Anal. chem. 1991, 63,2526-2529
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CORRESPONDENCE Quadrupole Fourier Transform Mass Spectrometry of Oligosaccharides Sir: The investigation of bioorganic molecules in Fourier transform mass spectrometry (FTMS) has been greatly advanced by the addition of the external source (1-8). Separating the ion source from the analyzer allows the use of ion sources that generate pressures generally much higher than those necessary to operate the FTMS system (1-8). Thus, the use of liquid secondary ion mass spectrometry (LSIMS) (9) in the FTMS system has been made possible only with an external source (4, 10, 11). Hunt and McIver first showed the capability of liquid secondary ion mass spectrometry (LSIMS)/FTMS on peptides (11). A detection limit of 10 pmole and resolution as high as 20000 for a peptide with a mass m / z 1758 was obtained (11). Subsequent studies by Hunt and others show that peptides larger than m / z 10000 can be generated and observed (12). The trapped-ion technique has several inherent advantages. Collisionally induced dissociation can be performed without loss in resolution due to kinetic energy release (13). Ions are all simultaneously sampled during the analysis (1, 2). Photodissociation of trapped ions is far simpler, particularly with low duty time lasers such as excimers (14). Thus, the photodissociation of peptides has been performed in the FTMS system to yield useful sequence information (14-16). Photodissociation has been performed on a 30-pmole sample of a peptide derived from spinach chloroplasts, yielding full sequence information (15). A comparison of FAB fragmentation and photodissociation of peptides shows that photodissociation produces many of the ions found in FAB alone as well as other fragments not observed in the FAB spectra (16). The general performance of FTMS is also enhanced with an external source (4, 17). For example, masses as large as m / z 31 830 corresponding to the cesium iodide cluster (CsI)&s+ have been observed as well as resolution of 53000 for m / z 9746 corresponding to the cluster (CSI)~,CS+ (4, 17). Despite its general utility, QFTMS has been used with a relatively few classes of bioorganic compounds. We have begun the study of oligosaccharides using a QFTMS instrument recently built in our laboratory. No studies exist on LSIMSfFTMS of this class of compounds although the laser desorption/ FTMS of oligosaccharides has been reported (18). In this report we show the ideal compatibility of these compounds with LSIMSfFTMS. In contrast to the lack of fragmentation, the presence of pseudomolecular peaks, and the overabundance of matrix ions reported for the FAB spectra of oligosaccharides in sector instruments, we find the spectra of these compounds to be very nearly free of matrix ions with large abundances of both parent and fragment ions (19).
EXPERIMENTAL SECTION
All experimentshave been performed on the UC Davis QFTMS instrument, which has been described in an earlier publication (20).Ions are produced in a SIMS source fitted with an Antek Cs+ gun operating between 5 and 10 kV. The primary beam is pulsed and accelerated to a copper probe tip that contains the sample in a glycerol or glycerol/thioglycerol matrix. The resulting secondary ion beam is extracted and guided by a 119 cm long quadrupole ion guide into the analyzer cell contained in the homogeneous region of a superconducting 3-T magnet from CryomagneticsInc. The ion current has been monitored on the 0003-2700/91/0363-2526$02.50/0
rear trapping plate of the analyzer cell and is usually between 1and 10 nA. The ion production/injection time is variable and is typically between 5 and 500 ms although pulses as narrow as 1ms are sufficient to obtain signals. The quadrupole, operated in an rf-only mode, functions only as an ion guide with typical frequencies ranging between 0.5 and 1.5 MHz, depending on the mass range desired (21). Pulsing the trapping plate is not necessary to trap the ions. Ions can be collected linearly by increasing the length of the injection until saturation (space charge limit) is reached. Saturation can be anywhere between 100 and lo00 ms, depending on the strength of the signal. To maintain differential pumping, a turbo pump with a pumping speed of 170 L/s operates on the source. Two APD cryopumps each with a pumping speed of 2000 L/s (for N2) operate on the ion transport region and on the analyzer region. Pressures in the source during an experiment are typically in the range 104-10-6 Torr, while pressures in the analyzer region are maintained at 10-e-lO-lo Torr. All the compounds used are commercially available (Sigma and Aldrich) and are used without further purification. For the low molecular weight oligosaccharides such as the di- and trisaccharides, impurities of higher molecular weight oligosaccharides are observed. Sample preparation involves dissolving the oligosaccharide in water or dilute acid (CH3C02Hor HC1) to form an approximately M solution. A 1-5-wL portion of the solution is placed on a copper probe tip that contains 2 pL of matrix material. For the disaccharides,pure glycerol is used. Mixtures of glycerol and thioglycerol are used for the larger oligomers. Before the sample is introduced into the ion source, it is briefly degassed to remove most of the water. When the sample probe is in place, it is grounded and bombarded by a Cs+beam produced from an Antek ion gun with acceleration energies typically between 5 and 10 kV. A series of scans are accumulated for each spectra. The number of accumulated scans varies; for long ion injection time (e.g. 500 ms) only 10 scans are accumulated, while for the short (e.g. 5 ms) injection time up to 100 scans are accumulated. A routine analysis usually lasta several minutes.
