Anal. Chem. 1997, 69, 1815-1819
Minimizing Peak Coalescence: High-Resolution Separation of Isotope Peaks in Partially Deamidated Peptides by Matrix-Assisted Laser Desorption/Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry John T. Stults
Protein Chemistry Department, Genentech, Inc., 460 Point San Bruno Boulevard, South San Francisco, California 94080
Resolution of greater than 100 000 is routinely achieved by MALDI-FT-ICR, based on measured peak widths. However, the ability to separate peaks that require this resolution is difficult to obtain in practice due to peak coalescence, a result of coupling of the cyclotron motion of ions with similar frequencies. This phenomenon is accentuated for high space charge, high trapping plate voltages, and high mass. Very low trapping plate voltages with properly chosen transient measurement times are shown here to yield ultrahigh-resolution separation of closely spaced peaks in peptide mixtures. Measurements of the isotope peaks for partially deamidated preparations of substance P or partially reduced/partially deamidated Ala-Gly-[Arg]8-vasopressin show as many as six isotope peaks at one nominal mass. In one example, the 13C isotope peak was separated from the 15N isotope, a separation that required a resolution in excess of 180 000. Measurements were made with an external source MALDIFT-ICR mass spectrometer with a 4.7 T magnet. These data suggest the need for high-resolution measurements for the determination of exact masses for peptide mixtures.
Matrix-assisted laser desorption/ionization (MALDI) with Fourier transform ion cyclotron resonance mass spectrometry (FTICR) provides an excellent technique for the analysis of peptides.1-13 The mating of this ionization technique and mass analyzer takes advantage of the positive features of each technique. MALDI (1) Hettich, R. L.; Buchanan, M. V. J. Am. Soc. Mass Spectrom. 1991, 2, 2228. (2) McIver, R. T.; Li, Y.; Hunter, R. L. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 4801-4805. (3) Strupat, K.; Karas, M.; Hillenkamp, F.; Eckerskorn, C.; Lottspeich, F. Anal. Chem. 1994, 66, 464-470. (4) Castoro, J. A.; Koster, C.; Wilkins, C. Rapid Commun. Mass Spectrom. 1992, 6, 239-241. (5) Sheng, L.-S.; Covey, J. E.; Shew, S. L.; Winger, B. E.; Campana, J. E. Rapid Commun. Mass Spectrom. 1994, 8, 498-500. (6) Castoro, J. A.; Wilkins, C. L. Anal. Chem. 1993, 65, 2621-2627. (7) Castoro, J. A.; Wilkins, C. L.; Woods, A. S.; Cotter, R. J. Biol. Mass Spectrom. 1995, 30, 94-98. (8) Li, Y.; McIver, R. T.; Hunter, R. L. Anal. Chem. 1994, 66, 2077-2083. (9) Li, Y.; McIver, R. T. Rapid Commun. Mass Spectrom. 1994, 8, 743-749. (10) Solouki, T.; Gillig, K. J.; Russel, D. H. Rapid Commun. Mass Spectrom. 1994, 8, 26-31. (11) Pasa-Tolic, L.; Huang, Y.; Guan, S.; Kim, H. S.; Marshall, A. G. J. Mass Spectrom. 1995, 30, 825-833. S0003-2700(97)00155-8 CCC: $14.00
© 1997 American Chemical Society
produces ions in a pulsed fashion, over a broad mass range, with straightforward sample preparation.14,15 The FT-ICR analyzer measures ion masses with high sensitivity, mass accuracy, and resolution.16-18 In fact, one of the hallmarks of Fourier transform ion cyclotron resonance mass spectrometry is ultrahigh resolution. Since the technique was developed over twenty years ago, the ability to measure cyclotron frequencies with high precision and to translate those signals into astonishingly narrow peaks on the mass axis has been achieved routinely. This characteristic continues to be observed with the more recent addition of ionization techniques that permit the ion formation of large, nonvolatile biomolecules. For example, resolution (m/∆m) of 1 500 000 has been measured for peptides generated by MALDI FT-ICR.11 High-resolution measurements are traditionally also associated with high mass accuracy and precision. Exact mass determinations are predicated upon the measurement of masses of pure components. High resolution is typically used for exact mass measurements due to the narrow peaks, which provide more precise mass values, and to