Exceptionally high mass resolving power, high

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1 Structural Validation of Saccharomicins by High Resolution and High Mass Accuracy Fourier Transform-Ion Cyclotron Resonance- Mass Spectrometry and Infrared Multiphoton Dissociation Tandem Mass Spectrometry Stone D.-H. Shi, Christopher L. Hendrickson, and Alan G. Marshall Center for Interdisciplinary Magnetic Resonance, National High Magnetic Field Laboratory, and Department of Chemistry, Florida State University, Tallahassee, Florida, USA Marshall M. Siegel, Fangming Kong, and Guy T. Carter Wyeth-Ayerst Research, Lederle Laboratories, Pearl River, New York, USA Exceptionally high mass resolving power and mass accuracy combined with tandem mass spectrometry (MS n ) capability make Fourier transform ion cyclotron resonance mass spectrometry a powerful tool for structure verification and determination of biological macromolecules. By means of local internal calibration and electron mass correction, mass accuracy better than 0.5 ppm was achieved for two oligosaccharide antibiotics, Saccharomicins A and B, consistent with the proposed elemental compositions based upon NMR data. High resolution and high mass accuracy MS/MS data were obtained for both oligosaccharides by use of infrared multiphoton dissociation (IRMPD) with a 40 W continuous-wave CO 2 laser. The spectra were charge-state deconvolved by the Z-score algorithm to yield much simpler mass-only spectra. Sequences of 15 sugar residues could be confirmed from the charge state deconvolved accurate mass MS/MS spectra for Saccharomicins A and B, even without use of traditional prior permethylation. A fragment corresponding to an internal sugar loss rearrangement was observed by IRMPD and studied by collision activated dissociation MS 4. (J Am Soc Mass Spectrom 1999, 10, ) 1999 American Society for Mass Spectrometry Exceptionally high mass resolving power, high mass accuracy, and tandem mass spectrometry (MS n ) capability make Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry [1 4] a powerful tool for structure verification and determination of biological macromolecules. Structurally informative fragments may be produced by various dissociation techniques, such as collision activated dissociation (CAD), blackbody infrared radiative dissociation, and infrared multiphoton dissociation (IRMPD). Originally developed for fragmentation kinetics of low-mass ions [5, 6], IRMPD has evolved into a versatile dissociation technique for biological macromolecules [7], for both protein [8, 9] and DNA [10, 11] sequencing, as well as adduct removal in nucleotide analysis [12]. Using a 9.4 tesla electrospray FT-ICR mass spectrometer, together with IRMPD as the dissociation technique, we have performed accurate-mass measurements and MS/MS on the two oligosaccharides, Saccharomicins A and B, Address reprint requests to Alan G. Marshall, Director, Ion Cyclotron Resonance Program, National High Magnetic Field Laboratory, Florida State University, 1800 East Paul Dirac Drive, Tallahassee, FL marshall@magnet.fsu.edu with nominal molecular weights of 2794 and 2778 Da. Both heptadecasaccharides are active antibiotics against multiply resistant pathogenic bacteria [13]. The mass spectrometry measurements described herein confirm the structures originally proposed mainly from NMR and chemical degradation studies. Experimental The antibiotic complex was isolated from fermentation broth generated by Saccharothrix espanaensis bacteria. Two components, Saccharomicin A and B, were isolated by reverse-phase HPLC, then desalted, concentrated, and lyophilized [13]. Solutions of the antibiotics ( 50 g/ L) were prepared for electrospray FT-ICR experiments in 50:50 (v/v) water:methanol with 2.5% acetic acid. All mass spectrometry measurements were performed in positive-ion mode with a homebuilt 9.4 tesla Fourier transform ion cyclotron resonance mass spectrometer [14]. The ions generated from a microelectrospray source [15] were externally accumulated [14] in a short (45 cm) rf-only octopole for 5 10 s before transfer 1999 American Society for Mass Spectrometry. Published by Elsevier Science Inc. Received May 3, /99/$20.00 Revised August 2, 1999 PII S (99) Accepted August 4, 1999

