- Email: [email protected]
Electrospray Ionization–Mass Spectrometry Characterization of Heterotetrameric Sarcosine Oxidase
Electrospray Ionization–Mass Spectrometry Characterization of Heterotetrameric Sarcosine Oxidase Ljiljana Pasˇa Tolic´, Amy C. Harms, Gordon A. Anderson, and Richard D. Smith Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, Washington, USA
Annie Willie and Marilyn Schuman Jorns Department of Biochemistry, Allegheny University of the Health Sciences, Philadelphia, Pennsylvania, USA
Electrospray ionization (ESI) Fourier transform ion cyclotron resonance (FTICR) mass spectrometry has been used to characterize heterotetrameric corynebacterial sarcosine oxidase. By using a conventional quadrupole mass spectrometer, no spectra for the intact complex could be obtained (i.e., electrospraying protein at neutral pH), but spectra showing the four protein subunits were obtained when electrospraying from acidic solution. Initial low resolution ESI-FTICR mass spectra of the intact heterotetramer revealed a typical narrow charge state distribution in the range 6000 , m/z , 9000, consistent with retention of a compact structure in the gas phase, and gave a mass measurement about 1000 u higher than predicted. Efficient in-trap clean up, based upon low energy collisionally induced dissociation of adducts, allowed significant improvement in mass measurement accuracy. The present results represent the largest heteromultimeric protein complex successfully analyzed using FTICR mass spectrometry, and clearly illustrate the importance of sample clean up methods for large molecule characterization. (J Am Soc Mass Spectrom 1998, 9, 510 –515) © 1998 American Society for Mass Spectrometry
E
lectrospray ionization mass spectrometry (ESIMS) [1] is now broadly utilized for biomolecule characterization. Most applications, however, involve biopolymers with molecular masses M r , 50,000 u. Although Fourier transform ion cyclotron resonance (FTICR) mass spectrometry [2–5] holds advantages for high M r analyses in terms of its high resolution and mass measurement precision, studies of high mass-tocharge ratio species produced by ESI [or matrix-assisted laser desorption ionization (MALDI)] have generally been problematic, and few results have been reported [6]. The major difficulties have included the reduced trapping efficiency of these larger ions, detection problems arising from imperfect electric fields due to the nonideal FTICR trap and, to some extent, the weaker confinement provided by the magnetic field (B) at higher mass-to-charge ratio. These factors combine to result in an upper mass-to-charge ratio limit that is often significantly lower than the predicted “critical mass-to-charge ratio,” i.e., highest mass-to-charge ratio that can be effectively trapped for given experimental Address correspondence to Richard D. Smith, Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, P.O. Box 999, Mail Stop K8-98, Richland, WA 99352. E-mail: [email protected]
conditions [5]. A major problem is that the number of species with different mass-to-charge ratio increases rapidly with molecular mass. The large number of trapped charges that is required to define the charge state distribution and enable M r determination leads to Coulomb-induced frequency shifts, peak coalescence, and rapid loss of ion packet coherence. Such ion cloud instability problems preclude high resolution and mass measurement accuracy for high molecular mass species (i.e., M r . 80,000 for a 7-tesla magnetic field strength) [7, 8]. Recently, we have obtained ESI-FTICR mass spectra of Starburst™ polyamidoamine dendrimers with masses up to ;1,000,000 u [9], representing the highest molecular mass species yet detected by FTICR mass spectrometry, with the exception of results based upon individual ion detection [10 –12]. ESI-FTICR mass spectra obtained with dendrimers, together with the spectra acquired with dendrimer/IgG immunoconjugates, highlight the current limitations of FTICR technology. Because most aspects of FTICR performance are enhanced with increasing B [13], it is reasonable to expect significant improvements in applicability of FTICR mass spectrometry to biological problems as higher magnetic field instruments become available. An illus-
© 1998 American Society for Mass Spectrometry. Published by Elsevier Science Inc. 1044-0305/98/$19.00 PII S1044-0305(98)00011-7
Received August 18, 1997 Revised December 12, 1997 Accepted December 20, 1997
J Am Soc Mass Spectrom 1998, 9, 510 –515
trative example is the unit resolution of 112,000 u molecules, with 3 u accuracy, recently achieved with a 9.4-tesla FTICR mass spectrometer [14], employing collisional focusing of electrosprayed ions in an octopole [15]. It should be noted that new approaches based on individual ion detection aim to circumvent the trapped ion density limitations arising at higher molecular masses, but present a different set of challenges [8, 10 –12]. The limited mass-to-charge ratio range is particularly problematic for studies of noncovalent complexes under physiologically relevant conditions where lower charge states are observed because of the typically more compact structures. There is now a substantial body of evidence that demonstrates that noncovalent biologically relevant complexes can be observed by ESI-MS [16 –29]. A recent demonstration that viruses can “survive” (e.g., preserve ability to infect) ESI-MS offers strong evidence that at least some native structure of biomolecules can be conserved through the electrospray process and mass analysis [30]. The utility of ESI-MS in studies of self-assembling processes of noncovalent complexes, i.e., subunit stoichiometry, which is essential for a better understanding of different cellular functions at the molecular level, is increasingly evident [31]. Although ESI-TOF instrumentation has proven its utility in characterization of noncovalent complexes, the ESI-FTICR combination, with its high resolution and multistage MSn capabilities, also holds considerable promise for such applications. However, further improvement is needed to significantly extend the routinely achievable mass and mass-to-charge ratio range, without sacrificing the impressive capabilities already realized in the lower mass-to-charge ratio range. ESI-MS of biological material is often additionally complicated by the presence of noncovalent adducts, associated mostly with the usage of different biological buffers. The adduction generally becomes worse with the increase in the M r of electrosprayed species. Numerous sample clean up procedures, applicable prior to ESI-MS analysis, have been reported [32–37]. Moreover, “in-trap” removal of adducted impurities from large protein ions, employing infrared multiphoton dissociation (IRMPD) or low energy sustained off-resonance irradiation collisionally induced dissociation (SORICID), has recently been demonstrated [38 – 40]. To aid in our studies of high mass-to-charge ratio noncovalent complexes, we have recently interfaced a custom ESI source with an FTICR spectrometer by employing an rf-quadrupole ion guide for collisional focusing of electrosprayed ions. The rf-quadrupole has significantly extended achievable mass-to-charge ratio range and increased the sensitivity of our 7-tesla FTICR mass spectrometer by reducing observed kinetic energy spread of ions, making it more suitable for studies of large noncovalent complexes. As a result, we succeeded in obtaining FTICR mass spectra for the intact heterotetrameric corynebacterial sarcosine oxidase (theoretical
ESI OF HETEROTETRAMERIC SARCOSINE
511
molecular mass: M r,th 5 180,602 u). A significant improvement in mass measurements accuracy, based upon efficient in-trap clean up by the use of low energy SORI-CID, is also demonstrated. Interestingly, “in-trap” cleanup is shown to be feasible without dissociation of the large noncovalent complex.