the ability to resolve closely spaced peaks that may overlap and skew the mass. It is not surprising, then, that exact mass measurements have also been achieved with MALDI FT-ICR, although not without careful control of the experimental parameters. Mass is inversely proportional to the cyclotron frequency. The measured cyclotron frequency is influenced by several factors, most notably the number of ions that occupy the ICR cell (space charge) and the electric field gradients due to the trapping plate voltages that hold ions in the trap. If these factors are carefully controlled or their effects properly compensated, mass accuracies of 6 months at 4 °C. Samples were prepared for MALDI by mixing a 1-µL aliquot of the peptide solution (10 pmol/µL) with 1 µL of 2,5-dihydroxybenzoic acid (DHB; 1 M in ethanol) on the sample target. Spectra were acquired with an IonSpec (Irvine, CA) HiResMALDI Fourier transform mass spectrometer equipped with an external source, a quadrupole ion guide, an orthorhombic (elongated cubic) cell, a 4.7 T magnet, a 230 L/s turbomolecular (20) Naito, Y.; Inoue, M. J. Mass Spectrom. Soc. Jpn. 1994, 42, 1-8. (21) Huang, J.; Tiedemann, P. W.; Land, D. P.; McIver, R. T.; Hemminger, J. C. Int. J. Mass Spectrom. Ion Processes 1994, 134, 11-21. (22) Mitchell, D. W.; Smith, R. D. Phys. Rev. E 1995, 52, 4366-4386. (23) Mitchell, D. W.; Smith, R. D. J. Mass Spectrom. 1996, 31, 771-790. (24) (a) Marshall, A. G.; Guan, S. Rapid Commun. Mass Spectrom. 1996, 10, 1819-1823. (b) Anderson, J. S.; Laude, D. A. Int. J. Mass Spectrom. Ion Processes 1996, 157/158, 163-174. (c) Solouki, T.; Emmett, M. R.; Guan, S.; Marshall, A. G. Anal. Chem. 1997, 69, 1163-1168. (25) Aswad, D. W. Deamidation anad Isoaspartate Formation in Peptides and Proteins; CRC Press: Boca Raton, FL, 1995.
1816 Analytical Chemistry, Vol. 69, No. 10, May 15, 1997
Figure 1. Protonated molecule region of the mass spectrum of two cyclic peptides that differ by a Gln/Lys substitution. The small letter amino acid indicates a D stereoisomer. The spectrum was acquired as a wide-band, 1024K transient. The measured resolution is 130 000.
pump on the source, and one cryopump each on the transfer quadrupole and ICR cell. The pulse sequence was as follows: 0 mssa -30-V trapping plate pulse to clear the cell; 100 mssthe laser was triggered; 100.5 mssthe plate closest to the source, initially at 0 V, was raised to 20 V to match the opposite plate (the time to raise the trapping plate voltage depends on the mass range of the ions to be trapped and is calculated by the data system to correspond to the flight times of the ions from the source); 110 mssa 2-ms pulse to the pulsed valve for argon introduction into the cell. After the argon was pumped away (3 s), the trapping plate voltage was normally lowered from 20 to 0.5 V in 1.5 s. Ions were excited with a 17.0-V chirp (400-4500 u), then detected in heterodyne mode at an ADC rate of 20 kHz with a 20-kHz filter. To reduce space charge, the trapping voltage in later experiments was lowered to 0.11 V. The mass axis was calibrated with broadband acquisition using a mixture of peptides. Due to differences in pulse sequence parameters and trapping plate voltages, a slight shift in the calibration is observed in heterodyne mode. To compensate, the calibration was corrected for the known mass of the 12C component in each spectrum, which can be determined with high accuracy in the broad-band mode. Instrument control, data acquisition, and analysis were done with an IonSpec Omega data system. RESULTS A series of peptide mixtures were analyzed to optimize and then demonstrate the separation of peaks. Mixtures are traditionally considered to be molecules of differing elemental composition. However, even single components, when examined at high resolution, are mixtures alsosmixtures of different isotopic species. Both types of mixtures will be considered in this study. Figure 1 shows the MALDI spectrum of a mixture of two cyclic peptides in which a glutamine in one has been substituted for lysine. These