2 1286 SHI ET AL. J Am Soc Mass Spectrom 1999, 10, through a 200 cm long rf-only octopole into the Penning trap. A continuous-wave 40 W CO 2 laser (Synrad E , Bothell, WA) provided IR irradiation that passed through a BaF 2 window into the vacuum chamber [15] for IRMPD experiments. Mass resolving power ranged from 100,000 m/ m 50% 200,000 for accurate-mass measurements and 20,000 m/ m 50% 40,000 for IRMPD MS/MS experiments, in which m 50% is the magnitude mass spectral peak full-width at half-maximum peak height. For accurate mass measurements, an internal mass calibrant, poly(ethylene glycol) bis(carboxymethyl) ether (PEG BCME, number average molecular weight, M n 600, Aldrich, Milwaukee, WI) was added to the sample solution. The concentration of the calibrant was adjusted to match the calibrant ion abundance to that of the analyte. The analyte ions, together with the two nearest calibrant ions, were isolated by stored waveform inverse Fourier transform (SWIFT) [16, 17] massselective ejection. The number of ions in the ICR cell was reduced by dropping the trapping voltage to 0.5 V to avoid degradation of mass measurement accuracy due to space charge effects. The ions were excited by dipolar frequency sweep (chirp), 130 Hz/ s at 78.5 V p-p, followed by heterodyne detection at a Nyquist bandwidth of 50 khz and a reference frequency of 272 khz (corresponding to a mass-to-charge ratio window, 530 m/z 580). The obtained 64 Kword transient (K 1024) was Hanning apodized, zero-filled, and Fourier transformed to give a magnitude mode mass spectrum. Each peak centroid was interpolated by use of a quadratic fit to the three highest-magnitude data points. Five replicate measurements were performed for each sample. For IRMPD MS/MS, parent ions were SWIFT isolated and then exposed to IR laser irradiation, typically for 0.2 s. After a chirp excitation, 300 Hz/ s at V p-p, the transient was detected in direct mode at a Nyquist bandwidth of 1.45 MHz (corresponding to m/z 200). The resulting 512 Kword transient was then Hanning apodized, zero-filled, and Fourier transformed to give a magnitude mode mass spectrum. The MS/MS spectra were charge-state deconvolved by use of the Z-score algorithm [18] to yield mass-only spectra. If the deconvolved masses from two or more different charge states agreed to within 20 ppm, their isotopic distributions were combined. For MS n experiments with CAD, the parent ions were SWIFT isolated. N 2 gas was introduced into the vacuum system to raise the pressure to torr. Sustained off-resonance irradiation (SORI) [19] excitation was applied at a frequency 1% lower than the parent ion cyclotron frequency. The isolation/fragmentation process was repeated as needed to perform MS n experiments. A standard chirp excitation (same as above) and detection, followed by standard data processing (same as IRMPD MS/MS, but without Z-score deconvolution) yielded MS n spectra. Figure 1. ESI FT-ICR expanded mass spectral segments of Saccharomicin A (top) and B (bottom), with PEG BCME 600 as an internal mass calibrant. Accurate mass measurement was based on SWIFT isolation and narrow-band heterodyne detection of the analyte ions (M 5H) 5, together with the nearest available calibrant ions, at a mass resolving power of 150,000. Results and Discussion Accurate Mass Measurement Accurate mass measurement of each analyte was made possible by addition of an internal calibrant, PEG BCME 600. The relative abundances of the two calibrant ions, one slightly higher and one slightly lower in mass than the analyte ions, were adjusted to match approximately the analyte ion abundance (Figure 1). Because the m/z range for analyte ions is bracketed so closely and narrowly by calibrant ions, any nonideality in the standard calibration function [20] is minimized. By reducing the number of ions in the cell, we also minimize space charge effects that shift the cyclotron frequency. Under the present circumstances, in which signal-to-noise ratio is not high, high mass resolving power is especially critical for accurate mass determination [21]. From internal narrow-range mass calibration [and correction for the mass of the missing electron(s) in positively charged ions] [22], the masses of the antibiotics were computed from five repeated measurements to be Da for Saccharomicin A, and Da for Saccharomicin B (see Table 1). From the elemental compositions predicted from NMR, the exact mass (of the monoisotopic peak) for the two analytes is Da (C 121 H 207 N 9 O 59 S 2 ) for Saccharomicin A and Da (C 121 H 207 N 9 O 58 S 2 ) for Saccharomicin B [13]. Thus, we have achieved a