RESULTS Representative QFTMS spectra of several oligosaccharides are shown in Figures 1-4. Abundant protonated molecular (MH+) and fragment ions are observed with all the oligosaccharides we have investigated. a-Lactose ( 4 - 0 - p ~ galactopyranosyl-cy-Dglumse)and sucrose (a-Dglucopyranosyl P-D-fructofuranoside) are representatives of the disaccharide samples. The sucrose sample we obtained contained trisaccharide impurities, which are observed as a trio of peaks having m/z 487,505, and 523 (Figure 1). The protonated molecular parent of sucrose is readily identifiable, forming the base peak of the spectrum. An adduct of water (MH + H20)+is observed for both the disaccharide (m/z 361) and the trisaccharide impurity ( m / z 523). Major fragmentation due to the cleavage of the glycosidic bond is also observed (m/z 163) as well as losses of water molecules from both the parent and the fragment ions. The cleavage of the glycosidic linkage ( m / z 163) is equivalent to that commonly reported in the mass spectrometry of oligosaccharides (19). Fragmentation to form the protonated monosaccharide ( m / z 181) is not observed. A matrix background corresponding to a peak at every mass unit is observed up to around m/z 325. These signals are, however, small with intensities less than 10% and usually below 5% of the base peak. The largest matrix signal is due to pro@ 1991 American Chemical Society
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Figure 1. Liquid SIMSIFTMS of sucrose with glycerol matrix in M jbb7 broad-band mode. The base peak is the protonated molecular Ion. I1 1 Unidentifiable matrix ions are present at every mass unit with the IO0 200 300 400 500 600 700 eo0 m/i n largest matrix Mnal (mlz 93) due to the protonated glycerol l ~ (a+). Fragment ions of sucrose are labeled by their masses with the corFigure 3. Liqukl SIMSIFTMS of maltotetraosewith glycerd/thkglycerd responding fragmentation illustrated in the structure above. The ions (1:l) matrix In the broad-band mode. Fragment ions are labeled by having mlz 505 and 523 correspond to oligosaccharide Impurities. their masses. The corresponding fragmentation is illustrated In the structure above. 619
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Figure 2. Liquid SIMSlFTMS of lactose wkh a 5-ms injection width and 1-ms detection delay (top spectrum) and a 5-ms injection wldth and l0-m~detection delay (bottom spectnnn)In the broabband mode. Two prominent matrix peaks are labeled Corresponding to protonated and the protonated dimer ( m / z 185, G,H+). glycerol (mlz 92, &I+) The dimer decomposes at the longer detection delay time (bottom). Fragment Ions are labeled by their masses with the corresponding fragmentation illustrated in the structure above. Both spectra are normalized to the protonated glycerol peak.