Experimental Materials Recombinant corynebacterial sarcosine oxidase was expressed in Escherichia coli and isolated as previously described [41]. Sample aliquots were desalted by the use of an off-line microdialysis procedure, described elsewhere [36, 37], and ;5-mM solutions were electrosprayed from 2.5% acetic acid and 10-mM ammonium acetate.
Finnigan Triple Quadrupole A Finnigan TSQ 7000 triple-quadrupole mass spectrometer (San Jose, CA) with sheathless microscale ESI source was used for the characterization of sarcosine oxidase subunits. The mass spectrometer inlet capillary temperature was 150 °C, and the spectra were obtained by signal averaging for 2 min at a scan rate of 3 s/scan (800 , m/z , 2597).
ESI-FTICR The 7-tesla FTICR mass spectrometer has been described elsewhere [42], as well as the custom ESI source and interface incorporating rf-quadrupole ion transfer region for collisional focusing of electrosprayed ions [43]. Mass spectra were obtained using selected ion accumulation (SIA) [44, 45] base upon either broadband (BB) or single frequency quadrupolar excitation (SFQE). Colored noise QE waveforms [46], generated by use of a PC board (PCIP-AWFG, 5 MHz, 12 bit, Keithley Metrabyte, CA) were sequentially repeated during each QE event, and typically involved 20 and 1 Vpp quadrupolar fields for BBQE and SFQE, respectively. Ion accumulation and CID were accomplished at ;1025 torr of N2, injected into the trap via a piezoelectric pulse valve (Lasertechniques, Albuquerque, NM). SORI, at the frequency ;1 kHz lower than the reduced ICR frequency of the selected ions, has been used for ion activation in CID experiments. An Odyssey data station (Finnigan, Madison, WI) provided ICR trap control, data acquisition, and storage.
Results and Discussion Sarcosine oxidases catalyze the oxidative demethylation of sarcosine (N-methylglycine) to yield glycine, formaldehyde, and hydrogen peroxide. Two major classes of bacterial sarcosine oxidases have been identified: heterotetrameric enzymes (abgd), containing subunits
512
PASA TOLIC ET AL.
J Am Soc Mass Spectrom 1998, 9, 510 –515
Figure 1. ESI mass spectrum of corynebacterial sarcosine oxidase in 2.5% acetic acid solution, obtained using a quadrupole mass spectrometer (Finnigan TSQ 7000), and showing that each of the four subunits (a, b, g, and d) can be detected under denaturing conditions.
ranging in size from ;10,000 to ;100,000 u, and monomeric enzymes that are similar in size to the b subunit from heterotetramers (40,000 – 45,000 u). Sarcosine oxidase from Corynebacterium sp. P-1 is a heterotetramer, having the following masses calculated based upon the gene sequence: M r ( a ) 5 102,633 u, M r ( b ) 5 43,854 u, M r ( g ) 5 20,898 u, and M r ( d ) 5 11,314 u. In addition, the complex also contains two noncovalently associated cofactors: flavin adenine nucleotide (FAD) (M r 5 786 u) and nicotinamide adenine dinucleotide (NAD1) (M r 5 663 u), as well as flavin mononucleotide (FMN) that is covalently bound to the enzyme’s b subunit [i.e., 8-a-(N3-histidyl)FMN, M r 5 454 u] [47– 49]. An ADP binding motif is found near the NH2-terminus of the b subunit and the a subunit; these motifs may contribute to the binding sites for the noncovalently bound FAD and NAD1. Although the stoichiometry of the complex is known, limited information is available concerning the complex quaternary structure of the enzyme, the role of each of the subunits, or the location of the binding sites for the enzyme’s various coenzyme and substrates. Knowledge of the assembly of sarcosine oxidase subunits may provide an insight into the assembly of more complex multisubunit, multiredox systems, e.g., enzymes involved in mitochondrial electron transport. All four subunits of corynebacterial sarcosine oxidase (a, b, g, and d) were observed when electrosprayed under acidic conditions using a conventional quadrupole mass spectrometer (Figure 1). Molecular masses of the individual subunits are in excellent agreement with
those previously published [41], and also consistent with values previously estimated by SDS-gel electrophoresis. The molecular masses obtained for a, g, and d subunits are also in good agreement with values estimated from the gene sequence, whereas the value obtained for b subunit includes a contribution because of covalently bound FMN. Initial studies at pH 8 (data not shown) showed lower mass subunits and smaller complexes (i.e., g1d) in low abundance, and suggested that the heterotetramer may be intact after ESI of solutions at pH 8, but not detectable with our quadrupole instrumentation. We speculated that these initial studies have been unsuccessful mostly because of the high mass-to-charge ratio of the compact heterotetrameric ions. Indeed, multiply charged ions corresponding to the intact corynebacterial sarcosine oxidase complex have been successfully detected (Figure 2) using our 7-tesla mass spectrometer. Although the low resolution results gave a mass measurement about 1000 u higher than calculated, the intact complex is clearly observed. Heterotetramer, with M r,th 5 180,602 u {a1b1g1d1FAD1NAD11[8-a-(N3-histidyl]FMN, where the molecular masses of the subunits are calculated based upon the gene sequence}, is detected in the range 6000 , m/z , 9000, in both positive and negative ion mode. These spectra illustrate the narrow charge state distribution, associated with the retention of the compact structure in the gas phase, typically observed for large noncovalent complexes. In order to obtain isotopic resolution, a second “beat” in the transient must be detected [50]; based on high M r and
J Am Soc Mass Spectrom 1998, 9, 510 –515
Figure 2. Positive and negative ion ESI-FTICR mass spectra of the intact heterotetrameric protein corynebacterial sarcosine oxidase at pH 8 in 10 mM ammonium acetate solution, shows the narrow charge distribution and relatively high mass-to-charge ratio indicative of a compact structure for the intact complex.