3 J Am Soc Mass Spectrom 1999, 10, SACCHAROMICINS BY FT-ICR-MS AND IRMPD 1287 Table 1. Accurate mass measurements, reported as monoisotopic mass of the neutral molecule, for Saccharomicin A and B Saccharomicin A Saccharomicin B Experimental Average Theoretical Error (Da) Error (ppm) mass accuracy of better than 0.5 ppm at nearly 3000 Da for both oligosaccharides, thereby confirming the chemical formula. The measured mass difference between Saccharomicin A and B is , unequivocally identifying a difference of one oxygen atom ( ), consistent with the proposed difference in elemental composition of sugar 10 between the two oligosaccharides (see Figures 2 4 for the molecular structures). MS/MS From the charge-state deconvolved MS/IRMPD/MS data (Figures 3 and 4), sequence information for each of the two oligosaccharides, Saccharomicins A and B, can be deduced. We limited our attention to underivatized oligosaccharides rather than the traditional permethylated compounds, because the issue here is sequence validation, not de novo sequencing. Numerous glycosidic cleavages from the nonreducing end are observed for the underivatized oligosaccharides [23, 24 ]. Various laser powers (from 16 up to 40 W) were applied to perform IRMPD. At low laser power (16 24 W), fragment ions are observed over the whole mass range with abundant structurally significant ions from 2250 to 2775 Figure 3. FT-ICR IRMPD charge state deconvolved mass spectrum of the Saccharomicin A parent ion. Composite spectra were obtained at laser duration of 0.2 s, with power ranging from 16 to 40 W at intervals of 8 W. Da. At higher laser power (32 40 W), fragment ions from 2250 to 2775 Da are absent, but additional lower mass fragment ions are observed over the range from 40 to 2200 Da. Some fragment ions result from multiple H 2 O losses and the facile losses of SO 3. The SO 3 loss probably comes from F16 rather than the ArSO 3 end group, because the O linkage is more fragile than the C linkage. Because IRMPD at different laser powers gives complementary structural information, we combined IRMPD mass spectra obtained at 16, 24, 32, and 40 W for each oligosaccharide, to yield the composite mass spectra shown in Figures 3 and 4. The dominant fragmentation pattern in the IRMPD spectrum of Saccharomicin A (Figure 3) is loss of the sugar residue on the nonreducing end, with the oxygen atom retained on the charged moiety (namely, Y ions, according to the fragmentation nomenclature proposed Figure 2. Molecular structures (and shorthand notations) for the sugar residues and the end group (reducing end of the oligosaccharides) found in the two oligosaccharide antibiotics, Saccharomicins A and B. Figure 4. FT-ICR IRMPD charge state deconvolved mass spectrum of the Saccharomicin B parent ion. Composite spectra were obtained at laser duration of 0.2 s, with power ranging from 16 to 40 W at intervals of 8 W.

4 1288 SHI ET AL. J Am Soc Mass Spectrom 1999, 10, by Domon and Costello [25]). Sequential loss of additional sugar residues produced fragment ions at mass (not m/z, because of the spectrum is charge state deconvolved), 2651 Da (Y 15 ) down to 576 Da (Y 2 ). Most of the oligosaccharide sequence, with the exception of the first two sugars at the reducing end, can therefore be validated. We assign the abundant ion at low mass ([435 Da as a double cleavage product that has one amino sugar (A or V) and two F (or R, which has exactly the same mass)]. The possible combinations can be F 12 A 11 R 10 (Y 11 /B 8 ), A 7 R 6 F 5 (Y 7 /B 12 ), R 6 F 5 A 4 (Y 6 /B 13 ), F 3 A 2 F 1 (Y 3 /B 16 ), etc. The IRMPD FT-ICR mass spectrum of Saccharomicin B is shown in Figure 4. Again, Y ions are the dominant fragments. The observed series of Y ions, from Y 15 down to Y 2 can be used to validate the sequence proposed by chemical degradation and 2D-NMR. By comparing Figures 3 and 4 in the low mass region, we infer that a large contribution at 435 Da in the IRMPD mass spectrum of Saccharomicin A is due to the Y 11 / B 8 fragment. The conclusion is based on the decreased ion abundance at 435 Da and increased ion abundance at 419 Da (corresponding