two residues differ by 0.037 u. The resolution required to separate these peptides is 15 800. The peaks are easily resolved and the measured resolution based on the peak widths is 130 000. This resolution and peak separation are readily achieved in this mass range with broad-band excitation and a 1024K-point transient acquisition. At higher mass, the separation of multiple components of the same nominal mass, especially peaks having smaller mass differences, is considerably more challenging. Peptides that contain sulfur (in methionine or cysteine residues) have a substantial A + 2 peak intensity due to the 34S isotope. This isotope should be resolved from the 13C2 peak with resolution easily obtained by FT-ICR. Figure 2 shows the protonated molecule region of the
Table 1. Identities of Isotope Peaks Observed for Ala-Gly-[Arg]8-vasopressin: AGCYFQNCPRG-NH2 (C51H74N17O14S2) m/z (theoretical)
isotope difference from monoisotopic peak
1212.5043
12C, 32S
1213.5076
13C, 32S
1214.5000 1214.5110 1214.5199
12C, 34S
(monoisotopic)
13C
32 2, S 12C, 32S (SH/SH)
Figure 2. Protonated molecule region of the MALDI FT-ICR spectrum of Ala-Gly-[Arg]8-vasopressin (AGCYFQNCPRG-NH2), taken in heterodyne mode. The inset shows an expanded view of the A + 2 peak. The measured resolution (fwhm) is 350 000. No separation of 13C2/34S is observed.
Figure 3. Mass spectrum of Ala-Gly-[Arg]8-vasopressin, taken from the same sample as used for Figure 2, but optimized to reduce peak coalescence. The identities of the peaks are given in Table 1.
Ala-Gly-[Arg]8-vasopressin ([M + H]+ ) 1212.5043) spectrum, a peptide with two cysteine residues in a disulfide bond. This spectrum, taken in heterodyne mode, is expected to show two peaks at the A + 2 peak, m/z 1214.5110 for the 13C2 peak and m/z 1214.5000 for the 34S peak. As the expanded region of the figure shows, only a single peak is observed. The peak width indicates a resolution of 350 000, yet the pair of peaks should require only a resolution of 110 000 for their separation. These peaks have merged into one, a manifestation of the coalescence or coupling of their closely spaced frequencies. In order to avoid coalescence, several parameters were optimized to reduce space charge. The trapping plate voltages were reduced from 0.5 to 0.11 V. The very low voltage minimizes the contribution of the electric field gradient to the cyclotron motion and reduces the space charge. Only a subset of the ion population has sufficiently low kinetic energy to remain in this shallow potential well. The ions were excited to the maximum radius allowed by the cell dimensions by increasing the excitation voltage to 17.0 V. Finally, it was observed that the transient lost coherence after ∼12 s, so the detection time was limited to this duration. These adjustments together permitted separation of the 13C /34S isotope peaks for Ala-Gly-[Arg]8-vasopressin, as shown 2 in Figure 3. A third peak was also observed which corresponds to a small amount of reduced peptide in which the disulfide bond was reduced (12C + 2H). The measured resolution is 390 000. The identities of the observed peaks and their predicted masses are given in Table 1. Peaks that correspond to other isotopes
Figure 4. Protonated molecule region of the MALDI FT-ICR mass spectrum of a partially deamidated sample of Ala-Gly-[Arg]8-vasopressin (A), taken in heterodyne mode. The A + 1 peak (m/z 1213) shows the higher abundance 12C peak of the deamidated form plus the smaller 13C peak of the nondeamidated form. The A + 2 (B) and A + 3 (C) peak clusters are mixtures of a number of isotope peaks of different deamidated forms. The identities of the peaks are given in Table 2.
were not observed due to the low abundance (abundance expected relative to the 12C peak: 33S, 1.6%; 15N, 5.9%; 18O, 3.1%) and the even higher resolving power required for their separation.26 The utility of this very high resolution for the analysis of deamidation can be seen in Figure 4. This sample is a degraded (26) Werlen, R. C. Rapid Commun. Mass Spectrom. 1994, 8, 976-980.