mass-to-charge ratio of the complex, this second beat is expected to occur at ;12 s. Therefore, at 7 tesla, a transient of .12 s would be required to obtain such a high resolution mass measurement. However, as described in the Introduction, high M r species display a rapid loss of ion packet coherence that is believed to cause short transients normally observed, presently precluding high resolution and mass measurement accuracy. The mass spectrum shown in Figure 3A, obtained employing BBQE, yielded a mass measurement: Mr,exp 5 181,500 6 600 u (95% confidence limit). Selected-ion accumulation of the 272 charge state using SFQE (Figure 3B) resulted in even worse agreement with a value calculated based upon gene sequence: experimental mass, M r,exp 5 182,000 6 1000 u, versus theoretical mass, M r,th 5 180,602 u, has been obtained (for SFQE spectra, error is reported as the 95% confidence limit of the mean obtained from different measurements acquired under identical conditions). Addition of a SORI clean up event immediately after selected-ion accumulation (Figure 3C), and increasing the amplitude of irradiation to ;40 Vpp (Figure 3D), resulted in a significantly narrower peak and a shift to lower mass-to-charge ratio, i.e., improved mass measurement accuracy (M r,exp 5 180,700 6 300 u versus
ESI OF HETEROTETRAMERIC SARCOSINE
513
Figure 3. Negative ion ESI-FTICR mass spectra of corynebacterial sarcosine oxidase: (A) spectrum obtained employing BBQE (1000 , m/z , 10,000), (B) SFQE at m/z ; 6700, corresponding to [M227H]272 molecular ion of the intact complex, (C) SFQE (m/z ; 6700) followed by mild SORI-CID (1 kHz off-resonance, ;40 Vpp ), and (D) SFQE (m/z ; 6700) followed by harsher SORI-CID (1 kHz off-resonance, ;50 Vpp ). The peak at m/z ; 8650 in C and D is noise introduced by our cryopump system.
M r,th 5 180,602 u), presumably due to the removal of noncovalently adducted impurities. Residual solvation or buffer related species might be a factor contributing to the slightly higher than expected mass obtained. However, we note that residual solvation is seldom observed for smaller proteins by FTICR mass spectrometry, presumably because of the longer time delay between ion injection and detection events compared with conventional instrumentation. Thus, although it is possible that this large complex may retain qualitatively larger amount of solvent [22], experience with smaller proteins suggests that the major source are buffer and other solution impurities. Further increase in SORI amplitude beyond that shown in Figure 3D (e.g., V pp . 50 V), eventually leads to the loss of all signal. Attempts to obtain SORI-CID spectra resulted in extensive to essentially complete “disappearance” of these large ions, but did not give detectable product ions in these initial studies. We speculate that the most likely reasons for this are insufficient energy deposition into the parent ion and/or dissociation to form species not effectively detected. In an ICR trap, CID experiments with large
514
PASA TOLIC ET AL.
molecules have most successfully used SORI, since, as well known, on-resonance excitation often leads to the ejection of off-axis generated daughter ions, due to the magnetron expansion [51]. As shown recently, daughter ions may be effectively axialized during (off- or onresonance excitation) CID experiments [52, 53], but axialization becomes progressively less effective at higher mass-to-charge ratio. In addition, the level of SORI excitation required increases with mass-to-charge ratio, and it is possible that ion losses accompany dissociation for the mass-to-charge ratio of interest. Collisional ion cooling and quadrupolar axialization during the SORI-CID event did not improve fragment ion trapping for the present studies with sarcosine oxidase. Higher power axialization combined with higher average kinetic energy deposited into the parent ion by the use of higher magnetic field and/or large format trap designs are likely to facilitate effective dissociation studies of such large gas-phase complexes, and this potential is currently being explored.
Conclusion Intact heterotetrameric corynebacterial sarcosine oxidase with its associated cofactors has been detected using ESI with a 7-tesla FTICR mass spectrometer. Although these results represent the highest M r (and mass-to-charge ratio) heteromultimeric noncovalent complex yet observed by ESI-FTICR mass spectrometry, they also illustrate limitations upon the performance presently achievable for the measurement of high M r and/or high mass-to-charge ratio species with commonly used FTICR instrumentation (i.e., B # 7 tesla). Regardless of these limitations that preclude isotopic resolution, efficient in-trap SORI clean up has been found to significantly improved mass measurement accuracy. The addition of a SORI clean-up event immediately after selected-ion accumulation yielded M r,exp 5 180,700 6 300 u, in good agreement with the mass calculated (based upon the gene sequence) for the intact complex, M r,th 5 180,602 u. Initial attempts to obtain SORI-CID spectra were not successful (i.e., application of harsher SORI-CID resulted in the loss of molecular ions, but no fragment ions were detected). Although obtaining the isotopic resolution will have to await implementation of advanced FTICR methodology (i.e., higher magnetic field and/or large format trap designs), the mass measurement accuracy obtained in the present study is sufficient for many purposes and potentially can be further improved.
Acknowledgments This work was supported in part by Grants GM 31704 (MSJ) and GM 53558 [Pacific Northwest National Laboratory (PNNL)] from the National Institutes of Health. PNNL is a multiprogram national laboratory operated by Battelle Memorial Institute for the U.S. Department of Energy under Contract DE-AC06-76RLO 1830.
