Tandem mass spectrometry of very large molecules. 2. Dissociation of

Anal. Chem. 1003, 65, 425-438

425

Tandem Mass Spectrometry of Very Large Molecules. 2. Dissociation of Multiply Charged Proline-Containing Proteins from Electrospray Ionization Joseph A. Loo,? Charles G. Edmonds, and Richard D. Smith'

Chemical Sciences Department, Pacific Northwest Laboratory, Richland, Washington 99352

Collklonal dlrroclatlon tandem ma88 spectra have been obtalned for multlply charged molecules produced by electrospray lonlzatlonfor a varlety of prollne-contalnlngprotelns extendlng up to 22 000 molecular welght. Interpretatlon of llmlted m/z range, low-reeolutlontandem ma88 spectra from multlply charged precursors can present dMlcultlesdue to the posdblllty of multlply charged product Ion8 and the lack of unembtgmusc h a m a t e Informath. Methodsusedto guide the rpectralInterpretatlonpr-88 under these circumstances are discussed. Proline k a unlque amino acld con8tltuent b e c a w 118 side chain k bonded to the tertlary nHrogen In a cydlc pyrrolkwne rlng. For largepolypeptlckscontainlngprdlne reddues, we haveohservedthat fragmentatlond w to cleavage of the amlde bond to proline I8 often dominant. Such proilna dkected proce88e8 are often the only dlsrociatlon pathway8 obeerved for large protelns. Thk Is attrlbuted to the quaslthermal nature of large molecule coilldonal actlvatlon/ dlrsoclatlon processes and the lower dl88oclatlon energles for peptlde bonds near prollne reddues. The present rewnr a h 8uggest podble effect8 on the dlscloclatlon processes for large molecuksdue to charge locationand perhapsprotein conformation.

INTRODUCTION

A significant reseaich effort has been expended since the 1950'sto develop mass spectrometry (MS) as a useful tool for application in the biological sciences. As new ionization methods have been developed to transfer larger ihd larger compounds into the gas phase with minimum sample degradation, a parallel effort has proceeded in developing mass analyzers and detectors to accommodate compounds of increasing molecular mass. From amino acids, to peptides, and finally to proteins, ionization methods such as field desorption (FD),' plasma desorption (PD),2 and fast atom bombardment (FAB)3 have enabled mass spectrometers to detect and determine molecular weights for biomolecules extending up to -40 kDa. The sensitive detection and accurate relative molecular mass (M,) determination of proteins as single components, or more typically as mixtures of closely related (microheterogeneous)proteins differing by as few as one amino acid residue, is an analytical challenge that has stimulated the development of new approaches. For example,the determination of trace impurities in recombinant products is important in establishing regulatory certification ~

~~

~

~

~

Current address: Parke-Davis Pharmaceutical Research Division, 2800 Plymouth Rd., Ann Arbor, MI 48105. (1) Beckey, H.-D. Principles of Field Ionization and Field Desorption Mass Spectrometry; Pergamon: Oxford, U.K., 1977. (2) Sundqvist, B.; Macfarlane, R. D. Mass Spectrom. Reu. 1985, 4, 4_m - --m - -. (3) Barber, M.; Bordoli, R. S.;Elliott, G. J.; Sedgwick, R. D.; Tyler, A. N. Anal. Chem. 1982,54,645A-657A. (4) Briggs, J.; Panfili, P. R. Anal. Chem. 1991,63, 850-859. +

0003-2700/93/0365-0425$04.00/0

and product quality and effectiveness in the biotechnology industry.4 Recently,two new techniques for the desorptiontionization of even larger biomolecules have emerged. Laser desorption with an organic matrix encorporated with the analyte, pioneered by Hillenkamp and co-workers,6has demonstrated the capability for ion formation for biomolecules greater than 200 kDa.596 Electrospray ionization (ESI)?-l4 which forms multiply charged gas-phase ions from highly charged liquid droplets from atmospheric pressure, was resurrected after a period of dormancy by Fenn's groupl1J2as a viable analysis method for compounds of biochemical interest. Examples of ESI-generated protein ions of over 100-kDarelative molecular mass include @-galactosidase(118 kDa),15 bovine albumin dimer (133 kDa),16-18 IgG class monoclonal antibodies (150 kDa),lg human cr2-macroglobulin subunit (186 kDa),l9 and human complement C4 (197 kDa).lg It is generally accepted that the molecular structure of a globular protein is ultimately determined by its amino acid sequence, within the constraints imposed by its chemical environment, and that the molecular structure determines its biological function.20 The specificity and activity of a protein depends on obtaining the correct sequence of amino acids (i.e., primary structure) comprising the polypeptide chain, the presence or absence of specific posttranslational modifications, and the folding of the polypeptide chain to a (5) Karas, M.; Bahr,U.; Ingendoh, A.; Hillenkamp, F. Angew. Chem., Int. Ed. Engl. 1989,28, 760-761. (6) Chan,T.-W.D.;Colburn,A.W.;Derrick, P. J. 0rg.MassSpectrom. 1992,27, 53-56. (7) Dole, M.; Mack, L. L.; Hines, R. L.; Mobley, R. C.; Ferguson, L. D.; Alice, M. B. J. Chern. Phys. 1968,49,2240-2249. (8) Iribarne,J. V.; Thomson, B. A. J.Chem.Phys. 1976,64,2287-2294. (9) Aleksandrov, M. L.; Gall', L. N.; Krasnov, N. V.; Nikolaev, V. I.; Shkurov, V. A. Zh.Anal. Khim. 1985,40, 1570-1580. (10) Covey, T. R.; Bonner, R. F.; Shushan, B. I.; Henion, J. Rapid Commun. Mass Spectrom. 1988,2, 249-256. (11) Whitehouse, C. M.; Dreyer, R. N.; Yamashita, M.; Fenn, J. B. Anal. Chern. 1985,57,675-679. (12) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Science 1989,246,64-71. (13) Loo, J. A.; Udseth, H. R.; Smith, R. D. Anal. Biochem. 1989,179, 404-412. (14) (a) Smith, R. D.; Loo, J. A.; Edmonds, C. G.; Barinaga, C. J.; Udseth, H. R. Anal. Chem. 1990,62,882-899. (b) Smith, R. D.; Loo, J. A.; Ogorzalek Loo, R. R.; Busman, M.; Udseth, H. R. Mass Spectrorn. Reu. iwi, IO, 359-451. (15) Jardine, I.;Hail, M.;Lewis, S.;Zhou, J.;Schwartz, J.; Whitehouse, C. Proceedinas of the 38th ASMS Conference on Mass Snectrometrv and Allied Tipi&, Tuscon, AZ, 1990; Ehevier: New York,*l990;pp l& 17. (16) Loo, J. A.; Edmonds, C. G.; Udseth, H. R.; Smith, R. D. Anal. Chem. 1990,62, 693-698. (17) Gallagher,R. T.; Chapman,J. R.; Barton, E. C. Proceedings ofthe 39th ASMS Conference on Mass Spectrometry and Allied Topics, Nashville, TN, 1991; Elsevier: New York, 1991; pp 242-243. (18) Bateman, R. H.; Major, H. J.; Woolfitt, A. R. Proceedings of the 39th ASMS Conference on Mass Spectrometry and Allied Topics, Nashville, TN, 1991; Elsevier: New York, 1991; pp 244-245. (19) Feng, R.; Konishi, Y. Anal. Chem. 1992,64, 2090-2095. (20) Creighton, T. E. Proteins, Structures and Molecular Principles; W. H. Freeman and Co.: New York, 1984. 0 1993 American Chemical Soclety

