously applied, permits the evaluation of complex peptide mixtures and often provides a semiquantitative overview. We have found that the availability of this method has enabled the provision of highquality peptide reagents for use in the local research environment. The integration of this methodology into our peptide synthesis facility has also enabled and encouraged us to undertake more challenging synthetic problems such as phosphopeptide synthesis, peptide cyclizations, and peptide modification chemistries that would not ordinarily be offered if the laboratory lacked this technology. MALDI-MS is one of the more versatile and readily integrable mass spectrometric methods that can be incorporated into the average peptide synthesis laboratory. Acknowledgments The author thanks Dr. John D. Lambris of the Department of Pathology and Laboratory Medicine, The School of Medicine, University of Pennsylvania, Philadelphia, for continual support and encouragement and for realizing the value of mass sPectrometry and fostering the inclusion of this technology at the Protein Chemistry Laboratory, and Ms. Lynn A. Spruce for expert and conscientious technical assistance in all the areas of peptide synthesis. The Association of Biomolecular Resource Facilities (ABRF) Peptide Synthesis Research Committee (PSRC) is also acknowledged for inspiring solutions to some well-designed and testable problem cases in solid-phase peptide synthesis.