J Am Soc Mass Spectrom 1998, 9, 510 –515
References 1. Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Mass Spectrom. Rev. 1990, 9, 37–70. 2. Comisarow, M. B.; Marshall, A. G. Chem. Phys. Lett. 1974, 25, 282–283. 3. Comisarow, M. B.; Marshall, A. G. Chem. Phys. Lett. 1974, 6, 489 – 490. 4. Marshall, A. G.; Verdun, F. R. Fourier Transforms in NMR, Optical, and Mass Spectrometry; Elsevier: New York, 1990. 5. Marshall, A. G.; Grosshans, P. B. Anal. Chem. 1991, 63, A215– A229. 6. Holliman, C. L.; Rempel, D. L.; Gross, M. L. Mass Spectrom. Rev. 1994, 13, 105–132. 7. Mitchell, D. W.; Smith, R. D. J. Mass Spectrom. 1996, 31, 771–790. 8. Bruce, J. E.; Anderson, G. A.; Udseth, H. R.; Smith, R. D. Anal. Chem., in press. 9. Pasa Tolic, L.; Anderson, G. A.; Smith, R. D. Int. J. Mass Spectrom. Ion Processes 1997, 156/157, 405– 418. 10. Smith, R. D.; Cheng, X.; Bruce, J. E.; Hofstadler, S. A.; Anderson, G. A. Nature 1994, 369, 137–139. 11. Chen, R. D.; Wu, Q.; Mitchell, D. W.; Hofstadler, S. A.; Rockwood, A. L.; Smith, R. D. Anal. Chem. 1994, 66, 3964 –3969. 12. Chen, R. D.; Cheng, X. H.; Mitchell, D. W.; Hofstadler, S. A.; Wu, Q.; Rockwood, A. L.; Sherman, M. G.; Smith, R. D. Anal. Chem. 1995, 67, 1159 –1163. 13. Marshall, A. G.; Guan, S. H. Rapid Commun. Mass Spectrom. 1996, 10, 1819 –1823. 14. Kelleher, N. L.; Senko, M. W.; Siegel, M. M.; McLafferty, F. W. J. Am. Soc. Mass Spectrom. 1997, 8, 380 –383. 15. Senko, M. W., Hendrickson, C. L.; Emmett, M. R.; Shi, S. D. H.; Marshall, A. G. J. Am. Soc. Mass Spectrom. 1997, 8, 970 –976. 16. Loo, J. A.; Loo, R. R. O.; Udseth, H. R.; Edmonds, C. G.; Smith, R. D. Rapid Commun. Mass Spectrom. 1991, 5, 101–105. 17. Smith, R. D.; Light-Wahl, K. J.; Winger, B. E.; Loo, J. A. Org. Mass Spectrom. 1992, 27, 811– 821. 18. Smith, R. D.; Light-Wahl, K. J. Biol. Mass Spectrom. 1993, 22, 493–501. 19. Winger, B. E.; Light-Wahl, K. J.; Loo, R. R. O.; Udseth, H. R.; Smith, R. D. J. Am. Soc. Mass Spectrom. 1993, 4, 536 –545. 20. Li, Y. T.; Hsieh, Y. L.; Henion, J. D.; Ganem, B. J. Am. Soc. Mass Spectrom. 1993, 4, 631– 637. 21. Light-Wahl, K. J.; Schwartz, B. L.; Smith, R. D. J. Am. Chem. Soc. 1994, 116, 5271–5278. 22. Loo, J. A. J. Mass Spectrom. 1995, 30, 180 –183. 23. Schwartz, B. L.; Gale, D. C.; Smith, R. D.; Chilkoti, A.; Stayton, P. S. J. Mass Spectrom. 1995, 30, 1095–1102. 24. Schwartz, B. L.; Bruce, J. E.; Anderson, G. A.; Hofstadler, S. A.; Rockwood, A. L.; Smith, R. D.; Chilkoti, A.; Stayton, P. S. J. Am. Soc. Mass Spectrom. 1995, 6, 459 – 465. 25. Smith, V. F.; Schwartz, B. L.; Randall, L. L.; Smith, R. D. Protein Sci. 1996, 5, 488 – 494. 26. Fitzgerald, M. C.; Chernushevich, I. V.; Standing, K. G.; Kent, S. B. H.; Whitman, C. P. J. Am. Chem. Soc. 1995, 117, 11075–11080. 27. Fitzgerald, M. C.; Chernushevich, I. V.; Standing, K. G.; Whitman, C. P.; Kent, S. B. H. Proc. Natl. Acad. Sci. USA 1996, 93, 6851– 6856. 28. Tang, X.-J.; Brewer, C. F.; Saha, S.; Chernushevich, I. V.; Ens, W.; Standing, K. G. Rapid Commun. Mass Spectrom. 1994, 8, 750 –754. 29. Chowdhury, S. K.; Katta, V.; Chait, B. T. Electrospray Ionization Mass Spectrometric Analysis of Proteins in Methods and Mechanisms for Producing Ions from Large Molecules Chowdhury; Standing, K. G.; Ens, W., Eds.; Plenum: New York, 1991; pp 201–210. 30. Siuzdak, G.; Bothner, B.; Yeager, M.; Brugkdou, C.; Faquet, C. M.; Hoey, K.; Chang, C. M. Chem. Biol. 1996, 3, 45– 48.
J Am Soc Mass Spectrom 1998, 9, 510 –515
31. Bruce, J. E.; Smith, V. F.; Liu, C.; Smith, R. D.; Randall, L. L., unpublished. 32. Stults, J. T.; Marsters, J. C. Rapid Commun. Mass Spectrom. 1991, 5, 359 –363. 33. Little, D. P.; Chorush, R. A.; Speir, J. P.; Senko, M. W.; Kelleher, N. L.; McLafferty, F. W. J. Am. Chem. Soc. 1994, 116, 4893– 4897. 34. Greig, M.; Griffey, R. H. Rapid Commun. Mass Spectrom. 1995, 9, 97–102. 35. Muddiman, D. C.; Cheng, X. H.; Udseth, H. R.; Smith, R. D. J. Am. Soc. Mass Spectrom. 1996, 7, 697–706. 36. Wu, Q.; Liu, C. L.; Smith, R. D. Rapid Commun. Mass Spectrom. 1996, 10, 835– 838. 37. Liu, C.; Wu, Q.; Harms, A. C.; Smith, R. D. Anal. Chem. 1996, 68, 3295–3299. 38. Little, D. P.; McLafferty, F. W. J. Am. Soc. Mass Spectrom. 1996, 7, 209 –210. 39. Speir, J. P.; Senko, M. W.; Little, D. P.; Loo, J. A.; McLafferty, F. W. J. Mass Spectrom. 1995, 30, 39 – 42. 40. Pasa Tolic, L.; Bruce, J. E.; Paula Lei, Q.; Anderson, G. A.; Smith, R. D. Anal. Chem., in press. 41. Chlumsky, L. J.; Zhang, L.; Ramsey, A. J.; Jorns, M. S. Biochem. 1993, 32, 11132–11142. 42. Winger, B. E.; Hofstadler, S. A.; Bruce, J. E.; Udseth, H. R.; Smith, R. D. J. Am. Soc. Mass Spectrom. 1993, 4, 566 –577.
ESI OF HETEROTETRAMERIC SARCOSINE
515
43. Pasa Tolic, L.; Anderson, G. A.; Smith, R. D. unpublished results. 44. Bruce, J. E.; Van Orden, S. L.; Anderson, G. A.; Hofstadler, S. A.; Sherman, M. G.; Rockwood, A. L.; Smith, R. D. J. Mass Spectrom. 1995, 30, 124 –133. 45. Bruce, J. E.; Anderson, G. A.; Hofstadler, S. A.; Van Orden, S. L.; Sherman, M. S.; Rockwood, A. L.; Smith, R. D. Rapid Commun. Mass Spectrom. 1993, 7, 914 –919. 46. Bruce, J. E.; Anderson, G. A.; Smith, R. D. Anal. Chem. 1996, 68, 534 –541. 47. Kvalnes-Krick, K.; Jorns, M. S. Biochemistry 1986, 25, 6061– 6069. 48. Willie, A.; Jorns, M. S. Biochemistry 1995, 34, 16703–16707. 49. Willie, A.; Edmondson, D. E.; Jorns, M. S. Biochemistry 1996, 35, 5292–5299. 50. Hofstadler, S. A.; Bruce, J. E.; Rockwood, A. L.; Anderson, G. A.; Winger, B. E.; Smith, R. D. Int. J. Mass Spectrom. Ion Processes 1994, 132, 109 –127. 51. Guan, S. H.; Kim, H. S.; Marshall, A. G.; Wahl, M. C.; Wood, T. D.; Xiang, X. Z. Chem. Rev. 1994, 94, 2161–2182. 52. Huang, Y. L.; Pasa Tolic, L.; Guan, S. H.; Marshall, A. G. Anal. Chem. 1994, 66, 4385– 4389. 53. Solouki, T.; Pasa Tolic, L.; Jackson, G. S.; Guan, S. G.; Marshall, A. G. Anal. Chem. 1996, 68, 3718 –3725.