tonated glycerol (mlz 931, which is 70% of the base peak. A signal due to Cs+ ( m / z 133) is often present in the QFTMS spectrum. Cs+ from the cesium gun is sometimes extracted from the source and injected into the analyzer cell, albeit in small amounts. We find that the intensity of the Cs+ signal rises as a function of time the sample is in the instrument. Initially, very little or no Cs+ is observed and, as the sample becomes "old", the Cs+ intensity increases. This phenomenon may be due to the increase of reflecting collisions between the primary beam and the metal probe tip as the sample and the matrix are depleted. The gun is mounted 4 5 O to the probe tip surface, with the extraction aperture forming an equivalent reflection angle to the Cs+ gun. When the sample is in place the primary beam is deflected away from the extraction aperture. As the sample is depleted and the metal surface of the probe becomes exposed, reflecting collisions direct the primary beam toward the extraction aperture. This is confirmed by the sole presence of Cs+ ions after the sample is nearly depleted. Two spectra of the disaccharide lactose were obtained to illustrate the effects of varying the detection delay time on
FIgure 4. Liquid SIMS/FTMS of makoheptaose with glyceroilthioglycerol (31) matrix in the broad-band mode. It is not always p i b k to totally eliminate matrix ions, but the oligosaccharide signals are clearly identifiable. Signals due to the matrix ions are labeled as G,H+. Fragment ions are labeled by the their masses wlth the corresponding fragmentation illustrated in the structure above. the general appearance of the spectrum. Figure 2 consists of spectra produced with an ion injection time of 5 ms with a detection delay time of 1ms (top spectrum) and a detection delay time of 10 ms (bottom spectrum) after the ion injection. Large abundances of protonated glycerol ( m / z 93,97% relative abundance) as well as the protonated dimer (m/z 185, base peak) are observed at the shorter detection delay time. With the longer detection delay time the dimer ion abundance decreases relative to the protonated glycerol. The monoglycerol signal increases as the diglycerol cluster decreases but is not observed in this comparison because both spectra have been normalized to the protonated glycerol base peak. Interestingly, the intensity of the saccharide fragment ions also increases slightly with respect to the parent. Weak abundances of background matrix ions are also observed with larger oligosaccharides (Figures 3 and 4). The spectrum for maltotetraose (Figure 3), for example, is nearly devoid of matrix ions. No attempts have been made in the experiments to filter out these ions using the quadrupole rode in any of the spectra shown. Although matrix ions can usually be minimized, it is not yet possible to totally eliminate them, as evidenced by the peaks labeled G,H+ in the maltoheptaose spectrum. Both examples, maltotetraose and maltoheptaose
2528 la
ANALYTICAL CHEMISTRY, VOL. 63, NO. 21, NOVEMBER 1, 1991 OH*
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ZAB-2F instrument. Identifiable signals due to matrix and matrixlanatyte complex ions are labeled accordingly (e.g.G,H+ or [MH + G,]'). A parent ion is obtained as well as two major fragment ions, which are also observed in the QFTMS spectra, i.e. m l z 325 and 487, respectively.
yield large fragment ion abundances. The spectrum of maltotetraose is significant in that underivatized samples of this compound have been reported to produce little fragmentation in sector instruments (22). For comparison, we have obtained the spectrum of this compound using a VG ZAB-PF double-sector instrument (Figure 5). The prevalence of matrix ions in this spectrum is apparent as well as the formation of complexes between analyte and matrix molecules (M + G, + H)+ (23). Fragment ions are formed, however, corresponding to the loss of one and two glycoside units. The major fragment peak observed in both instruments corresponds to the same ionic species ( m / z 325). The relative abundance of the parent is greater in the sector than in the QFTMS instrument. The large abundances of fragment ions as well as the lack of matrix and matrix/analyte clusters may be attributed to the significantly different time scales between the two MS methods. The time between ion production and ion detection for sector instruments is in the order of microseconds,while for the FTMS system it ranges from milliseconds to even minutes. We propose that sufficient internal energy is obtained by the ions during LSIMS and FAB ionization to produce the unimolecular fragmentation of both the oligosaccharide and matrix cluster ions. However, only the longer experimental time scale of FTMS allows the ions sufficient time to undergo a greater degree of fragmentation. This effect is somewhat illustrated by the two spectra of lactose, where the detection times are varied. It is further evidenced by the similarity in fragment ions observed in both the QFTMS and ZAB spectra. Thus, by varying the detection delay time with the QFTMS instrument, it is possible to vary the relative abundance of