426

ANALYTICAL CHEMISTRY, VOL. 65, NO. 4, FEBRUARY 15, 1993

information from intact species more rapidly and sensitively precise three-dimensional structure. Mass spectrometry than with conventional techniques. In contrast to the offers the advantages of speed and sensitivity in protein conventional time-of-flight mass analyzers employed with primary structure determination and also has a unique and laser desorption/ionization, ESI with quadrupole MS is wellimportant role in the determination of structural changes suited to tandem mass spectrometry experiments.l4,28,35-37In introduced into proteins by posttranslational modifications addition, CAD efficiencies of such multiply charged ions can (such as glycosylation, methylation, phosphorylation, and be very high due to the effective deposition of internal energy disulfide bonding),features that DNA sequencesalone cannot reveal.21-24 The combination of tandem mass spectrometry (i.e., "preheating") in the ESI atmosphere/vacuum interface (MS/MS),25 in conjunction with an arsenal of selective region.38 These efficiencies may be further enhanced by proteolytic enzymes, provides a powerful method for amino Coulombicrepulsive forces,making highly charged molecules acid sequence elucidationfor small polypeptidesand proteins, more susceptible to dissociation.39 suchas thioredoxin (11.6 kDa),26asdemonstrated by Biemann Previously published reports40341 have demonstrated CAD and co-workers. Confirmation of the primary structure for of a doubly charged peptide molecule (by FAB) in the 1-2proteins as large as bovine serum albumin (66 kDa) can be kDa molecular mass range, and Neumann and Derrick42 achieved in this rnanner.27~28 For example, the 583 amino demonstrated MS/MS of doubly charged bradykinin (M, acid sequence for bovine albumin has been recently 1060) molecules produced by field desorption. The first based on three residue corrections determined by MS and tandem MS studies of more highly charged polypeptides were MS/MS. Additional possible errors in the NHz-terminal reported by our laboratory.35 Large multiply charged olisequences of other albumin proteins have also been noted.28 gopeptide and protein ions can be efficiently dissociated by The primary structure of polypeptides with M,up to 3000 collisions with a neutral gas target, but interpretation of (and in some cases 4000, depending upon molecular structure tandem mass spectra presents special difficulties due to the and instrumentation) can be addressed directly by collisionally fact that mass spectrometry (with relatively low-resolution activated dissociation (CAD) with conventional ionization instrumentation) does not directly provide information on methods that produce primarily singly charged s p e ~ i e s . ~ ~ product ~ ~ ~ + ion ~ ~ charge state. The principal limitations of these tandem mass spectrometric This complication is largely avoided for doubly charged methods arise from the combined effects of the decreasing ions, and especially tryptic peptides. Researchers have efficiency with which large M,ions are formed and the lower exploited the multiple charging phenomenon of electrospray efficiency for dissociation of these singly charged ionization for application in protein characterization after Photodissociation,32surface-induced dissociation,33and the proteolytic digestion with tryp~in~~3-4' Such doubly charged use of ion-trapping techniques (which allow very large peptides result due to trypsin's activity on the COOHnumbers of collisions)34 are alternatives being investigated terminus of lysine and arginine residues, leaving a positive to overcome these limitations. charge on both the COOH-terminal amino acid and the It is conceivable that the production of large multiply primary amine on the NH2-terminus under the acidic solution charged molecules by ESI followed by collisionally activated conditions generally used for ESI. (Additional charging may dissociation may be exploited to obtain protein sequence arise when a histidine residue is present, often increasing the maximum charge state of the molecule by 1.) CAD of such (21) Biemann, K.; Scoble, H. A. Science 1987, 237, 992-998. (22) Biemann, K. Biomed. Enuiron. Mass Spectrom. 1988,16,99-111. doubly charged molecules primarily results in singly charged (23) Hunt, D. F.; Yates, J. R., 111;Shabanowitz, J.; Winston, S.; Hauer, product ions. ESI-MS/MS of the tryptic peptides combined C. R. R o c . Natl. Acad. Sci. U.S.A. 1986,83,6233-6237. with on-line separation methods such as liquid chromatog(24) Alexander, J. E.; Hunt, D. F.; Lee, M. K.; Shabanowitz, J.; Michel, H.; Berlin, S. C.; Macdonald, T. L.; Sundberg, R. J.; Rebhun, L. I.; raphy and capillary electrophoresis has been demonstrated Frankfurter, A. Proc. Natl. Acad. Sci. U.S.A. 1991,88, 4685-4689. to be a powerful method for protein analysis.45-47 (25) (a) Tandem Mass Spectrometry; McLafferty, F. W., Ed.; Wiley-Interscience: New York, 1983. (b) Busch, K. L.; Glish, G. L.; Much of our recent research efforts have focused on McLuckey, S. A. Mass SpectrometrylMass Spectrometry. Techniques understanding the fundamental constraints and developing and Applications ojTandem Mass Spectr0metry;VCH: New York, 1988. practical methods aimed at obtaining structurally related (26) (a) Johnson, R. S.; Biemann, K. Biochemistry 1987, 26, 12091214. (b) Johnson, R. S.; Mathews, W. R.; Biemann, K.; Hopper, S. J. information from collisional dissociation of large, highly

Biol. Chem. 1988,263,9589-9697. (27) Hirayama, K.; Akashi, S.; Furuya, M.; Fukuhara, K. Biochem.

Biophys. Res. Commun. 1990,173, 639-646. (28) Loo, J. A,; Edmonds, C. G.; Smith, R. D. Anal. Chem. 1991,63, 2488-2499. (29) (a) Johnson, R. S.; Martin, S. A,; Biemann, K. Int. J. Mass Spectrom. Zon. Processes 1988, 86, 137-154. (b) Carr, S. A.; Green, B. N.; Hemling, M. E.; Roberte, G. D.; Anderegg,R. J.; Vickers,R.Proceedings ojthe 35th ASMS Conference on Mass Spectrometry and Allied Topics, Denver, CO, 1987; Elsevier: New York, 1987; pp 830-831. (30) Gross, M. L.; Tomer, K. B.; Cerny, R. L.; Giblin, D. E. In Mass Spectrometry in the Analysis of Large Molecules; McNeal, C. J., Ed.; John Wiley & Sons: Chichester, U.K., 1986; pp 171-190. (31) Neumann, G. M.; Sheil, M. M.; Derrick, P. J. 2. Naturjorsch. 1984,39A, 584-592. (32) (a) Hunt, D. F.; Shabanowitz, J.; Yates, J. R., 111. J. Chem. SOC., Chem. Commun. 1987, 548-550. (b) Lebrilla, C. B.; Wang, D. T.-S.; Mizoguchi, T. J.; McIver, R. T., Jr. J. Am. Chem. SOC.1989,111, 85938598. ( c ) Tecklenburg, R. E., Jr.; Russell, D. H. Mass Spectrom. Rev. 1990,9,405-451. (d) Martin, S. A.; Hill, J. A.; Kittrell, C.; Biemann, K. J.Am. SOC.Mass Spectrom. 1990, I, 107-109. (e) Gorman, G . S.;Cornett, D. S.;Amster, I. J. Proceedings of the 39th ASMS Conference on Mass Spectrometry and Allied Topics, Nashville, TN, 1991; Elsevier: New York, 1991; pp 849-850. (33) (a) Mabud, M. A.; DeKrey, M. J.; Cooks, R. G. Znt. J. Muss Spectrom. Zon Processes 1985,67,285-294. (b) Williams, E. R.; Henry, K. D.; McLafferty, F. W.; Shabanowitz, J.; Hunt, D. F. J. Am. SOC.Mass Spectrom. 1990, I, 413-416. (c) McCormack, A. L.; Jones, J. L.; Wysocki, V. H. J. Am. SOC.Mass Spectrom. 1992,3, 859-862. (34) Cooks, R. G.; Kaiser, R. E., Jr. Acc. Chem. Res. 1990,23,213-219.

(35) Barinaga, C. J.; Edmonds, C. G.; Udesth, H. R.; Smith, R. D. Rapid Commun. Mass Spectrom. 1989,3,160-164. (36) Loo, J. A.; Edmonds, C. G.; Smith, R. D. Science 1990,248,201204. (37) Smith, R. D.; Loo, J. A.; Barinaga, C. J.; Edmonds, C. G.; Udseth, H. R. J. Am. SOC.Mass Spectrom. 1990, 1, 53-65. (38) Smith, R. D.; Barinaga, C. J. Rapid Commun.Mass Spectrom. 1990,4, 54-57. (39) (a) Rockwood, A. L.; Busman, M.; Smith, R. D. Int. J. Mass Spectrom,ZonProcesses 1991,111,103-129. (b)Busman,M.;Rockwood, A. L.; Smith, R. D. J. Phys. Chem. 1992,96, 2397-2400. (40) Barber, M.; Bell, D. J.; Morris, M.; Tetler, L. W.; Woods, M. D.; Monaghan, J. J.; Morden, W. E. Org. Mass Spectrom. 1989,24,504-510. (41) Hunt, D. F.; Zhu, N.-Z.; Shabanowitz, J. Rapid Commun. Mass Spectrom. 1989, 3, 122-124. (42) Neumann, G.; Derrick, P. J. A u t . J. Chem. 1984,37,2261-2277. (43) Edmonds, C. G.; Loo, J. A.; Ogorzalek Loo, R. R.; Smith, R. D. In Techniques inProtein Chemistry: ZI;Villafranca, J. J., Eds.; Academic Press: San Diego, CA, 1991; pp 487-496. (44) Chowdhury, S. K.; Katta, V.; Chait, B. T. Biochem. Biophys. Res. Commun.1990,167,686-692. (45) Hail,M.;Lewie,S.; Jardine, L;Liu, J.; Novotny,M. J.Microcolumn Sep. 1990,2, 285-292. (46) (a) Covey, T. R.; Huang, E. C.; Henion, J. D. Anal. Chem. 1991, 63,1193-1200. (b) Smith,R. D.;Udseth,H. R.; Barinaga,C. J.;Edmonds, C. G. J. Chromatogr. 1991, 559,197-208. (47) Huang, E. C.; Henion, J. D. J. Am. SOC.Mass Spectrorn. 1990, I , 158-165.