Annie Willie and Marilyn Schuman Jorns Department of Biochemistry, Allegheny University of the Health Sciences, Philadelphia, Pennsylvania, USA
Electrospray ionization (ESI) Fourier transform ion cyclotron resonance (FTICR) mass spectrometry has been used to characterize heterotetrameric corynebacterial sarcosine oxidase. By using a conventional quadrupole mass spectrometer, no spectra for the intact complex could be obtained (i.e., electrospraying protein at neutral pH), but spectra showing the four protein subunits were obtained when electrospraying from acidic solution. Initial low resolution ESI-FTICR mass spectra of the intact heterotetramer revealed a typical narrow charge state distribution in the range 6000 , m/z , 9000, consistent with retention of a compact structure in the gas phase, and gave a mass measurement about 1000 u higher than predicted. Efficient in-trap clean up, based upon low energy collisionally induced dissociation of adducts, allowed significant improvement in mass measurement accuracy. The present results represent the largest heteromultimeric protein complex successfully analyzed using FTICR mass spectrometry, and clearly illustrate the importance of sample clean up methods for large molecule characterization. (J Am Soc Mass Spectrom 1998, 9, 510 –515) © 1998 American Society for Mass Spectrometry
E
lectrospray ionization mass spectrometry (ESIMS) [1] is now broadly utilized for biomolecule characterization. Most applications, however, involve biopolymers with molecular masses M r , 50,000 u. Although Fourier transform ion cyclotron resonance (FTICR) mass spectrometry [2–5] holds advantages for high M r analyses in terms of its high resolution and mass measurement precision, studies of high mass-tocharge ratio species produced by ESI [or matrix-assisted laser desorption ionization (MALDI)] have generally been problematic, and few results have been reported [6]. The major difficulties have included the reduced trapping efficiency of these larger ions, detection problems arising from imperfect electric fields due to the nonideal FTICR trap and, to some extent, the weaker confinement provided by the magnetic field (B) at higher mass-to-charge ratio. These factors combine to result in an upper mass-to-charge ratio limit that is often significantly lower than the predicted “critical mass-to-charge ratio,” i.e., highest mass-to-charge ratio that can be effectively trapped for given experimental Address correspondence to Richard D. Smith, Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, P.O. Box 999, Mail Stop K8-98, Richland, WA 99352. E-mail: [email protected]
conditions [5]. A major problem is that the number of species with different mass-to-charge ratio increases rapidly with molecular mass. The large number of trapped charges that is required to define the charge state distribution and enable M r determination leads to Coulomb-induced frequency shifts, peak coalescence, and rapid loss of ion packet coherence. Such ion cloud instability problems preclude high resolution and mass measurement accuracy for high molecular mass species (i.e., M r . 80,000 for a 7-tesla magnetic field strength) [7, 8]. Recently, we have obtained ESI-FTICR mass spectra of Starburst™ polyamidoamine dendrimers with masses up to ;1,000,000 u [9], representing the highest molecular mass species yet detected by FTICR mass spectrometry, with the exception of results based upon individual ion detection [10 –12]. ESI-FTICR mass spectra obtained with dendrimers, together with the spectra acquired with dendrimer/IgG immunoconjugates, highlight the current limitations of FTICR technology. Because most aspects of FTICR performance are enhanced with increasing B [13], it is reasonable to expect significant improvements in applicability of FTICR mass spectrometry to biological problems as higher magnetic field instruments become available. An illus-
© 1998 American Society for Mass Spectrometry. Published by Elsevier Science Inc. 1044-0305/98/$19.00 PII S1044-0305(98)00011-7
Received August 18, 1997 Revised December 12, 1997 Accepted December 20, 1997
J Am Soc Mass Spectrom 1998, 9, 510 –515
trative example is the unit resolution of 112,000 u molecules, with 3 u accuracy, recently achieved with a 9.4-tesla FTICR mass spectrometer [14], employing collisional focusing of electrosprayed ions in an octopole [15]. It should be noted that new approaches based on individual ion detection aim to circumvent the trapped ion density limitations arising at higher molecular masses, but present a different set of challenges [8, 10 –12]. The limited mass-to-charge ratio range is particularly problematic for studies of noncovalent complexes under physiologically relevant conditions where lower charge states are observed because of the typically more compact structures. There is now a substantial body of evidence that demonstrates that noncovalent biologically relevant complexes can be observed by ESI-MS [16 –29]. A recent demonstration that viruses can “survive” (e.g., preserve ability to infect) ESI-MS offers strong evidence that at least some native structure of biomolecules can be conserved through the electrospray process and mass analysis [30]. The utility of ESI-MS in studies of self-assembling processes of noncovalent complexes, i.e., subunit stoichiometry, which is essential for a better understanding of different cellular functions at the molecular level, is increasingly evident [31]. Although ESI-TOF instrumentation has proven its utility in characterization of noncovalent complexes, the ESI-FTICR combination, with its high resolution and multistage MSn capabilities, also holds considerable promise for such applications. However, further improvement is needed to significantly extend the routinely achievable mass and mass-to-charge ratio range, without sacrificing the impressive capabilities already realized in the lower mass-to-charge ratio range. ESI-MS of biological material is often additionally complicated by the presence of noncovalent adducts, associated mostly with the usage of different biological buffers. The adduction generally becomes worse with the increase in the M r of electrosprayed species. Numerous sample clean up procedures, applicable prior to ESI-MS analysis, have been reported [32–37]. Moreover, “in-trap” removal of adducted impurities from large protein ions, employing infrared multiphoton dissociation (IRMPD) or low energy sustained off-resonance irradiation collisionally induced dissociation (SORICID), has recently been demonstrated [38 – 40]. To aid in our studies of high mass-to-charge ratio noncovalent complexes, we have recently interfaced a custom ESI source with an FTICR spectrometer by employing an rf-quadrupole ion guide for collisional focusing of electrosprayed ions. The rf-quadrupole has significantly extended achievable mass-to-charge ratio range and increased the sensitivity of our 7-tesla FTICR mass spectrometer by reducing observed kinetic energy spread of ions, making it more suitable for studies of large noncovalent complexes. As a result, we succeeded in obtaining FTICR mass spectra for the intact heterotetrameric corynebacterial sarcosine oxidase (theoretical
ESI OF HETEROTETRAMERIC SARCOSINE
511
molecular mass: M r,th 5 180,602 u). A significant improvement in mass measurements accuracy, based upon efficient in-trap clean up by the use of low energy SORI-CID, is also demonstrated. Interestingly, “in-trap” cleanup is shown to be feasible without dissociation of the large noncovalent complex.