Analytical Chemistry, Vol. 69, No. 10, May 15, 1997
1817
Figure 5. Protonated molecule region of the MALDI FT-ICR mass spectrum of a partially deamidated sample of substance P, taken in heterodyne mode. The peak intensities of the A + 2 peaks indicate that the abundance of the deamidated form is ∼50% of the nondeamidated peptide. The A + 3 peaks (inset) show the presence of a minor amount of a second deamidated form. The peak identities are given in Table 3. Table 2. Identities of Isotope Peaks Observed for Partially Deamidated Ala-Gly-[Arg]8-vasopressin: AGCYFQNCPRG-NH2 (C51H74N17O14S2) m/z (theoretical)
isotope difference from monoisotopic peak
Table 3. Identities of Isotope Peaks Observed for Partially Deamidated Substance P: RPKPQQFFGLM-NH2 (C63H99N18O13S1) m/z (theoretical)
isotope difference from monoisotopic peak
1212.5043
12C, 32S
(monoisotopic)
1347.7360
12C, 32S
(monoisotopic)
1213.4883 1213.5076
12C, 32S
(NH2 > OH)
1348.7200 1348.7394
12C, 32S
(NH2 > OH)
1214.4723 1214.4853 1214.4918 1214.5000 1214.5110 1214.5199
12C, 32S
1349.7040 1349.7234 1349.7317 1349.7428
12C, 32S
1215.4757 1215.4842 1215.4952 1215.5039
13C, 32S
13C, 32S
(NH2 > OH)2 (NH2 > OH) 13C, 32S (NH > OH) 2 12C, 34S 13C , 32S 2 12C, 32S (SH/SH) 12C, 32S, 15N
(NH2 > OH)2 (NH2 > OH) 13C , 32S (NH > OH) 2 2 12C, 32S (NH > OH) (SH/SH) 2 12C, 34S
form of Ala-Gly-[Arg]8-vasopressin that shows deamidation. Three sites are potentially available for deamidation, an asparagine residue, a glutamine residue, and the C-terminal amide. The isotope peaks indicate that at least two deamidation reactions have occurred. These data do not indicate which sites have been modified. A list of the peaks and their identities is given in Table 2. Expanded views of the A + 2 and A + 3 peaks are shown in parts B and C of Figure 4, respectively. Note that two of the peaks (m/z 1214.4853 and 1214.4918) differ by only 0.0065 u and require a resolution of 187 000 to be separated. Furthermore, the 15N isotope peak is observed for the most abundant component, which has not been observed previously for peptides of this mass by FT-ICR or any other mass spectral technique due to its low natural abundance and the high resolution required to observe it. The lack of a resolved 15N peak at 1213.5013 may be attributed to a larger number of ions with nominal mass 1213 that lead to an earlier onset of coalescence or the slightly higher resolution required to separate this peak (m/∆m ∼ 192 000). This reasoning may also explain the lack of resolution of the 15N peak in Figure 3. 1818 Analytical Chemistry, Vol. 69, No. 10, May 15, 1997
13C, 32S
13C, 32S
(NH2 > OH)2 (NH2 > OH)
12C, 34S 13C
2,
32S
These data show clearly that at least five molecular species contribute to this isotope cluster: the expected peptide, the peptide with one and two deamidations, and the reduced forms of the nondeamidated and singly deamidated forms. The reduced form of the doubly deamidated peptide would appear at m/z 1216 and was below noise level of the spectrum. Due to the very similar composition of these peptides, one would expect that the relative ion abundances of the isotopes are representative of the relative amounts of each component in the sample. Independent verification of the quantitation has not been made. However, if the observed isotope peaks are in the process of coalescing, Mitchell and Smith22 have observed that the measured abundance ratios do not reflect the true ratio. The utility of very high resolving power to show minor deamidated species is demonstrated for a degraded form of another peptide, substance P, in Figure 5. A list of the peaks is given in Table 3. The abundance of the 34S component is smaller for this peptide in comparison with the earlier example due to the single sulfur atom on the lone methionine residue. As in the previous peptide mixture, two deamidated forms are observed. There are three potential deamidation sites: two glutamine residues and the C-terminal amide. Detection of the second deamidated form (∼3% of the major component) in the presence of the nondeamidated and singly deamidated forms, and its identification on the basis of its exact mass, could not be