427

charged molecules. Tandem MS of large intact species (>3-4 104Torr, respectively. The triple-quadrupoleMS (TAGA6000E, Sciex, Thornhill, Ontario, Canada) has a m/z limit of 1400. kDa) would, in principle, provide a basis for a more rapid sequencing method, without the additional steps of chemical Polypeptidesamples were commercially obtained from Sigma or enzymaticdigestion and preseparation. We have previously Chemical Co. (St. Louis, MO), with the exception of epidermal growth factor (Serva, Westbury, NY), bovine ribonuclease A reported the CAD-ESI mass spectra of polypeptides ranging (Professors F. W. McLafferty and H. A. Scheraga, Cornell from substance P (MI1348),@melittin (M, 2845),%a7adrenoUniversity),human growth hormone (hGH) (Dr. R. M. Riggin, corticotropic hormone (1-39, MI 4567),14and human parEli Lilly), methionyl-hGH and thioredoxin (Dr. Ian Jardine, athyroid hormone (1-44, MI5040)37to proteins such as bovine Finnigan MAT), and interleukin-2 (Dr. D.Liberato, Hoffmannribonuclease A (MI13 682),36equine myoglobin (M,16 951),37 LaRoche) and were used without further purification. Analyte and serum albumins (M, -66 000).14928Our general obsersolutions (100 pmol pL-l) were prepared in distilled water with vation from these studies is that the formation of product 5% acetic acid or a mixture of 1:l methanol/5% aqueous acetic ions with high relative abundances occurs for dissociation of acid. Tryptic digests were prepared at a 1/20 (w/w) ratio of peptide bonds in relatively limited portions of the molecule. enzyme (sequencinggrade, Boehringer Mannheim) to protein in 50 mM NH4HC03, pH 8.2 buffer. In our earlier CAD studies of oligopeptides, we have encountered several examples in which dissociation of the amide For CAD studies in the collision quadrupole (Q2) region, the bond to a proline residue is seemingly highly favored.35-37 argon collision gas target thickness was maintained at approximately 1X 1014molecules cm-2. The laboratory-framecollision This phenomenon has been observed before in both highenergy (Elah)is estimated from the product of the charge state energy tandem double-focusing CAD s t ~ d i e s ~ and ~ s a with low collision energy q ~ a d r u p o l and e ~ ~hybrid ~ ~ ~ ~ ~ of~the parent species and the voltage difference between Q2 and the skimmer. instrumentation51-53for relatively small peptides. Proline is unique among the amino acids in that the end of the side RESULTS AND DISCUSSION chain is covalently bound to the preceding a-amide nitrogen. The five-membered ring prevents N-C rotation and thus Interpretation of Tandem Mass Spectra of Multiply may have significant effects on the conformation of the Charged Ions. For larger molecules, the more highly charged polypeptide backbone. In this report, we present CAD results ions are more susceptible to dissociation.37-39 Because collision for a number of multiply charged peptides and proteins energy is proportional to the number of charges at a given extending to -22 kDa in which dissociation adjacent to a mlz, it is reasonable to assume that the greater translational proline residue is predominant. We show that dissociation energies for ions with a greater number of charges is a primary processes due to the presence of more labile structural reason for an increased CAD efficiency. In addition, efficiency elements, such as apparently induced by proline residues, for MSIMS of multiply charged ions may be enhanced due become increasingly significant as molecular mass increases to electrostatic repulsive forces.39 We have previously demand dominate CAD processesfor the larger moleculesstudied. onstrated the production of sequence specific fragment ions from the multiply charged molecules of oligopeptides larger EXPERIMENTAL SECTION than might be efficiently dissociated as singly charged species and that multiply charged product ions observed in such The electrosprayionizationsourceand triple-quadrupolemass experiments can depend upon the parent ion charge state. spectrometer have been previously de~cribed.~~ The analyte solution, in a 100-pm-i.d. fused-silica capillary, mixes with a For example, collisional dissociation of the (M + 3H)3+to (M flowing liquid methanol sheath electrode at the tip of the ESI 6H)6+molecular ions of melittin (M, 2845) from ESI yields source. A potential of +4 kV is applied to the sheath electrode multiply charged product ions (to 4+) that can be readily to produce a fine mist of highly charged droplets. The flow rates ascribed to the known sequence of the polypeptide.36~37~65 of the analyte solution and liquid sheath are independently Dramatic differences among the spectra of the various charge controlled by separate syringepumps (HarvardApparatus,South states and the large number of fragment ions with variable Natick, MA, and Sage Instruments, Cambridge, MA) at flow charge states were observed. rates of 0.2-0.5 and 2.5-3.0 pL min-l, respectively. Ions enter the intrument through a differentiallypumped nozzle/skimmer Although CAD of multiply charged ions from larger interface of our design, similar to that present on our prototype peptides yields product ions that can be readily correlated to single-quadrupolein~trument.~~ A lens element,typically at +700 the sequence, complete sequence assignments are generally V, mounted in front of the nozzle/skimmer assembly, improves not obtained with quadrupole mass spectrometers. Studies the ion-sampling efficiency. Desolvation of the droplets is to date indicate that a progressively smaller portion of the accomplished by a countercurrent stream of nitrogen gas and total molecule is observed to be susceptible to low-energy through collisions in the interface region. The energy of the CAD with increasing analyte size. For example, human collisions is crudely controlled by varying the nozzle/skimmer parathyroid hormone (1-44, MI 5064)37yielded multiply voltage bias (ANS);54a typical bias used is +135 to +200 V (with charged molecules from 4+ to 9+ charge states. Collisional the skimmer held at +65 V) before dissociation of the analyte molecule occurs. The pressure in the atmosphere/vacuum indissociation analysis of these parent ions, producing primarily terface and in the mass spectrometeris typically 2.1 Torr and 6 X singly charged b, and y, sequence ions from both COOHand "2-termini, affords sequence information from approximately one-third of the molecule. (Nomenclature used (48) Edmonds, C. G.; Loo, J. A.; Fields, S.M.; Barinaga, C. J.; Udseth, H. R.; Smith, R. D. In Biological Mass Spectrometry; Burlingame, A. L., for the oligopeptide fragmentation pattern is based on McCloskey, J. A,, Eds.; Elsevier: Amsterdam, 1990; pp 77-100. conventional notation.21822956 Subscripts are used to denote (49) Martin, S. A.; Biemann, K. Znt. J.Mass Spectrom. Zon Processes the residue position, counting from the NHz-terminus for a,, 1987, 78, 213-228. (50) Vestling, M.; Hua, S.; Murphy, C.; Orlando, R.; Wu,2.;Fenselau, b,, and c, ions and from the COOH-terminus for x,, yn, and C. Proceedings of the 39th ASMS Conference on Mass Spectrometry z, product ions. A superscript is added to indicate the and Allied Topics, Nashville, TN, 1991; Elsevier: New York, 1991; pp fragment ion charge state. Lack of a superscript denotes a 1473-1474. (51) Gaskell, S. J.; Reilly, M. H. Rapid Commun. Mass Spectrom. singly charged fragment.)

+

1988,2,18&191. (52) Bean, M. F.; Carr, S. A.; Thorne, G. C.; Reilly, M. H.; Gaskell, S. J. Anal. Chem. 1991,63, 1473-1481. (53) Schwartz, B. L.: Bursev, M. M. Bioi. Mass Spectrom. 1992, 21, 92-96. (54) Loo. J. A,: Udseth. H. R.: Smith. R. D. Rapid Commun.Mass Spectrom. isas, i,207-2io. '

(55) (a) McLuckey, S. A.; Glish, G. L.; Van Berkel, G. J. Anal. Chem. 1991,63,1971-1978. (b)Van Berkel, G. J.; Glish, G. L.; McLuckey, S. A. Anal. Chem. 1990,62, 1284-1295. (56) Roepstorff, P.; Fohlman, J. Biomed. Mass Spectrom. 1984, 11, 601.

428


Even in cases in which the amino acid sequence is known, interpretation of the resulting product ion spectrum can be difficult because the charge states of the fragment ions are uncertain, especially in our present experiments with relatively low resolution quadrupole instrumentation. With higher resolution methods, such as double-focusing mass spectrometers57 and ion-trapping instruments, such as the ion trap mass spectrometer (ITMS)58 and the Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR-MS),5w1 measurement of the 12C/13Cisotopic separation reveals the charge state. Henry et al.60r61 have demonstrated resolving powers of >60 000 by ESIlFT-ICRMS with a 2.8-T superconducting magnet, easily allowing resolution of the isotopic envelope for the 18+ charge state of equine myoglobin and the 16+ charge state of equine cytochrome e, providing both high precision and mass accuracy (0.1 Da). However, even with low-resolution instrumentation, the interpretation of multiply charged product ion mass spectra for molecules of known structures can be aided by careful inspection. For example, for each peak in the product ion mass spectrum, all possible MI values may be calculated (the observed rnlz multiplied by all possible charge states (up to parent charge)) and compared to all possible "b" and "y" mode fragment ions from the known sequence. More probable assignments are initially selected by searching for features that appear to be common for most of the MSlMS spectra we have previously examined, such as progressions of product ion charge states (e.g., ~ 2 5 ~y+~, 5 ~ + , y254+,etc.) and product ion series (e.g., y2,j2+,~ 2 7 ~etc.). +, In addition, by energetic collisions in the atmospheric pressure/vacuum interface, we are able to produce a fragment ion species for further dissociation in Q2, a so-called "MSl MSJMS" e ~ p e r i m e n t . ~This ~ ~ ~can ~ , be ~ ~used to confirm product ion assignments and often provides additional sequence information by probing molecular regions not examined in conventional MS/MS experiments. Finally, comparison of CAD mass spectra from structurally related molecules, such as molecules with only a few amino acid modifications (e.g., variants, proteins from different species with high homology or amino acid sequence alignment) or chemical derivatives (e.g., carboxymethylation of free cysteines, acetylation of the "2-terminus, oxidation of a methionine residue, etc.), helps to identify the approximate location of dissociation and thus the assignment of the product ion due to the expected shifts in mlz. It is likely that a multiply charged macromolecule undergoing a single dissociation event would produce two charged product species. The presence of these complementary ion pairs,36whichin sum (mass and charge) account for the entire molecule, alleviates some of the interpretation problems. Simple algorithms can be written to determine whether two product ions are possibly complementary. [Dissociative electron capture from the collision gas62 (e.g., ABn++ T A(n-1)+ B T+, where T is the collision target species) is