Experimental Materials Recombinant corynebacterial sarcosine oxidase was expressed in Escherichia coli and isolated as previously described [41]. Sample aliquots were desalted by the use of an off-line microdialysis procedure, described elsewhere [36, 37], and ;5-mM solutions were electrosprayed from 2.5% acetic acid and 10-mM ammonium acetate.
Finnigan Triple Quadrupole A Finnigan TSQ 7000 triple-quadrupole mass spectrometer (San Jose, CA) with sheathless microscale ESI source was used for the characterization of sarcosine oxidase subunits. The mass spectrometer inlet capillary temperature was 150 °C, and the spectra were obtained by signal averaging for 2 min at a scan rate of 3 s/scan (800 , m/z , 2597).
ESI-FTICR The 7-tesla FTICR mass spectrometer has been described elsewhere [42], as well as the custom ESI source and interface incorporating rf-quadrupole ion transfer region for collisional focusing of electrosprayed ions [43]. Mass spectra were obtained using selected ion accumulation (SIA) [44, 45] base upon either broadband (BB) or single frequency quadrupolar excitation (SFQE). Colored noise QE waveforms [46], generated by use of a PC board (PCIP-AWFG, 5 MHz, 12 bit, Keithley Metrabyte, CA) were sequentially repeated during each QE event, and typically involved 20 and 1 Vpp quadrupolar fields for BBQE and SFQE, respectively. Ion accumulation and CID were accomplished at ;1025 torr of N2, injected into the trap via a piezoelectric pulse valve (Lasertechniques, Albuquerque, NM). SORI, at the frequency ;1 kHz lower than the reduced ICR frequency of the selected ions, has been used for ion activation in CID experiments. An Odyssey data station (Finnigan, Madison, WI) provided ICR trap control, data acquisition, and storage.
Results and Discussion Sarcosine oxidases catalyze the oxidative demethylation of sarcosine (N-methylglycine) to yield glycine, formaldehyde, and hydrogen peroxide. Two major classes of bacterial sarcosine oxidases have been identified: heterotetrameric enzymes (abgd), containing subunits
512
PASA TOLIC ET AL.
J Am Soc Mass Spectrom 1998, 9, 510 –515
Figure 1. ESI mass spectrum of corynebacterial sarcosine oxidase in 2.5% acetic acid solution, obtained using a quadrupole mass spectrometer (Finnigan TSQ 7000), and showing that each of the four subunits (a, b, g, and d) can be detected under denaturing conditions.
ranging in size from ;10,000 to ;100,000 u, and monomeric enzymes that are similar in size to the b subunit from heterotetramers (40,000 – 45,000 u). Sarcosine oxidase from Corynebacterium sp. P-1 is a heterotetramer, having the following masses calculated based upon the gene sequence: M r ( a ) 5 102,633 u, M r ( b ) 5 43,854 u, M r ( g ) 5 20,898 u, and M r ( d ) 5 11,314 u. In addition, the complex also contains two noncovalently associated cofactors: flavin adenine nucleotide (FAD) (M r 5 786 u) and nicotinamide adenine dinucleotide (NAD1) (M r 5 663 u), as well as flavin mononucleotide (FMN) that is covalently bound to the enzyme’s b subunit [i.e., 8-a-(N3-histidyl)FMN, M r 5 454 u] [47– 49]. An ADP binding motif is found near the NH2-terminus of the b subunit and the a subunit; these motifs may contribute to the binding sites for the noncovalently bound FAD and NAD1. Although the stoichiometry of the complex is known, limited information is available concerning the complex quaternary structure of the enzyme, the role of each of the subunits, or the location of the binding sites for the enzyme’s various coenzyme and substrates. Knowledge of the assembly of sarcosine oxidase subunits may provide an insight into the assembly of more complex multisubunit, multiredox systems, e.g., enzymes involved in mitochondrial electron transport. All four subunits of corynebacterial sarcosine oxidase (a, b, g, and d) were observed when electrosprayed under acidic conditions using a conventional quadrupole mass spectrometer (Figure 1). Molecular masses of the individual subunits are in excellent agreement with
those previously published [41], and also consistent with values previously estimated by SDS-gel electrophoresis. The molecular masses obtained for a, g, and d subunits are also in good agreement with values estimated from the gene sequence, whereas the value obtained for b subunit includes a contribution because of covalently bound FMN. Initial studies at pH 8 (data not shown) showed lower mass subunits and smaller complexes (i.e., g1d) in low abundance, and suggested that the heterotetramer may be intact after ESI of solutions at pH 8, but not detectable with our quadrupole instrumentation. We speculated that these initial studies have been unsuccessful mostly because of the high mass-to-charge ratio of the compact heterotetrameric ions. Indeed, multiply charged ions corresponding to the intact corynebacterial sarcosine oxidase complex have been successfully detected (Figure 2) using our 7-tesla mass spectrometer. Although the low resolution results gave a mass measurement about 1000 u higher than calculated, the intact complex is clearly observed. Heterotetramer, with M r,th 5 180,602 u {a1b1g1d1FAD1NAD11[8-a-(N3-histidyl]FMN, where the molecular masses of the subunits are calculated based upon the gene sequence}, is detected in the range 6000 , m/z , 9000, in both positive and negative ion mode. These spectra illustrate the narrow charge state distribution, associated with the retention of the compact structure in the gas phase, typically observed for large noncovalent complexes. In order to obtain isotopic resolution, a second “beat” in the transient must be detected [50]; based on high M r and
J Am Soc Mass Spectrom 1998, 9, 510 –515
Figure 2. Positive and negative ion ESI-FTICR mass spectra of the intact heterotetrameric protein corynebacterial sarcosine oxidase at pH 8 in 10 mM ammonium acetate solution, shows the narrow charge distribution and relatively high mass-to-charge ratio indicative of a compact structure for the intact complex.
mass-to-charge ratio of the complex, this second beat is expected to occur at ;12 s. Therefore, at 7 tesla, a transient of .12 s would be required to obtain such a high resolution mass measurement. However, as described in the Introduction, high M r species display a rapid loss of ion packet coherence that is believed to cause short transients normally observed, presently precluding high resolution and mass measurement accuracy. The mass spectrum shown in Figure 3A, obtained employing BBQE, yielded a mass measurement: Mr,exp 5 181,500 6 600 u (95% confidence limit). Selected-ion accumulation of the 272 charge state using SFQE (Figure 3B) resulted in even worse agreement with a value calculated based upon gene sequence: experimental mass, M r,exp 5 182,000 6 1000 u, versus theoretical mass, M r,th 5 180,602 u, has been obtained (for SFQE spectra, error is reported as the 95% confidence limit of the mean obtained from different measurements acquired under identical conditions). Addition of a SORI clean up event immediately after selected-ion accumulation (Figure 3C), and increasing the amplitude of irradiation to ;40 Vpp (Figure 3D), resulted in a significantly narrower peak and a shift to lower mass-to-charge ratio, i.e., improved mass measurement accuracy (M r,exp 5 180,700 6 300 u versus
ESI OF HETEROTETRAMERIC SARCOSINE
513
Figure 3. Negative ion ESI-FTICR mass spectra of corynebacterial sarcosine oxidase: (A) spectrum obtained employing BBQE (1000 , m/z , 10,000), (B) SFQE at m/z ; 6700, corresponding to [M227H]272 molecular ion of the intact complex, (C) SFQE (m/z ; 6700) followed by mild SORI-CID (1 kHz off-resonance, ;40 Vpp ), and (D) SFQE (m/z ; 6700) followed by harsher SORI-CID (1 kHz off-resonance, ;50 Vpp ). The peak at m/z ; 8650 in C and D is noise introduced by our cryopump system.