the parent and fragment ions (24). In sector instruments, derivatization is often used to increase the yields of fragmentation. However, derivatization significantly increases the mass of the compound and represents an added step (25). Other complications occur during derivatization such as noncompletion and even degradation of naturally occurring, partially derivatized oligosaccharides (26). Thus, in general, fragmentation without derivatization is clearly desirable. By far the major cleavage reactions observed with all the oligosaccharideswe have investigated in the QFTMS instrument correspond to the same as those observed for the disaccharides. These cleavages occur strictly along interglycosidic linkages, producing the fragments shown earlier. The fragmentation features of maltohexaose and malto-
heptaose differ slightly from those of smaller oligomers in that sequence peaks appear as doublets starting with the tetrasaccharide fragment ions of the larger oligomers up to the parent peak. The doublets correspond to dissociations occurring on both sides of the connecting oxygen atom (Figure 4). A recent report has shown that surfaceinduced dissociation (SLD)occurs when ions are transported by the quadrupole rods and collided with a 90% transmittance wire mesh, which forms the front trapping plate (i.e. the trapping plate closest to the quadrupole rods) of the analyzer cell (26). We must emphasize that the UCD instrument contains a solid stainless steel plate with a 2-mm aperture as a front trapping plate. Ions enter unhindered through the hole into the analyzer cell. SID may occur if the injected ions collide with a solid rear trapping plate of the analyzer cell. However, the rear trapping plate of our instrument has a 13-mm hole covered with a high-transmittance gold wire mesh (90% optical transmittance), which is used as part of the internal electron impact source. The trapping plate is maintained between 1.5 and 3.5 V, thus allowing high translational energy ions to go through the mesh and become lost. In addition, SID on metal surfaces is a low-efficiency process due to the competition with neutralization reactions so that the few back-scattered particles are most likely neutralized. It is, therefore, unlikely that SID occurs on the rear trapping plate of our instrument. Similar conclusions have been reported by Williams et al. on their QFTMS instrument (26). A source for vibrational excitation could be collisions between the ions and the background neutral either during ion transport or after the ions have been trapped in the analyzer cell. This mode of excitation is unlikely due to the low background pressure (6 X 10-loTorr) in the analyzer chamber during the experiments. Hence, we are fairly confident that ions acquire sufficient energy for decomposition during ion formation and decompose unimolecularly in the analyzer cell of the FTMS instrument. Further investigations are underway to probe the magnitude and the nature of the vibrational excitation. Cleavages within the glycosidic rings are not observed with any of the oligosaccharides in the study. This means that no informationon the linkage positions is obtainable. This result is not atypical for the FABMS of underivatized oligosaccharides. However, the CID of the trapped ions or better yet, the photodissociation, may yield fragmentation of the glycosidic ring, thus providing linkage information. The large fragment ion abundances and the low matrix background means that MS/MS experiments can also be performed on the daughter ions, yielding further structural information. The detection limit of the instrument for oligosaccharides has been determined by using maltotriose. At least 6 pg (12 nmol) of material is needed to obtain a spectrum complete with parent and fragment ions. With this amount of sample, a signal/noise ratio of roughly 4 is obtained for the parent (m/z 505) and about 20 for the most abundant disaccharide fragment ( m / z 325). This limit corresponds well to those reported for sector instruments, which is between 1and 5 pg (27). Further optimization, however, is still being performed on the instrument. Satisfactory agreement is obtained between the observed masses and exact mass for parent and fragment ions despite the large mass range. An average deviation of 56 ppm between exact mass and observed mass is obtained for the ions produced from maltohexaose. This mass range includes the smallest fragment with mlt 163 to the protonated parent with m / z 991. This kind of mass deviation is not yet a t the best limit reported for FTMS instruments. Average mass deviation of less then 10 ppm has been reported for the laser desorption/FTMS of several oligosaccharides (28). The resulting
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Anal. Chem. 1991, 63,2529-2532
resolution at the high mass value is a limitation of the data system and can be corrected by using faster digitization rates. High resolution is, nonetheless, still obtainable with the current system by using the heterodyne narrow-band mode. In this mode, a resolution corresponding to 16000 for sodiated maltotetraose ( m / z 689) is obtained.