+ +

-

(57) (a) Dobberstein, P.;Giessmann, U.; Schroeder,E. Proceedings of the 39th ASMS Conference on Mass Spectrometry and Allied Topics, Nashville, TN, 1991; Elsevier: New York, 1991; pp 238-239. (b) Larsen, Mass Spectrom. 1991,2, 205-211. B. S.; McEwen, C. N. J. Am. SOC. (58) (a) Schwartz, J. C.; Syka, J. E. P.; Jardine, I. J. Am. SOC. Mass Spectrom. 1991,2, 198-204. (b) Williams, J. D.; Cox, K. A.; Cooks, R. G.; Kaiser, R. E., Jr.; Schwartz, J. C. Rapid Commun. Mass Spectrom. 1991,5,327-329. (59) (a) Henry,K. D.;Williams,E. R.; Wang,B. H.;McLafferty,F. W.; Shabanowitz,J.;Hunt,D. F.Proc.Natl.Acad. Sci. U.S.A. 1989,86,90759078. (b) Henry, K. D.; McLafferty, F. W. Org. Mass Spectrom. 1990, 25, 49C-492. (60) Henry, K. D.;Quinn, J. P.; McLafferty, F. W. J.Am. Chem. SOC. 1991, 113, 5447-5449. (61) Loo, J. A.;Quinn, J. P.; Ryu, S.I.; Henry, K. D.; Senko, M. W.; McLafferty, F. W. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 286-289. (62) Waddell, D. S.;Boyd, R. K. Int. J.Mass Spectrom. Ion Processes 1989,93, 337-358.

not treated in our example. In general, we do not observe a significantly abundant rnlz 40 species for Ar+,but small contributions from the dissociative electron capture mechanism cannot be rigorously ruled out.] In principle, a set of possible complementary ions, (m/z)b,exist for each peak in the CAD spectrum (rnlz),. Because MI and the total charge or parent ion charge (zt) are known from the distribution of multiply charged ions in the initial ESI mass spectrum, then

and

(mlz),= (ma+ 1.0079za)/z,

(3)

(m/z)b= (m, + 1.0079zb)/z, (4) if we assume that charging is due to multiple protonation. Because there are four unknown quantities (ma,mb, zs,and Zb) and three known parameters (MI,zt, and (mlz),),there is no unique solution for (m/z)b.Rearrangement of relationships 1 through 4 yields zt- z, = [MI+ 1.0079(zt)- z, (rnlz),ll(ml~)~ ( 5 ) However, since za and Zb are constrained to integer values, there are only a finite number of possible (m/z)bsolutions, where z, has integer values ranging from 1 to zt - 1 (assuming that both products are charged). Thus, the possible values for the complementary ion to (rnlz), is a set of zt - 1 values, not all of which are necessarily within the instrumental rnlz range or likely to correspond to chemically reasonable products. For example, the CAD mass spectrum of the (M + 17H)17+ ion for methionyl-human growth hormone (M, 22 256) (see later section) shows a prominent product ion at rnlz 279. The possible complementary ions constitutes a set of 16 possible rnlz values (and their corresponding charge): 1374.6 (16+ charge), 1447.7 (15+), 1531.2 (14+), ..., 9044.2 (2+), and 17 809.3 (l+). The spectrum would be examined for peaks at each of these mlz values within the rnlz constraints of the particular instrument (an upper limit of rnlz 1400 in the present case). Since a search based upon rnlz 279 yields a possible match (an abundant product ion at rnlz 13741, one can now calculate the mass and charge corresponding to each of the products to evaluate further the likelihood that these products are complementary on the basis of experimental rnlz measurement accuracy, likely dissociation products, amino acid sequence, and other available information. (Some cauton must be exercised in application of these methods, particularly due to the accuracy of rnlz measurements and for instruments of limited rnlz.) The possible complementary ion pairs can also be screened on the basis of the fragmentation products predicted for polypeptides, an approach which becomes increasingly useful as the amount of known sequence increases. Algorithms for interpretation of CAD mass spectra of peptides have been described in the literature63 but only deal with singly charged parent and product ions. Recent (63) (a) Johnson,R.S.; Biemann, K. Biomed.Enuiron.Mass Spectrom. 1989, 18, 945-957. (b) Bartels, C. Biomed. Enuiron. Mass Spectrom. 1990, 19, 363-368. (64) (a) Yates, J. R., III; Mao, Y.; Griffin, P. R.; Hood, L. E.; Zhou, J. X. Proceedings of the 39th ASMS Conference on Mass Spectrometry and Allied Topics, Nashville, TN, 1991; Elsevier: New York, 1991; pp 1221-1222. (b) Bonner, R.;Covey, T.; Shushan, B. Proceedings of the 39th ASMS Conference on Mass Spectrometry and Allied Topics, Nashville, TN, 1991; Elsevier: New York, 1991; pp 1394-1395. (c) Johnson, R.S.; Ericsson,L.; Walsh, K. A. Proceedings of the 39th ASMS Conference on Mass Spectrometry and Allied Topics, Nashville, TN, 1991; Elsevier: New York, 1991; pp 1233-1234. (d) Siegel, M. M. Proceedings of the 39th ASMS Conference on Mass Spectrometry and Allied Topics, Nashville, TN, 1991;Elsevier: New York, 1991; pp 11211122.


429

Table I. Proline-ContainingPolypeptides Analyzed by Electrospray Ionization Tandem Mass Spectrometry total residues

no. of proline residues

polypeptide

M,

[Glnel-LH-RH LH-RH phydaemin ACTH (18-39) melittin insulin B-chain (oxidized) ACTH (1-39) PTH (1-44) EGF (mouse) BPTI (disulfidereduced) ubiquitin (bovine) thioredoxin ribonuclease A (bovine) Met-interleukin-2 hemoglobin @-chain (human) hGH Met-hGH carbonic anhydrase (bovine)

1154 1182 1265 2466 2845 3496

22 26 30

3

4567 5064 6040 6519

39 44 53 58

4

8565 11673 13683

76

108

3 5

124

4

15547 15867

134 146

5

22125 22256 29021

191 192 259

8 8 19

10 10 11

1 1 1 1 1

2 2 3

active proline residue position" 9 not observed 4

19 14 28 36 40 4

8 250

7

19,37 40 114,117

750

1000

ndr

d+2H)*+

(b)

3 51 not observed 3 193,199,213,235

500

b8

x1

0

984

1

Y2 172

a Based upon enhanced dissociation adjacent to the indicated site(s).

i

b:

bi

work on extension of computer programs such as those for multiply charged ions show promise64 but need further refinement before sequencedeterminationof unknown species by ESI-MSIMS becomes routine. Although these methods Flgwr 1. Tandem MS spectra of the (M i-2H)2+ parent Ion of (a) do not fully answer the problem of interpretation of CAD LH-RH and (b) [ G l n e ] l M H . spectra of large multiply charged ions, they do contribute useful information toward this purpose. Scheme I Dissociation Adjacent to Proline Residues. Previous reporta for tandem mass spectrometry of peptides have often noted an unusually prominent product ion peak resulting pGlu - His Trp - Ser - Tyr - Gly - Leu - X Pro - Gly - NH2 from cleavage of the CO-NH backbone linkage to a proline residue.23,35147,49150,53Proline is unique as the only imino acid b8 found in mammalian protein sequences.65 Proline has been noted by Barinaga et al.35 for the CAD of the 3+ to 6+ implicated in drastically altering the conformation of small molecules of the 26-residue melittin peptide. Other ESIpeptides and thus the fragmentation pattern in MS CAD results have followed;47,5'Jin particular, Loo et al.3'3 have studies.66-68 In earlier studies, comparison of field desorpreported on the ESI-MS/MS of bovine ribonuclease A (124 tion,% FAB,67 and tandem mass spectra68 of N-benzyloxyaminoacids,13.9 kDa) and have ascribed most of the abundant carbonyl-protected tripeptide8 with and without a proline product ions to dissociation of the Val 116-Pro 117 bond. It amino acid at position 2 yielded significant differences in the is interesting that there are such marked and specific loss of benzyl alcohol. Conformational differences due to variations in relative dissociation rates for the many apparent proline were indicated. Williams and co-workers69 have dissociation routes available to molecules as large as proteins. reported particularly abundant proline-bearing fragmentions The special role of proline is implicated in dissociation of a in FAB mass spectra which they attribute to an enhanced of polypeptides and proteins, which we discuss stability of the proline iminium ion. Martin and B i e m a n ~ ~ ~ number ~ individually below (see also Table I). have observed the predominance of the cleavage of the "2CAD of Small Multiply Charged Peptides. Tandem terminal peptide bond of proline in high-energy CAD studies MS of the doubly charged luteinizing hormone releasing and appearance of internal fragment ions bearing the proline hormone molecule (LH-RH,pGlu-His-Trp-Ser-Tyr-Gly-Leuresidue at the NH2-terminalposition. Similarly Hunt et aLZ3 Arg-Pro-Gly-"2, M, 1182) produces abundant cleavage have observed this phenomenon in low-energy CAD with products ye, y7, and ye (in addition to their complementary quadrupole mass spectrometers. On the basis of thermoions, b4, b3, and bz, respectively) with only a very weak chemical arguments, Schwartz and Burseys3 have estimated contribution from the Arg &Pro 9 bond dissociation product, that a y-type ion with an "2-terminal proline residue is 32 b8 (Figure 1 and Scheme I). However, CAD of the [GlnEIkJ mol-' more stable than with NH-.derminal alanine. LH-RH analogue (Figure lb) yields an abundant be product The enhanced tendency of this dissociation pathway has ion along with smaller contributions from bs, by, and be2+. also been observed for multiply charged ions, as was initially Substitution of a neutral glutamine residue for a potentially positively charged arginine residue allows for the X &Pro 9 (65) Williams, K. A,; Deber, C. M. Biochemistry 1991,30,8919-8923. (66) Tsunematsu, H.; Hachiyama, S.;Isobe, R.; Ishida, E.; Kakoi, M.; dissociation process to be favored (see Scheme I). Dissociation Yamamoto,M. Biochem. Biophys. Res. Commun. 1987, 146, 907-911. near a basic and potential charge site appears to be less favored M.;Isobe, (67) Tsunematau,H.;Nakashima,S.;Yoahida,S.;Yamamoto, than charge remote-site fragmentation, especially for X-Pro R. Og. Mass Spectrom. 1991,26,147-150. bond cleavage. As a result, much greater abundance8 are (68) Schwartz, B. L.; Erickson, B. W.; Bursey, M. M. 39th ASMS Conference on Mass Spectrometry and Allied Topics, Nashville, TN, observed for the b-mode product ions. 1991; Elsevier: New York, 1991; pp 769-770. Such behavior was also observed for substance P peptide (69) Williams,D. H.;Bradley,C. V.;Santikam,S.;Bojesen,G.Biochem.