to the Y 11 /B 8 fragment) in Saccharomicin B, 16 Da lower because of the substitution of a rhamnose by digitoxose. The neutral losses are accurate to within 10 mda (i.e., to within 0.01% of the neutral mass loss). The mass accuracy of the fragment ions was about 30 ppm with standard external calibration [20]. Space charge compensation [26] with the parent ion as the lock mass improved the mass accuracy to 20 ppm. Introducing an internal calibrant into the MS/MS spectrum [27] can improve the mass accuracy to the low ppm level. Because the sugar residues are either isomers, with the same mass, or differ in mass by at least 3 Da, efforts to achieve higher mass accuracy in the MS/MS experiment are not warranted. A peak at mass 1380 Da (Y 7 ), found in both tandem mass spectra, is a product of sequential sugar loss. No unexpected fragment ion (such as 1383 Da) is found in that region. On the other hand, another peak at mass 1303 Da (Y* 7 SO 3 ), also observed in both IRMPD spectra, suggests a sequence reversal in the oligosaccharides at F 8 and A 7. No expected fragment ion at mass 1300 is found. These two fragments ions (1380 and 1303 Da) represent the same sequence region (A 7 F 8 ) but give different answers. We propose that one of the fragments results from internal sugar loss prior to loss of terminal sugar. Such internal residue loss in oligosaccharides was originally reported for chemical ionization [28] and later for CAD of oligosaccharide ions produced by fast-atom bombardment [29, 30], liquid secondary ionization mass spectrometry [31], and electrospray ionization (ESI) [32] for both permethylated [28, 29] and underivatized [29 32] oligosaccharides. To determine the origin of these fragment ions, and therefore the oligosaccharide sequence of the whole compound, we performed MS n (n 2 4) experiments on compound 6, a nonsaccharide derived from chemical Figure 5. FT-ICR IRMPD charge state deconvolved mass spectrum of compound 6 (a chemical degradation product from Saccharomicin B), obtained at a laser duration of 0.2 s and 16 W power. degradation (methanolysis in 0.5% HCl) of Saccharomicin B (Figure 5) [13]. CAD as well as IRMPD was used as the fragmentation method. CAD gave very similar fragment ions but higher abundance for high mass fragments, presumably because IRMPD deposits energy onto all ions, including parent ions and daughter ions, whereas CAD can mass-selectively energize parent ions only. Although IRMPD FT-ICR MS/MS of compound 6 gave exclusively m/z 1304 (i.e., neutral mass 1303 Da), SORI-CAD FT-ICR MS/MS of compound 6 gave an additional product at m/z 1301, the expected sequential fragmentation result. The presence of species at m/z 1301 and 1304 was also observed by CAD MS/MS (not shown) with a Q-TOF instrument. We can further elucidate the fragmentation pathway by SORI-CAD FT-ICR MS n (n 3, 4). The parent ion (doubly charged ion of 1669 Da equivalent neutral monoisotopic mass) gave MS 2 first-generation fragment ions at 1526 Da. Subsequently, singly charged MS 2 ions of 1526 Da gave second-generation (MS 3 ) ions of 1446 Da. Finally, additional isolation and fragmentation of (MS 3 ) ions of 146 Da gave nearly equal amounts of (MS 4 ) 1300 and 1303 Da ions at low abundance, suggesting either the presence of two fragmentation pathways or multiple original compounds. Finally, in separate experiments, MS 4 of compound 6 was performed by two pathways, each of which begins by SWIFT isolation of the parent ions of compound 6, followed by SORI-CAD to yield ions of 1526 Da, which are then SWIFT isolated and subjected to SORI-CAD to yield ions of 1300 and 1303 Da. SORI-CAD of SWIFTisolated ions of 1303 Da produced far fewer 1157 Da ions (i.e., the product of further loss of one more sugar residue) than SORI-CAD of simultaneously SWIFTisolated ions of 1303 and 1300 Da (Figure 6). These MS 4 experiments imply that fragment 1300 Da is the sequential product because it is more readily fragmented to the next product, 1157 Da. The rearrangement product,