CONCLUSION The external source QFTMS instrument is ideal for the analysis of oligosaccharides. Large abundances of parent as well as fragment ions are observed. As with sector instruments, sensitivity is not as good as that obtained for oligopeptides. However, unlike sector instruments, the spectra of oligosaccharides are clean without major interference from matrix ions. Furthermore, derivatization is not necessary to produce fragment ions. Further work is in progress to increase the sensitivity and to determine the upper mass limit of the instrument for oligosaccharides. In addition, collisionally induced dissociation and laser photodissociation studies are currently in progress to produce fragmentation of the glycosidic ring to yield positional information of the glycosidic bonds. Registry No. Sucrose, 57-50-1;lactose, 63-42-3;maltotetraose, 34612-38-9; maltoheptaose, 34620-78-5. LITERATURE CITED (1) Comlsarow, M. 8.; Marshall, A. G. Chem. phys. Lett. 1974, 2 5 , 282-283. (2) Comlsarow, M. B.; Marshall, A. 0. Chem. Phys. Lett. 1974, 2 6 , 489-490. (3) Mcive;,-R. T., Jr.; Hunter, R. L.; Bowers, W. D. Int. J. Mass Spectrom. Ion Processes 1985. 64. 67-77. (4) Lebrllla, C. 8.; Amster, I. J.; McIver, R. T., Jr. Int. J. Mass Spectrom. Ion Processes 1989,87, R7-Rl3. (5) Kofel, P.; Allemann, M.; Kellerhals, Hp.; Wanczek, K. P. Int. J . Mass Spectrom. Ion Processes 1988, 72, 53-61. (6) Kofel, P.; Allemann, M.; Kellerhals. Hp.; Wanczek, K. P. Int. J. Mass Spectrom. Ion Processes 1985,65, 97-103. (7)Alford, J. M.; Welss, F. D.; Laaksonen, R. T.; Smalley, R. E. J. phys. Chem. 1986, 90, 4480-4402. (8) McMahon, T. 8. Proceedlngs of the 37th ASMS Conference on Mass Spectrometry and Allied Topics, Miami Beach, FL, May 21-26,1989. Kofel, P.; McMahon, T. B. Int. J. Mass Spectrom. Ion Processes 1990, 98, 1160-1161. (9) Barber, M.; Bordoll, R. S.; Sedgwlck, R. D.; Tyler, A. N. J. Chem. Soc.. Chem. c 0 ” o n . 1981,325-327. (10) Ijames. C. F.; Wllkins. C. L. J. Am. Soc. Mass Spectrom. 1990, 7 ,
208-216. (11) Hunt, D. F.; Shabanowltz, J.; Yates, J. R., 111; McIver, R. T., Jr.;
Hunter, R. L.; Syka, J. E. P.; Amy, J. Anal. Chem. 1985, 57,
2728-2733.
(12) Hunt, D. F.; Shabanowltz, J.; Yates. J. R.. 111; Zhu, N.2.; Rwsel, D. H.; Castro, M. E. Roc. Mtl. Aced. Sci. U.S.A. 1987, 8 4 , 620-623. (13) For kinetic energy loss durlng CID In sector Instruments see: New mann, 0. M.; Derrick, P. J. &g. Mass Spectrom. 1984. 79, 165-170. (14)Bowers, W. D.; Delbert, S.S.; Hunter, R. L.; McIver, R. T., Jr. J . Am. Chem. SOC.1984. 106, 7280-7209. (15) Hunt, D. F.; Shabanowltz, J.; Yates, J. R., 111 J. Chem. Soc., Chem. Commun. 1987. 548-550. (16) Lebrllla, C. B.; Wang, D. T.-S.; Miroguchi, T. J.; Mclver, R. T., Jr. J. Am. Chem. Soc. 1989, 7 7 1 , 8593-8590. (17) Lebrllla, C. B.; Wang, D. T.-S.; Hunter, R. L.; McIver, R. T., Jr. Anal. Chem. 1990, 6 2 , 878-800. (18) Coates, M. L.; Wllkins, C. L. Anal. Chem. 1987, 5 9 , 197-200. Lam, 2.; Comisarow. M. B.; Dutton, G. G. S.; Parolis, H.; Parolls, L. A. S.;
Bjarnason, A.; Well, D. A. Anal. Chim. Act8 1990. 241, 107-199. (19) Dell, A. Adv. Carbohydr. Chem. Blochem. 1917, 45, 19-72. (20) McCullough, S. M.; Gard, E.; Lebrllla, C. 8. Int. J. Mass Spectrom. Ion Processes 1991, 107, 91-102. (21) McIver, R. T., Jr. Int. J. Mass Spectrom. Ion Rocesses 1990,98, 35-50. (22) Martin, W. B.; Silly, L.; Murphy, C. M.; Raley, T. J., Jr.; Bean, M. F.; Cotter, R. J. Int. J. Mass Spectrom. Ion Processes 1989. 92,