fY8

I

~~

J. 1981,201, 105-117.

(2-11) (Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-"2, M,

430

ANALYTICAL CHEMISTRY, VOL. 65, NO. 4, FEBRUARY 15, 1993 I

(M+6H)6+

PQQiKPQ

I

(M+H)+ x2

1 bI0

PQQFFGL

. 11

500

"

750

500

1250

loo0

750

m/z

mlz

1

1044

I

(M+2H)2+

6 5

I b9

yso

5

L 1250

750

250

lo00

mlz

miz

ESI tandem mass spectra of the (a) (M + H)+ and (b) (M + 2H)2+Ions of substance P (2-1 1). Single letter codes are used to denote the structures of the internal fragment ions (e.g., P = Pro, K = Lys, Q = Gln, F = h e , G = Gly, L = Leu).

Flgure 3. (a)ESI mass spectrum of mouse epidermal growth factor (A4 6040) and (b) CAD mass spectrum of the (M + 6H)6+ parent Ion with a laboratory-frame colllsbn energy of 930 eV. Peaks at mlr 988.6 and 1186.0 (labeled with 0 ) are due to the des-Asn EGF component.

1192). Consistent with previously published CAD data for the singly charged m ~ l e c u l eMS/MS , ~ ~ ~ ~of~the (M H)+ion yields internal fragment ions resulting from loss of the "2terminal Pro 1-Lys 2 residues, Le., cleavage of Lys 2-Pro 3 occurs readily followed by further dissociation along the polypeptide backbone (Figure 2a). A ys fragment is also present due to Lys 2-Pro 3 bond cleavage. Similar internal cleavage reactions were observed for interleukin-2 and MethGH (see latter sections). However, MSIMS of the (M + 2H)2+ ion (Figure 2b) produces mainly b, fragments originating near the COOH-terminus. Product ions ys and PQ due to Lys 2-Pro 3 bond dissociation are present, but only in relatively low abundance. For a doubly charged molecule, the Lys 2 residue is more likely to be positively charged and thus suppresses nearby bond cleavage. The lysine residue is less likely to be charged for the singly charged molecule; dissociation of the Lys 2-Pro 3 bond is more favored. If we speculate that the CAD results for small multiply charged peptides can be extrapolated to larger protein systems, then it appears likely that charge sites (i.e., protonation) adjacent to a proline residue could influence the extent of dissociation. The charge-directing influence of Arg and Lys for singly charged molecules resulting in fragmentation remote from the charge site has been noted by others.23,ma,"J Sirniar effects appear evident for multiply charged polypeptides. Epidermal Growth Factor. Mouse epidermal growth factor (EGF) is a 53 amino acid single-chainpolypeptide (M, 6040) with three disulfide bonds joining cysteine residues at positions 6-20,14-31, and 33-42. Fast atom bombardment

Scheme I1

of mouse EGF has been reported'l with a singly charged molecule present, as well as an ion of mass 114 Da lower, suggested to be a component missing the "2-terminal asparagine residue. Consistent with the FAB-MS data, the electrospray ionization mass spectrum of mouse EGF shows a (M + 5H)5+and (M + 6H)6+species for both the 53-residue peptide and the des-Asn component (Figure 3a). CAD of the (M + 6 H P ion generates mainly product ions resulting from cleavage of bonds from the first 4 amino acids in the sequence (Figure 3b). A series of 6+ product ions, y50-y52, arising from neutral losses and sizable contributions for y ~ 5and + ys15+are evident (see Scheme 11). A few singly charged ions with charge retention on the "2-terminal side, below mlz 400, such as b2 (mlz 202) and b3 (mlz3651, are also present. The unusually large intensity for the b3 and ym5+ sequence ions (i.e., complementaryion pair) is consistent with C-N bond cleavage adjacent to a proline residue from the "2-terminal side. No product ions were ascribed to regions bound by disulfide linkages, as expected because dissociation of at least two bonds must occur to liberate detectable fragment species. Ubiquitin. Ubiquitin is a single polypeptide chain of 76 amino acid residues (M, 8564.8) believed to be present in all eukaryotic cells and is one of the most highly conserved of

(70)Vath, J. E.; Biemann, K. Znt. J. Mass Spectrom. Zon Processes 1990,100,287-299.

(71) Cotter, R. J.; Larsen, B. S.;Heller, D. N.; Campana, J. E.; Fenselau, C . Anal. Chem. 1985,57, 1479-1480.

Flgure 2.

+

y50

+I-

Am, - Ser - Tyr Pro4 - Gly - Cys - Pro

~

...

b3


4S1

(M+ 1OH)lo+

- . x 2 I 934 y8;

I

I

1

(M+7H)7+

(M+7H)7+

500

250

750

1oM)

rnlz

+

Flgwe 5. ESIMS/MS spectrum of the (M 10H)IO+ Ion of bovine ubiquhln (laboratory-frame collision energy of 1150 eV).

8t

(M+lIH)"*

I

I

YS8

Scheme 111

fYS8

94

...Glu

I

I

C

x4

1

i

0

500

1000

750

-

Prolg- Ser - Asp Thr

- ... - Gly - Ile

f

Pro37- Pro - .,.

b18

I (M+7H)?*

y40

1250

mtz Flgwo 4. ESI mess spectra of bovine ubiquitin prepared In 48.75: 48.75:2.5 methanol:H20:acetlcacid with ANS of (a)+160 V, (b) +235 V, and (c) +310 V.

all eukaryotic proteins.72 The ESI mass spectrum of bovine ubiquitin (M, determined = 8564.7 f 0.4) shows multiple charging to the 13+ molecular ion state, consistent for a polypeptide with 13 basic sites (Figure 4a). The unusual bimodal charge state distribution observed in Figure 4a has been attributed to the effecta of solution-phasehigher order structure on the production of gas-phase multiply protonated oligopeptide molecules.73 The higher charge-state distribution is hypothesized to represent the more extended denatured conformation, while the low charge state representa the native form.73 Separate collisionally activated dissociation experiments of the 7+ to 13+ molecular ions (data not shown) reveal fragmentation primarily from the initial 20 residues (from the "2-terminal) with a weaker ion series originating near the Pro 37 residue. At constant laboratory-frame collision energies (approximately 1200 eV), a series of singly charged b, sequence ions to be is evidentfrom the CAD of lower charged parent ions. Doubly charged bn2+producta from b132+to b d + are also evident, in addition to more highly charged y, ions. (72) The Ubiquitin System; Schhinger, M., Hershko, A., Eds.;Cold Spring Harbor Laboratory: Cold Spring Harbor, NY, 1988. (73) Loo, J. A.; Ogorzalek Loo,R. R.; Udseth, H. R.; Edmonds, C. G.; Smith, R. D. Rapid Commun. MQSSSpectrom. 1991,5, 101-105.