M r,th 5 180,602 u), presumably due to the removal of noncovalently adducted impurities. Residual solvation or buffer related species might be a factor contributing to the slightly higher than expected mass obtained. However, we note that residual solvation is seldom observed for smaller proteins by FTICR mass spectrometry, presumably because of the longer time delay between ion injection and detection events compared with conventional instrumentation. Thus, although it is possible that this large complex may retain qualitatively larger amount of solvent [22], experience with smaller proteins suggests that the major source are buffer and other solution impurities. Further increase in SORI amplitude beyond that shown in Figure 3D (e.g., V pp . 50 V), eventually leads to the loss of all signal. Attempts to obtain SORI-CID spectra resulted in extensive to essentially complete “disappearance” of these large ions, but did not give detectable product ions in these initial studies. We speculate that the most likely reasons for this are insufficient energy deposition into the parent ion and/or dissociation to form species not effectively detected. In an ICR trap, CID experiments with large
514
PASA TOLIC ET AL.
molecules have most successfully used SORI, since, as well known, on-resonance excitation often leads to the ejection of off-axis generated daughter ions, due to the magnetron expansion [51]. As shown recently, daughter ions may be effectively axialized during (off- or onresonance excitation) CID experiments [52, 53], but axialization becomes progressively less effective at higher mass-to-charge ratio. In addition, the level of SORI excitation required increases with mass-to-charge ratio, and it is possible that ion losses accompany dissociation for the mass-to-charge ratio of interest. Collisional ion cooling and quadrupolar axialization during the SORI-CID event did not improve fragment ion trapping for the present studies with sarcosine oxidase. Higher power axialization combined with higher average kinetic energy deposited into the parent ion by the use of higher magnetic field and/or large format trap designs are likely to facilitate effective dissociation studies of such large gas-phase complexes, and this potential is currently being explored.
Conclusion Intact heterotetrameric corynebacterial sarcosine oxidase with its associated cofactors has been detected using ESI with a 7-tesla FTICR mass spectrometer. Although these results represent the highest M r (and mass-to-charge ratio) heteromultimeric noncovalent complex yet observed by ESI-FTICR mass spectrometry, they also illustrate limitations upon the performance presently achievable for the measurement of high M r and/or high mass-to-charge ratio species with commonly used FTICR instrumentation (i.e., B # 7 tesla). Regardless of these limitations that preclude isotopic resolution, efficient in-trap SORI clean up has been found to significantly improved mass measurement accuracy. The addition of a SORI clean-up event immediately after selected-ion accumulation yielded M r,exp 5 180,700 6 300 u, in good agreement with the mass calculated (based upon the gene sequence) for the intact complex, M r,th 5 180,602 u. Initial attempts to obtain SORI-CID spectra were not successful (i.e., application of harsher SORI-CID resulted in the loss of molecular ions, but no fragment ions were detected). Although obtaining the isotopic resolution will have to await implementation of advanced FTICR methodology (i.e., higher magnetic field and/or large format trap designs), the mass measurement accuracy obtained in the present study is sufficient for many purposes and potentially can be further improved.
Acknowledgments This work was supported in part by Grants GM 31704 (MSJ) and GM 53558 [Pacific Northwest National Laboratory (PNNL)] from the National Institutes of Health. PNNL is a multiprogram national laboratory operated by Battelle Memorial Institute for the U.S. Department of Energy under Contract DE-AC06-76RLO 1830.
J Am Soc Mass Spectrom 1998, 9, 510 –515
References 1. Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Mass Spectrom. Rev. 1990, 9, 37–70. 2. Comisarow, M. B.; Marshall, A. G. Chem. Phys. Lett. 1974, 25, 282–283. 3. Comisarow, M. B.; Marshall, A. G. Chem. Phys. Lett. 1974, 6, 489 – 490. 4. Marshall, A. G.; Verdun, F. R. Fourier Transforms in NMR, Optical, and Mass Spectrometry; Elsevier: New York, 1990. 5. Marshall, A. G.; Grosshans, P. B. Anal. Chem. 1991, 63, A215– A229. 6. Holliman, C. L.; Rempel, D. L.; Gross, M. L. Mass Spectrom. Rev. 1994, 13, 105–132. 7. Mitchell, D. W.; Smith, R. D. J. Mass Spectrom. 1996, 31, 771–790. 8. Bruce, J. E.; Anderson, G. A.; Udseth, H. R.; Smith, R. D. Anal. Chem., in press. 9. Pasa Tolic, L.; Anderson, G. A.; Smith, R. D. Int. J. Mass Spectrom. Ion Processes 1997, 156/157, 405– 418. 10. Smith, R. D.; Cheng, X.; Bruce, J. E.; Hofstadler, S. A.; Anderson, G. A. Nature 1994, 369, 137–139. 