243-265. (23) The use of flow FAB has recently been reporled to Increase the sensitlvity as well as diminish matrix ion abundances in sector Instruments. See for example: Caprioll. R. M.; Fan, T.; Cottrell, J. S. Anal. Chem. 1986,58, 2949-2954. (24)Ngoka, L.; Carroll, J. A.; Lebrllla. C. 8. Unpubllshed results. (25)Dell, A.; Tiller, P. R. Biochem. Blophys. Res. Commun. 1986, 135, 1 126-34. (26) Williams, E. R.; Henry, K. D.; McLafferty, F. W.; Shabanowltz, J.; Hunt, D. F. J. Am. Soc. Mass Spectrom. 1990, 1 , 413-410. (27) Dell, A.; Oates, J. E.; Morris, H. R.; Egge, H. Int. J. Mass Spectrom. Ion Phys. 1983,46, 415-418. (28) Lam, 2.; Comisarow. M. B.; Dutton, G. G. S.; Parolis, H.; Parolls, L. A. S.; Bjarnason, A.; Well, D. A. Anal. Chim. Acta 1990, 247, 167-199.
James A. Carroll Lambert Ngoka S6si McCullough Eric Gard A. Daniel Jones Carlito B. Lebrilla* Department of Chemistry and Facility for Advanced Instrumentation University of California Davis, California 95616
RECEIVED for review March 25,1991. Accepted July 17,1991. Financial support has been provided by the Chemistry Department of the University of California, Davis, Faculty Research Grants, and the Petroleum Research Fund. E.G. wishes to thank the U. C. President’s Undergraduate Fellowship Program for the award.
Separation and Determination of Copper, Zinc, Palladium, Iron, and Manganese with meso-Tetrakis(3-bromo-4-sulfophenyl)porphine and Reversed-Phase Ion-Pair Liquid Chromatography Sir: Porphyrins are highly sensitive reagents for the spectrophotometricdetermination of trace amounts of metals. Nevertheless, the overlapping of the major absorption peaks of the metal complexes seriously interferes with the spectrophotometric determination of metals. Separation of these species by HPLC permits the metalloporphyrins to be determined a t one wavelength, thds the application of HPLC to the separation and determination of metalloporphyrins is a highly sensitive, selective, and effective analytical method. Previous HPLC studies with metalloporphyrins only include meso-tetraphenylporphyrin (TPP) (1,2), meso-tetrakis(4toly1)porphyrin (TTP) (31, meso-tetrakis(N-methyl-4pyridinium)porphine (TMPyP) (4), meso-tetrakis(4hydroxypheny1)porphine (THPP) (5), and meso-tetrakis(4-
carboxypheny1)porphine (TCPP) (6). In those porphyrin reagents only TPP and THPP have been applied to the quantitative determination of metals in the practice samples. TPP was used to determine Cu, Zn, and Ni in NBS bovine liver, and limits of detection (5 pL injected) were 125 pg for every metal. THPP was used to determine Cu and Zn in tap water, and limits of detection (0.1 mL injected) were 70 pg for Cu and 9150 pg for Zn. We synthesized a new porphyrin reagent-meso-tetrakis( 3-bromo-4-sulfopheny1)porphine (meso-BrTPPS,, Figure 1). The five complexes of Mn(II), Fe(III), Zn(II), Pd(II), and Cu(I1) were successfully separated by reversed-phaseion-pair HPLC using meso-BrTPPS4as the precolumn chelating reagent. This method was successfully applied to the determination of the metals in the peach leaves
0003-2700/91/0363-2529$02.50/00 1991 American Chemical Society