For example, a relatively abundant ion at rnlz 1308 in the CAD spectrum for the (M + 7HI7+molecule73can be attributed to the y685+product, which would be the complement ion of the rnlz 1018b1a2+product ion. The unusually large intensity of these product ions is qualitatively consistent with facile bond cleavage on the "2-terminal side of a proline residue (position 19). A weaker yn4+series originating near a proline site at position 37 (ym) is also present (see Scheme 111). In general, as M , increases, the probability of generating dissociation from interior regions of the molecule by CAD for multiply charged ions appears to decrease. However, the location of a proline residue within the middle of the polypeptide chain can apparently reverse such a trend and allow abundant fragmentation to occur. SimilarCADmaea spectra areobtainedfor the higher charge state molecules. As molecular charge state increases, the charge state for the y s and the y~ series increases, but the b, product ions remain largely the same. For example, MSI MS of the (M + 10H)lO+molecular ion (Figure 6) produces a distribution of multiply charged ym ions from 6+ (mlz 1089) to 9+ (mlz 727)with the ys7+(mlz 934)and ys8+ (mlz 817) as the most abundant producta. The ym ion series continues to increase in charge until the (M + 12H)12+charge state ie reached, where an 8+ to 10+ ( m / z 654) charge-state progression of y~ product ions are generated.73 The maximum charge for this sequence ion is also the lo+ charge state for CAD of the 13+ molecular ion. Because there are three basic residues (out of a totalof 13)on the "2-terminal side of Pro 19,the maximum charge for such a y s ion might be expected to be no more than lo+. Confiiation of the mass and charge stateoftheputativeymproductionwaeobtainedby separately reported high-resolution ESI-CAD-FTMS experiments,Bl in which the y68 isotopic contributions were resolved, revealing the charge identity. Likewise, the ion at rnlz 683was identified as the yzr4+product from high-resolution FTMS.6' Additional information can be obtained by MSIMS of fragment ions produced in the atmospherelvacuum ion sampling interface.37 We have previously demonstrated the ability to efficiently dissociate highly charged molecules in this initial stage of the mass spectrometeTM by control of the voltage bias on the nozzle-skimmer elementa (ANSI. Increasing this potential causes more highly charged molecules to dissociate more extensively since collision energy depends on charge state. For ANS of +235 V, fragment ions at rnlz

432


1

9:i

i-:

MSiMS (M+12H)I2+

Ubiquitin

(M+~zH)~*+

(a)

II

I

Bovine

727 9

Y58

1

IO Y58

2 a 2

654

P

C

Y14‘

Yeast

29

y9 2

1021

y12

1000

750

19: p c O ~ & 24: Glu -Asp 28. Ala *Ser

816

1

I

500

8

Y58

I

T

250

rcsrdve

i250

mi2

Fburr 6. CAD-MS/MS spectra of the (a) ysee+ product ion of bovine ubiqultinwith ANS = +235 V and Ehb(Q2)= 1000 eV and (b) the y14*+ Ion with ANS = +310 V, Ebb= 330 eV.

727 and 817 are generated, assigned as the y5a9+and y5a8+ sequence ions, respectively (Figure 4b). CAD of both these ions produces products ascribed as doubly charged ySle2+ from the COOH-terminus in addition to singly charged b3-5 products from the “2-terminus, as illustrated in Figure 6a for the MSIMS spectrum of the y588+ion. A relatively intense y405+ product originating near the proline residue is also present. (This proline residue is at position 37 for the intact ubiquitin molecule, corresponding to position 19 for the 58 residue fragment species.) CAD of such fragment ions allows regions to be probed that are unexamined by MSIMS of the Further increasingANS to +310 intact molecular V produces more extensive fragmentation of the higher charged molecules and fragment ions (Figure 4c) since sequential dissociation steps are now viable, particularly at lower mlz values. As observed from Figure 4a, CAD of y58 fragments yields lower charged ye15 ions, which contribute to the high ANS interface dissociation spectrum. The y142+ product ion was subjected to MSIMS analysis (Figure 6b), yielding additional sequence information from the COOHterminus. The products observed here possibly result from three sequential dissociation steps, two in the interface to yield the y142+product and a third in Q2 to yield the indicated products. The amino acid sequence of ubiquitin from Baker’s yeast differs from most eukaryotic forms by three residues, specifically Pro-to-Ser at position 19, Glu-to-Asp at position 24, and Ala-to-Serat position 28. The crystal structure of Baker’s yeast ubiquitin is quite similar to human ubiquitin (human and bovine ubiquitin have identical sequences).74 However, slight differences are evident in their CAD mass spectra. For bovine ubiquitin, most of the fragmentation processes are (74) Vijay-Kumar, S.; Bugg, C. E.; Wilkinson, K. D.; Vierstra, R. D.; Hatfield, P. M.; Cook, W. J. J.Biol. Chem. 1987,262,6396-6399.

500

600

700

800 mlz

900

1000

+

Flgurr 7. ESIMSlMS spectra of the (M 12H)12+parent ion of (a) bovineand (b) Baker’syeast ubiqumn. Peaks labeledwith the V symbol are due to a series of doubly charged y2+ fragment ions (ys2+ to yle2+), with a few assignments indicated on the figure.

focused near Pro-19, generating an intense y58 and its complement, bl8 (Figures 5 and 7a). Dissociation also occurs near position 19 (Ser-19)for Baker’s yeast ubiquitin (Figure 7b), but the relative abundance of the corresponding y58 ion is far less significant. Its complementary b18 ion is not present with discernable abundance in the spectra. A distribution of sequence ions from y52 to y s ~is observed, in addition to a set of doubly charged yg to yl6 ions not observed for the bovine form. Dissociation of the CO-NH bond on the COOHterminal side of a proline residue is typically unfavored; substitution of Ser for Pro allows for these fragmentation pathways (y52-57). Interestingly, dissociation processes are directed toward similar molecular regions, regardless of the sequence of these molecules, arguably reflecting their simihr higher order structures. However, a major portion of the fragmentation intensity is directed into the y58 ion upon the presence of a proline residue (bovine ubiquitin). Thus, while similar higher order structures may give rise to similar molecular dissociation sites, the relative abundance of the product ions is highly influenced by the nature of the individual residues. Discussions of the role of higher order structure in low-energy CAD must be considered speculative at this point due to the lack of knowledge concerning noncovalent interactions in the gas phase for these molecules and, in particular, their likely isomerization during ‘heating” from the multple collisions required for activation. Thioredoxin. Thioredoxin from Escherichia coli contains 108 amino acid residues and a disulfide bridge from Cys 32 11673). It is a small redox protein that binds to Cys 35 (M, electrons reversibly via reversible formation of a disulfide bond between two cysteine residues. The primary structure of thioredoxin from Chromatium uinosum and rabbit bone marrow has been determined chiefly by tandem mass spectrometry and through the use of specific proteolytic digests by Biemann and co-workers.26 The electrospray ionization


499

3

750

loo0

niiz

1250

mlz

(b)

(M+13H)I3'

2

20

YII

8 y69

Y8 872

616

r x4 9 y69 818

8 y70

.,

AI

YQ9

250

750

500

mlz 750

loo0 mlr

1250

Flgure 8. Tandem MS with Gbof 1560 eV of the (a) (M and (b) (M 13H)13+ parent ions of thloredoxin.

+

+ 12H)I2+

mass spectrum of recombinantthioredoxiis fromE. colishows multiple charging from 9+ to 15+ with an experimental M, of 11672.9 10.5 Da.14 MSIMS of the individual multiply charged molecular ions produces similar fragment ions with varying product ion charge states (Figure 8). Many of the product ions can be ascribed as multiply charged products originating from the first 40 residues from the "2-terminus. Series of 2+ and 3+ b,-mode sequence ions near residue 23 are present. As expected, dissociation of the bond to a proline residue (position 40), gelds a series of intense product ions (y69). For example, tandem mass spectrometry of the (M + 10H)l0+ion produces the ye#+ and yes7+ ions; however a distribution of 6+ to 9+ y69 ions are observed in the MSIMS spectrum for the (M + 12H)12+parent ion (Figure 8a), in addition to a series of ions clustered around the yes7+ and y6g8+ions, attributed to dissociation near Pro 40 (i.e., y65-71). A product ion found irl the dissociation spectra of higher charged state molecules at mlz 865 is assigned as a b395+ sequence ion, the complementary ion to y6g8+ in the (M + 12H)12+case. Slightly less intense y7+ba8and y71-b37 complementary ion pairs h e also evident in the MSIMS spectra. Most of the dissociation products are formed in regions adjacent to a protrusion determined by X-ray cyrstallogra~ h y , including 7~ residues 29-37 (and thus, also includes the qctive center disulfide bridge). Although possibly coincidental, it is interesting to speculate whether observed dissociation at such sites may be augmentedby the constrainta imposed by the cystine-bridged ring structure. Ribonuclease A and 5. In general, the portion of the polypeptide chain accessible by collisionally activated dis(75) (a) Dyson, H.J.; Gippert, G. P.; Case, D. A,;Holmgren,A.; Wright, P. E. Biochemistry 1990,29,4129-4136. (b) Holmgren, A.; Soderberg, B.-O.;Eklund, H.; Branden, C.-1.Proc. Natl. Acad. Sci. U.S.A. 1985,72, 2305-2309.

+

+

Flgure 9. ESI-MSIMS spectra of the (a)(M 14H)I4+and (b) (M 15H)I5+ions from the dlsuitlde-reduced form of bovine ribonuclease A.