5 J Am Soc Mass Spectrom 1999, 10, SACCHAROMICINS BY FT-ICR-MS AND IRMPD ), Florida State University, and the National High Magnetic Field Laboratory in Tallahassee, FL. References Figure 6. MS 4 spectra of compound 6B produced by CAD with each of two different third stage isolations. Top: fragmentation of ions at m/z 1304 (Y* 7 SO 3 H) only. Bottom: fragmentation of ions at m/z 1304 (Y* 7 SO 3 H) and 1301 (Y 7 SO 3 H). fragment 1303 Da, resulting from internal loss of A 7,is presumably more stable than fragment 1300 Da, the product of sequential loss of F 8. Conclusions By means of local internal calibration and electron mass correction, mass accuracy better than 0.5 ppm has been achieved for each of the two oligosaccharides, Saccharomicins A and B, consistent with their proposed elemental compositions based upon prior NMR data. High resolution and high mass accuracy MS/MS data were obtained by IRMPD with a 40 W continuous-wave CO 2 laser for the oligosaccharides. The spectra were charge-state deconvolved by the Z-score algorithm [18] to yield much simpler mass-only spectra. Accurate mass measurement of the neutral losses from each parent ion serves to identify the sequential losses of sugar residues. Sequences of 15 sugar residues could be confirmed from charge state deconvolved MS/MS spectra Saccharomicins A and B. Internal sugar loss has been observed in IRMPD and studied by CAD MS 4. Acknowledgments The authors thank Forest M. White, Catherine E. Costello, and Bruce B. Reinhold for helpful discussions. This work was supported by Wyeth-Ayerst Research, and by grants from the NSF National High Field FT-ICR Mass Spectrometry Facility (CHE Comisarow, M. B.; Marshall, A. G. Fourier Transform Ion Cyclotron Resonance Spectroscopy. Chem. Phys. Lett. 1974, 25, Comisarow, M. B.; Marshall, A. G. Frequency-Sweep Fourier Transform Ion Cyclotron Resonance Spectroscopy. Chem. Phys. Lett. 1974, 26, Amster, I. J. A Tutorial on Fourier Transform Mass Spectrometry. J. Mass Spectrom. 1996, 31, Marshall, A. G.; Hendrickson, C. L.; Jackson, G. S. Fourier Transform Ion Cyclotron Resonance Mass Spectrometry: A Primer. Mass Spectrom. Rev. 1998, 17, Campbell, S.; Beauchamp, J. L. Infrared Laser Multiphoton Dissociation of Proton Bound Dimers of Biomolecules: A New Method to Probe the Acid Base Properties of Local Sites in Complex Molecules. Proc. SPIE-Int. Soc. Opt. Eng. 1992, 1636, Baer, S.; Brauman, J. I. Infrared Multiple Photon Studies of Alkoxide Alcohol Complexes. J. Am. Chem. Soc. 1992, 114, Little, D. P.; Speir, J. P.; Senko, M. W.; O Cornnor, P. B.; McLafferty, F. W. Infrared Multiphoton Dissociation of Large Multiply-charged Ions for Biomolecule Sequencing. Anal. Chem. 1994, 66, Aaserud, D. J.; Little, D. P.; O Connor, P. B.; McLafferty, F. W. Distinguishing N-Terminus and C-Terminus Ions for Mass- Spectrometry Sequencing of Proteins without Prior Degradation. Rapid Commun. Mass Spectrom. 1996, 9, Dufresne, C. P.; Wood, T. D.; Hendrickson, C. L. Highresolution Electrospray Ionization Fourier Transform Mass Spectrometry with Infrared Multiphoton Dissocation of Glucokinase from Bacillus Stearothermophilus. J. Am. Soc. Mass Spectrom. 1998, 9, Little, D. P.; McLafferty, F. W. Sequencing 50-mer DNAs Using Electrospray Tandem Mass Spectroscopy and Complementary Fragmentation Methods. J. Am. Chem. Soc 1995, 117, Little, D. P.; Aaserud, D. J.; Valaskovic, G. A.; McLafferty, F. W. Sequence Information from mer DNAs (Complete for a 50-mer) by Tandem Mass Spectrometry. J. Am. Chem. Soc. 1996, 118, Little, D. P.; McLafferty, F. W. Infrared Photodissociation of Noncovalent Adducts of Electrosprayed Nucleotide Ions. J. Am. Soc. Mass Spectrom. 1996, 7, Kong, F.; Zhao, N.; Siegel, M. M.; Janota, K.; Ashcroft, J. S.; Koehn, F. E.; Borders, D. B.; Carter, G. T. Saccharomicins, Novel Heptadecaglycoside Antibiotics Effective Against Multidrug-Resistant Bacteria. J. Am. Chem. Soc. 1998, 120, Senko, M. W.; Hendrickson, C. L.; Emmett, M. R.; Shi, S. D.-H.; Marshall, A. G. External Accumulation of Ions for Enhanced Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry. J. Am. Soc. Mass Spectrom. 1997, 8, Emmett, M. R.; White, F. M.; Hendrickson, C. L.; Shi, S. D.-H.; Marshall, A. G. Application of Micro-Electrospray Liquid Chromatography Techniques to FT-ICR MS for High Sensitivity Biological Analysis. J. Am. Soc. Mass Spectrom. 1998, 9, Marshall, A. G.; Wang, T.-C. L.; Ricca, T. L. Tailored Excitation for Fourier Transform Ion Cyclotron Resonance Mass Spectrometry. J. Am. Chem. Soc. 1985, 107, Guan, S.; Marshall, A. G. Stored Waveform Inverse Fourier

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