11. Chen, R. D.; Wu, Q.; Mitchell, D. W.; Hofstadler, S. A.; Rockwood, A. L.; Smith, R. D. Anal. Chem. 1994, 66, 3964 –3969. 12. Chen, R. D.; Cheng, X. H.; Mitchell, D. W.; Hofstadler, S. A.; Wu, Q.; Rockwood, A. L.; Sherman, M. G.; Smith, R. D. Anal. Chem. 1995, 67, 1159 –1163. 13. Marshall, A. G.; Guan, S. H. Rapid Commun. Mass Spectrom. 1996, 10, 1819 –1823. 14. Kelleher, N. L.; Senko, M. W.; Siegel, M. M.; McLafferty, F. W. J. Am. Soc. Mass Spectrom. 1997, 8, 380 –383. 15. Senko, M. W., Hendrickson, C. L.; Emmett, M. R.; Shi, S. D. H.; Marshall, A. G. J. Am. Soc. Mass Spectrom. 1997, 8, 970 –976. 16. Loo, J. A.; Loo, R. R. O.; Udseth, H. R.; Edmonds, C. G.; Smith, R. D. Rapid Commun. Mass Spectrom. 1991, 5, 101–105. 17. Smith, R. D.; Light-Wahl, K. J.; Winger, B. E.; Loo, J. A. Org. Mass Spectrom. 1992, 27, 811– 821. 18. Smith, R. D.; Light-Wahl, K. J. Biol. Mass Spectrom. 1993, 22, 493–501. 19. Winger, B. E.; Light-Wahl, K. J.; Loo, R. R. O.; Udseth, H. R.; Smith, R. D. J. Am. Soc. Mass Spectrom. 1993, 4, 536 –545. 20. Li, Y. T.; Hsieh, Y. L.; Henion, J. D.; Ganem, B. J. Am. Soc. Mass Spectrom. 1993, 4, 631– 637. 21. Light-Wahl, K. J.; Schwartz, B. L.; Smith, R. D. J. Am. Chem. Soc. 1994, 116, 5271–5278. 22. Loo, J. A. J. Mass Spectrom. 1995, 30, 180 –183. 23. Schwartz, B. L.; Gale, D. C.; Smith, R. D.; Chilkoti, A.; Stayton, P. S. J. Mass Spectrom. 1995, 30, 1095–1102. 24. Schwartz, B. L.; Bruce, J. E.; Anderson, G. A.; Hofstadler, S. A.; Rockwood, A. L.; Smith, R. D.; Chilkoti, A.; Stayton, P. S. J. Am. Soc. Mass Spectrom. 1995, 6, 459 – 465. 25. Smith, V. F.; Schwartz, B. L.; Randall, L. L.; Smith, R. D. Protein Sci. 1996, 5, 488 – 494. 26. Fitzgerald, M. C.; Chernushevich, I. V.; Standing, K. G.; Kent, S. B. H.; Whitman, C. P. J. Am. Chem. Soc. 1995, 117, 11075–11080. 27. Fitzgerald, M. C.; Chernushevich, I. V.; Standing, K. G.; Whitman, C. P.; Kent, S. B. H. Proc. Natl. Acad. Sci. USA 1996, 93, 6851– 6856. 28. Tang, X.-J.; Brewer, C. F.; Saha, S.; Chernushevich, I. V.; Ens, W.; Standing, K. G. Rapid Commun. Mass Spectrom. 1994, 8, 750 –754. 29. Chowdhury, S. K.; Katta, V.; Chait, B. T. Electrospray Ionization Mass Spectrometric Analysis of Proteins in Methods and Mechanisms for Producing Ions from Large Molecules Chowdhury; Standing, K. G.; Ens, W., Eds.; Plenum: New York, 1991; pp 201–210. 30. Siuzdak, G.; Bothner, B.; Yeager, M.; Brugkdou, C.; Faquet, C. M.; Hoey, K.; Chang, C. M. Chem. Biol. 1996, 3, 45– 48.
J Am Soc Mass Spectrom 1998, 9, 510 –515
31. Bruce, J. E.; Smith, V. F.; Liu, C.; Smith, R. D.; Randall, L. L., unpublished. 32. Stults, J. T.; Marsters, J. C. Rapid Commun. Mass Spectrom. 1991, 5, 359 –363. 33. Little, D. P.; Chorush, R. A.; Speir, J. P.; Senko, M. W.; Kelleher, N. L.; McLafferty, F. W. J. Am. Chem. Soc. 1994, 116, 4893– 4897. 34. Greig, M.; Griffey, R. H. Rapid Commun. Mass Spectrom. 1995, 9, 97–102. 35. Muddiman, D. C.; Cheng, X. H.; Udseth, H. R.; Smith, R. D. J. Am. Soc. Mass Spectrom. 1996, 7, 697–706. 36. Wu, Q.; Liu, C. L.; Smith, R. D. Rapid Commun. Mass Spectrom. 1996, 10, 835– 838. 37. Liu, C.; Wu, Q.; Harms, A. C.; Smith, R. D. Anal. Chem. 1996, 68, 3295–3299. 38. Little, D. P.; McLafferty, F. W. J. Am. Soc. Mass Spectrom. 1996, 7, 209 –210. 39. Speir, J. P.; Senko, M. W.; Little, D. P.; Loo, J. A.; McLafferty, F. W. J. Mass Spectrom. 1995, 30, 39 – 42. 40. Pasa Tolic, L.; Bruce, J. E.; Paula Lei, Q.; Anderson, G. A.; Smith, R. D. Anal. Chem., in press. 41. Chlumsky, L. J.; Zhang, L.; Ramsey, A. J.; Jorns, M. S. Biochem. 1993, 32, 11132–11142. 42. Winger, B. E.; Hofstadler, S. A.; Bruce, J. E.; Udseth, H. R.; Smith, R. D. J. Am. Soc. Mass Spectrom. 1993, 4, 566 –577.
ESI OF HETEROTETRAMERIC SARCOSINE
515
43. Pasa Tolic, L.; Anderson, G. A.; Smith, R. D. unpublished results. 44. Bruce, J. E.; Van Orden, S. L.; Anderson, G. A.; Hofstadler, S. A.; Sherman, M. G.; Rockwood, A. L.; Smith, R. D. J. Mass Spectrom. 1995, 30, 124 –133. 45. Bruce, J. E.; Anderson, G. A.; Hofstadler, S. A.; Van Orden, S. L.; Sherman, M. S.; Rockwood, A. L.; Smith, R. D. Rapid Commun. Mass Spectrom. 1993, 7, 914 –919. 46. Bruce, J. E.; Anderson, G. A.; Smith, R. D. Anal. Chem. 1996, 68, 534 –541. 47. Kvalnes-Krick, K.; Jorns, M. S. Biochemistry 1986, 25, 6061– 6069. 48. Willie, A.; Jorns, M. S. Biochemistry 1995, 34, 16703–16707. 49. Willie, A.; Edmondson, D. E.; Jorns, M. S. Biochemistry 1996, 35, 5292–5299. 50. Hofstadler, S. A.; Bruce, J. E.; Rockwood, A. L.; Anderson, G. A.; Winger, B. E.; Smith, R. D. Int. J. Mass Spectrom. Ion Processes 1994, 132, 109 –127. 51. Guan, S. H.; Kim, H. S.; Marshall, A. G.; Wahl, M. C.; Wood, T. D.; Xiang, X. Z. Chem. Rev. 1994, 94, 2161–2182. 52. Huang, Y. L.; Pasa Tolic, L.; Guan, S. H.; Marshall, A. G. Anal. Chem. 1994, 66, 4385– 4389. 53. Solouki, T.; Pasa Tolic, L.; Jackson, G. S.; Guan, S. G.; Marshall, A. G. Anal. Chem. 1996, 68, 3718 –3725.