accomplished with any other type of mass spectrometer. The deamidation of glutamine is uncommon, however, so this result should be viewed as preliminary. DISCUSSION The measurement of high resolution as demonstrated here requires the maintenance of separate ion populations with very similar frequencies. The main criteria for achieving this resolution are maintenance of a very low space charge and of low trapping plate voltage, excitation to the maximum possible cyclotron radius, and careful selection of the transient acquisition time. The results were achieved with very low trapping plate voltages; only a small population of ions with very low kinetic energy are trapped under these conditions. The very low space charge has an added benefit for summing the transients from multiple laser shots. The shotto-shot variability observed in MALDI may produce widely varying ion intensities. This variability produces peaks that shift by as much as 5 ppm for subsequent laser shots. At high resolution, this shift is manifest in broadened or even separate peaks. The low space charge ensures that peaks from subsequent laser shots have identical cyclotron frequencies. Thus, the added benefit of low space charge and low trapping voltages is a much more reproducible calibration. The measurement of long transients is also a prerequisite for high resolution. Transient lifetimes can be limited by collisional dampening (thus the requirement for high vacuum) and unstable ions (high internal energy causes fragmentation). The requirement that ions be stable for a long period of time can be a limitation for MALDI FT-ICR. Many molecules acquire sufficient internal energy during the desorption/ionization process to undergo extensive fragmentation,27 sometimes to the point of complete disappearance of the [M + H]+ ion. It is necessary to take precautions to reduce fragmentation as much as possible through the use of low laser fluence, the proper analyte to matrix ratio, and the use of matrices that impart the least internal energy, e.g., DHB28 and DHB + sugar.4,29 These experiments were performed with a simple pulse sequence (ionization, trapping in the presence of a cooling gas, reduction in trapping voltage, excitation, detection). The use of an external source with a quadrupole ion guide places the ions in the center of the ICR cell to permit high-resolution measurement30 without the need for other techniques that have been required by others, such as quadrupolar axialization.31 Nonetheless, further improvements in S/N may be possible by remeasurement techniques that require axialization. These experiments are planned. The utility of very high resolution is demonstrated here with just a few synthetic peptides in which deamidation occurred. These measurements are equally applicable to many other synthetic peptides. Exact mass measurements (to yield elemental composition) have long been one of the tests for compound identity for smaller synthetic molecules. These measurements have less (27) Kaufmann, R.; Spengler, B.; Luetzenkirchen, F. Rapid Commun. Mass Spectrom. 1993, 7, 902-910. (28) Strupat, K.; Karas, M.; Hillenkamp, F. Int. J. Mass Spectrom. Ion Processes 1991, 111, 89-102. (29) Billeci, T. M.; Stults, J. T. Anal. Chem. 1993, 65, 1709-1716. (30) McIver, R. T.; Li, Y.; Hunter, R. L. Rapid Commun. Mass Spectrom. 1994, 8, 237-241. (31) Guan, S.; Marshall, A. G.; Wahl, M. C. Anal. Chem. 1994, 66, 1363-1367. (32) Jensen, O. N.; Podtelejnikov, A.; Mann, M. Rapid Commun. Mass Spectrom. 1996, 10, 1371-1378. (33) Winger, B. E.; Campana, J. E. Rapid Commun. Mass Spectrom. 1996, 10, 1811-1813.
frequently been applied to synthetic peptides due to difficulties in achieving adequate precision and accuracy (