Scheme IV y11 r+

Y8

J

...AsnliJ Pro - Tyr - Val b113

r-

Proll7 -Val - His - ...

b116

sociation decreases as the molecular size increases. As we have shown previously with tandem quadrupole instrumentation, only limited primary information can be obtained for multiply protonated proteins with M,> 10 O00 Da by MSI MS. For example, for bovine pancreatic ribonuclease A (RNase A, 124 residues, M,13 682 Da),36 a protein with four disuMde bonds, assignablefragmentationis limited to residues 20-26 from the "2-terminus and the last 11residues near the COOH-terminus, with the major dissociation process involving the bond cleavage from the "2-terminal side of a proline residue (shown for the 14+ and 15+ charge states of the disulfide-reduced protein in Figure 9). The y8 ion is formed by cleavage of the Val 116-Pro 117 peptide bond, with the charge retained on the COOH-terminal fragment. Dissociation of the y8 product ion generated in the nozzle/ skimmer interface (MS3) confirmed its assignment.36 Other intense peaks are assigned to cleavage of the "2-terminal amide bond to Pro 114 or yll (see Scheme IV). Complementary ion pairs are a major feature of the reduced RNase A CAD mass spectra, but not for the cystine-bridgedor native form.36 For example, for the (M + 14H)14+ion of disulfidereduced RNase A, the complement of the singly charged y8 ion is the 13+ species, b l l P product ion. The yll-b11313+ and ~11~+-b11312+ pairs are also prominent. Spectra for other charge states studied (up to 17+, data not shown) display peaks consistent with these assignments. A series of triply charged bI43+to bZ43+ions (Asp 14 to Asn 24) is observed between mlz 500 and 850.

1


434

-

1

(M+9H)9+

8

loo-

(Mt I3H)

(a)

1

w

50-

.-

-2

3t

141

75-

B

2

'

j

l7'

a

2

78

1

250

YJIYU

I

y9

17+

0

1250

750

500

2s.

So0

750

lo00

1250

lo00

1250

mlz

mlz

Flgure 10. ESI-MS/MS spectrum of the (M + 9H)9+ parent ion of RNase A (resMues 21-124) or RNase Sprotein.

750

m/z 200

600

400

+

800

1000

miz

the (M 4H)'+ parent ion of RNase A (residues 1-20) or RNase S-peptide. Peaks labeled with V are due to the loss of 18 Da from bn3+ product ions. Flgure 11. CAD of

We infer from the RNase A CAD studies that protein structure and charge location in the gas phase may be strongly interactive. At a minimum, changes in structurewill generally correspond to different distributions of charge sites. Thus, in the case of RNase A, the disulfide-reduced form is more likely to have protonation sites toward the center of the (initially) globular structure due to reduced electrostatic repulsion. Comparison of CAD maas spectra for similar charge states of the native and reduced forms reveals dissociation producta with different charge states. Whether higher order structure by itaelf influences the probability of cleavage at a particular site likely depends on whether structural differences are still meaningful after the multiple collisional excitation process. Regardless, in cases where structure is constrained by covalent disulfide bonds, significant differences in dissociation products can result. Additional supporting evidence for the fragmentation assignments is obtained from the CAD of ribonuclease S-protein and ribonuclease S-peptide. Enzymatic hydrolysis of RNase A with subtilisin resulta in cleavage of the Ala 20Ser 21 bond, resulting in a heterodimerconsistingof S-peptide (residues 1-20, MI2166) noncovalently bound to the remaining S-protein (residues 21-124, MI 11534hZ0 Full enzymatic activity is generally retained by the S-proteinls-peptide complex. At acidicpH, the S-peptideand S-proteindissociate. ESI-MSIMS spectra of S-protein from a sample of bovine ribonuclease S-protein76 and S-peptide from a ribonuclease S sample are shown in Figures 10 and 11, respectively. Additional impurities were also detected in both samples. (76)(a) Chowdhurv. S. K.: Katta.V.: Beavi8.R. C.: Chait.B. T.J. A;. Soc. Mass Spectrorn.-1990, i,382-388.' (b)L&, J. A.; Edmonds, C. G.; Smith, R. D.; Lacey, M. P.; Keough, T.Biorned. Enuiron.Mass Spectrorn. 1990,19, 286-294.

Flgure 12. ESI mass spectra of Met-interleukln-2 with ANS of (a) 4-110 V and (b) +260 V. Peaks labeled with 0 are assigned as (b, - MetAla)3+ products, with n = 17-25. For reference, m/z 708 Is the (b2, - MetAlaY+ fragment ion.

+

Dissociation of the (M 9H)g+molecular ion of S-protein (Figure 10)produces the same fragment ions near the proline residues (located close to the COOH-terminus)as found for ribonuclease A. An intense mlz 872 ye with the ha+ complementary counterpart and the y9-b9b8+ pair are the major fragment ions. Ions for a truncated S-protein species (residues 22-124, MI11447) are also observed in the ESI mass spectrum of S-protein (data not shown). MSIMS of its 9+ parent ion produces the bg58+-y8complementaryion pair. The b, product ions are shifted accordingly because of the missing Ser residue (residue 21 of RNase A). CAD of the (M + 4H)4+ ion for S-peptide (M,2166) (Figure 11) yields a distribution of triply charged bl4 to b19 sequence ions, as was observed for ribonuclease A (Figure 9) at the same mlz positions. Loss of 18 Da (H20)from the triply charged b,3+ product ions is readily observed, common for serine- and threonine-containing peptide~.~3 A peptide consistent with residues 1-19 (M, 2095) of RNase A was also detected in the RNase S sample (data not shown). Ita CAD spectrum is similar to S-peptide with the exception of the missing bl++ product ion. Thus, all the ribonuclease A product ions assigned near residue 20 from the "2-terminal can be produced by CAD of the S-peptide, while the y, ions and the more highly charged complementary b, product ions can be formed by dissociation of the S-protein. No additional assignable product ions from the "2-terminal region of the S-protein were found. These results may suggest that higher order structure of the RNase A protein molecule is retained by the S-peptideand S-protein molecular fragmentaand that the outcome of CAD processes may be influenced by such structural consideration. Interleukin-2.The ESI mass spectrum of recombinant interleukin-2 shows two distributions of multiply charged molecules to the 17+ state (Figure 12a). The more abundant


series is due to the protein with the initiator methionine residue present on the "2-terminus (134 residues, M,15 547). The much lower abundant distribution at lower m/z is indicative of the des-methionine form. CAD in the atmospherelvacuum region (ANS = +260V) causes nearly complete dissociation of the 13+ to 17+ molecules and yields a spectrum (Figure 12b) showing several series of multiply charged product ions. Adjacent to the intact molecular ions present, the (M + 12H)12+and (M + 13H)13+species, are intense y13312+ and Y133l3+ contributions, respectively, from loss of the NH2terminal methionine residue (although contribution due to the des-Met form originally present is not excluded). A complementarybl product ion is not observed in the low mlz region of the spectrum. Two overlapping triply charged product ion series are present between rnlz 500 and 1O00, a b1&+ series in which the b213+ and b2$+ ions are unusually intense, and a similar b17-253+set in which the first two residues from the "2-terminus of the protein, Met-Ala, have been removed (e.g., (b21-MetAlaP+). Again, the des-MetAla bz13+ and b2s3+ions are particularly prominent. A singly charged b2 ion at rnlz 203 (data not shown) is observed due to the facile cleavage at proline residue 3. In our studies to date, internal fragment ions, i.e., products from two-bond cleavage processes, can be observed for smaller cyclic peptides (e.g., somatostatin) and other peptides (see earlier section), but are unusual for the larger multiply charged peptides and proteins studied to date. It is likely that the multiple collision conditions which prevail enhance the probability of such reactions. These internal product ions could result from the intact molecule or from the bn3+ions, with subsequent loss of -Met&. However, the lability of the "2-terminal amide bond to a proline residue very near the NH&rminus (position 3) surely increases the chances for such internal fragmentation processes. Tandem mass spectrometry of the individual multiply charged molecular ions (12+ to E+)produces a similar set of triply charged b19-24 ions, a y133 ion with the same charge state as the parent species, and singly charged y24 sequence ions (Figure 13). For example, dissociation of the (M + 13H)13+parent ion yields an abundant y13313+ product ion from the loss of a neutral Met species, blr+243+ions, and weaker contributions from des-MetAla b21#. A bz ion for ionized MetAla is present (mlz 203), but its complementary y13212+ ion is not observed. For interleukin-2 CAD experiments, a proline residue at position 3 causes only internal fragment ions to be formed. Human Growth Hormone. Human growth hormone is a 191-residue protein (M,22 125) secreted by the anterior pituitary gland and plays a role in the promotion of longitudinal bone growth. Recombinant human growth hormone expressed in E. coli is the methionyl analog of hGH (MethGH, M,22 256). ESI mass spectra of hGH and Met-hGH have previously been published.14Je With ANS at +185 V, multiply charged molecules along with lower abundant y1w17-20+ fragment ions from the loss of the first two "2terminal residues (MetPhe)are observed (Figure 14a). These ylw product ions are not observed at ANS below +135 V. The mass spectrum of Met-hGH at a more elevated nozzleskimmer voltage bias (ANS = +260 V) shows more abundant fragmentation processes occurring (Figure 14b). As was observed for interleukin-2, the "2-terminal bond to the proline residue at position 3 is extremely labile. Multiply charged 3+, 4+,and 5+ sequence ions of type b25 to b35, in which the "2-terminal MetPhe residues have been lost, dominate the spectrum (e.g., (b28- MetPhe)3+at rnlz 1006) in addition to an ion at mlz 279 (b2) and ita potential complementary ion, ylgOls-l8+(see Scheme V). Also present at substantially lower abundance are the conventional b2,-3~~+

I

1187

496

lli

I

w

750 mlz

I250

Flgurr 19. ESI-MSIMS spectra of the (a) 13+ and (b) 14+ parent ions of Met-interleukin-2 (see Figure 12 caption).

I

171

I

100- (a) (M+lSH)'*'

I

26

500

700

900 m/Z

1100

1300

+

Flgurr 14. ESI mass spectra of methionyl-hQHwith ANS of (a) 185 V and (b) 4-260 V. Peeks labeled with V are due to ylwd product ions. Peaks labeled a8 4 products are due to Internal fragments from lose of -MetPhe (e.g., bt (ba MetPhe3+).

-

sequence ions (i.e., without the loss of MetPhe). Loss of the terminal methionine residue, observed in the interleukin-2 CAD data, was not observed for Met-hGH. The MS/MS spectra for Met-hGH contain most of the product ions found in the case of dissociation induced in the atmospheric pressurelvacuum interface, with the exception of the b27-32" sequence ions. Nearly all fragment ions are assigned as products resulting from prior cleavage of the Phe

436


12 Yl4l

Y

q

1374

(M+17H)I7

(b)

200

800

600

400

1000

600

1200

800

1000

Fbwe 15. ESIMS/MSspectraofthe(M+17H)17+ionof(a)methbnyC hGH and (b) hW (see Figure 14 Caption).

Scheme V

(4

Met, - Phe Pro - Thr - Ile - Pro - Leu b2 b27

1400

+

Tandem mass spectra of the (M 19H)lS+ ion of (a) methlonyl-hGH and (b) hGH (see Figure 14 caption). Flgure 16,

y190

s

1200

rnlz

m/Z

(M+2H)2+

2 Y7

392

... - A s p 4 Thr

'

2-Pro 3 bond. The bz ion is quite prominent in all tandem mass spectra, as well as ita complementary y1wn-'+ ion. However, formation of ylwn+from loss of a neutral MetPhe species is also pronounced. For example, MSIMS of the (M + 17H)17+molecular species produces a relatively abundant ion at mlz 1294(ylw17+)as well as at mlz 1374 (y1w16+)(Figure 15a). In addition, tandem mass spectrometry of the 18+ and 19+ molecules also produces a series of ions between mlz 1324and 1369,as shown in Figure 16a. These are tentatively assigned as y139-14412+ products that originate near Pro 49 and adjacent to a bridging cysteine residue (a disulfide bond bridges Cys 54 and Cys 166). Similar product ions might have been expected from CAD mass spectra of hGH, with all the "b" product ions shifted accordingly to lower mlz due to the missing "2-terminal methionine residue. However, the CAD results indicate that cleavage of the Phe 1-Pro 2 bond is not favored for hGH. No product ions were observed corresponding to the loss of the "2-terminal phenylalanine residue (e.g., (bze - Phe)3+). Product ions between rnlz 1324 and 1369 are still present in the CAD spectra for the 18+ and 19+ molecular ions of hGH, providing some confirmation for the assignments since these "y" ions were not expected to show a mlz shift. However, the conventional b26-30 ions of 3+, 4+, and 5+ charge states are the major products, with no evidence for ylwn+ ions in the CAD spectrum generated at high ANS and MSIMS of the individual parent ions (Figures 15b and 16b). Thus, we observe that the position of the proline residue has a more pronounced effect on the dissociation pathways for hGH proteins than for interleukin-2. As for the Met-hGH protein, CAD studies of the "2terminal tryptic peptide of Met-hGH (residues 1-9) also show

mlr

(b)

(M+2H)2+ y6 687 I

4 2

+

Flgure 17. ESI-MS/MS spectra of the (M 2H)2+ Ion terminal tryptk peptide from (a) Met-hGH and (b) hGH.

of the NHT

a propensity for the Phe-Pro bond cleavage. Dissociation of the doubly charged peptide, Met-Phe-Pro-Thr-Ile-Pro-LeuSer-Arg (M, 1061)from trypsin digestionof Met-hGH (Figure 17a)produces they7 and^^^+ fragments as the major products, due to cleavage at Phe 2-Pro 3 (see Scheme VI). (Internal fragment ions from loss of MetPhe with subsequent dissociation, observed for Met-hGH proteins, were not observed


Scheme VI Y7

- Thr - Ile - Pro - Leu - Ser - A r g for the "2-terminal tryptic peptide.) The MSIMS mass spectrum of Phe-Pro-Thr-Ile-Pro-Leu-Ser-Arg (M,930 Da) tryptic peptide from hGH (Figure 17b) yields the y6 ion as the most abundant product; the y7 a n d ' y ~ product ~+ ions are less favored. One possible explanation for this exception to the "proline rule" comes from an assessment of likely charge locations. The most likely charge sites for the NH2-terminal tryptic peptide are the COOH-terminal arginine and the "2terminal amino acid residues, although an additional charge site (for example, the proline residue) is necessary for the appearance of the doubly charged yy2+fragmention. Cleavage of the Phe 1-Pro 2 bond to produce y7 (for hGH) may be less likely due to its proximity to the NH2 terminal positive charge site, similar to that describedin the previous section for other small peptides (i.e., LH-RH peptides and substance P (211)).Thus, charge site locations in addition to positions of other important amino acids (i.e., proline) can be highly influential on CAD processes, especially for multiply charged polypeptides. CONCLUSIONS

The majority of polypeptide and protein CAD studies in our laboratory using quadrupole mass spectrometers have not indicated significant intensities for complementary ions; i.e., one ion of the pair is "lost". An interesting exception to this observationfor M,> -5000 Dais observed in cases where fragmentation at particular sites appears facile. The present results show peptide bonds in the vicinity of a proline residue are particularly facile. The fact that complementaryions are observed by mass spectrometryin conjunction with such facile dissociation is consistent with a lower activation energy for this dissociation pathway. Indeed, estimates from Schwartz and Bureey53 indicate a barrier lowering on the order of 0.35 eV for dissociation of the X-Pro bond. If one assumes that peptide bonds adjacent to proline residues cleave at lower levels of molecular excitation than required for other sites, a general explanationcan be advanced for both the observationof complementaryions in such cases, as well as the general absence of other observed dissociation products. The CAD process involves stepwise activation by a large number of low-energy collisions37.BJ7 to quite large levels of external excitation (on the order of 100-1000 eV for proteins on the basis of RRK or RRKM consideration^^^). The extent of "dissociative cooling" upon the first in a series of dissociation steps (toyield complementarypairs) becomes relatively small for large ions; i.e., the "temperature" of a large product ion will approach the same level as that of the parent ion at the large molecule limit. Due to the multiple collision conditions of large-molecule CAD experiments, further collisional activation of the initial (complementary) products will generally occur. If the activation energy for other dissociation processes is similar to that of the initial CAD process, then there should be a substantial probability that the "hot" CAD products will undergo subsequent dissociation. In such a w e , the observation of complementary products may be unlikely. However, if the initial CAD process yielding complementaryproducts is substantially more facile than other dissociation processes, the temperatures of the initial products will be lower. Under such conditions, as (77) Smith, R. D.; Barinaga, C. J.; Udseth, H. R. J.Phys. Chem. 1989, 93, 5019-5022.

437

postulated for dissociations in the vicinity of proline residues, Subsequent dissociation need not occur, since many more collisions will be required to raise the ions' temperatures to the level required for a second dissociation, and the intact complementaryions may then be observed. This explanation is generally consistent with the present experimental results but leaves unresolved the molecular level details as to why sites near certain proline residues are more labile and whether similar effects, perhaps less extreme, are induced by other residues or polypeptide structural elements (e.g., the relative lability of peptide and disulfide bonds). The effect of proline on protein solution-phase conformation has been extensively studied.78~79 Proline commonly occurs within the first three positions of an a-helix. It is less compatible with helical structures because the side chain is bonded to the backbone nitrogen atom, preventing its participation in hydrogen bonding to a neighboring carbonyl group.20 A proline-induced "kink" in an a-helix is often observed in globular proteins, bending a helix by as much as 30°.66 Proline is often involved in reverse turns or 6 turns; the polypeptide chain generally makes a rather sharp bend on the protein surface, reversing the direction of the polypeptide chain.20 Proline often appears near protein surfaces because its structure excludes it from occupying internal positions in a-helices and 6-structures.80 "Proline residues are therefore recognized as being of special significance in their effect on chain conformation and the process of protein folding."79 Clearly, the presence of proline residues has a pronounced effect on collisional dissociation processes of oligopeptides and proteins observed by mass spectrometry. Whether this effect may be influenced to any extent by possible higher order polypeptide conformation arising from noncovalent interactions remains speculative. Preliminary evidence for multiply (positively) charged molecules suggests that fragmentation often originates from regions distant from basic residues. A more systematicstudy is needed to fully elucidate this behavior. The current analytical utility of MSIMS of large (>lo kDa) polypeptides for sequence determinaton is relatively low, especially for species where no information is available (e.g., partial sequence,sequence homology). Large amounts of material, up to 500 pmol in one instance, were consumed to produce the CAD mass spectra shown for the >lO-kDa proteins, although much smaller amounts (

Tandem mass spectrometry of very large molecules. 2. Dissociation of

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