Dissociations of Disulfide-Linked Gaseous Polypeptide

Dissociations of Disulfide-Linked Gaseous Polypeptide/Protein Anions: Ion Chemistry with Implications for Protein Identification and Characterization Paul A. Chrisman and Scott A. McLuckey* 1393 Brown Laboratory, Department of Chemistry, Purdue University, West Lafayette, Indiana 47907-1393 Received August 16, 2002

Ion trap collisional activation of whole protein anions that contain disulfide bonds results in the cleavage of one of the bonds that comprises the disulfide linkage. The disulfide linkage can break at any of three possible locations, giving rise to several products with different partitioning of sulfur atoms. A facile second-generation dissociation occurs at the polypeptide backbone from products formed from cleavage of the nearest C-S bond of a disulfide linkage. This cleavage occurs exclusively at the N-terminal side of the cysteine residue, from which the C-S bond was cleaved, thereby yielding c and z-S type product ions. This secondary reaction is apparently a relatively low-energy reaction with relatively high entropy requirements because it is not observed to be a major process under beamtype collisional activation conditions, but is a major process under ion trap collisional activation conditions. The specificity of this cleavage, as well as the ability to distinguish it from other cleavages by the sulfur atom distribution, make it useful for the identification of unknown proteins via database searching. Furthermore, the pattern of disulfide cleavages can be useful in providing information about the location of post-translational modifications. Examples using bovine pancreatic trypsin inhibitor and ribonuclease A and B are given to illustrate these points. Keywords: negative protein ions • disulfide bonds • ion trap tandem mass spectrometry • top-down protein identification

Introduction Mass spectrometry is playing an increasingly important role in proteomics due to its capabilities for highly specific measurements on relatively low levels of material. The high specificity of mass spectrometry derives from its capability for the accurate measurement of the masses of intact proteins, as well as those of protein fragments. Furthermore, ion chemistry within the mass spectrometer, particularly when the chemistry takes place under well-defined tandem mass spectrometry conditions, can provide valuable information not available from the measurement of the mass of a protein alone. The most common reactions in most protein analyses involve the fragmentation of peptide ions, as in bottom-up protein identification via tandem mass spectrometry,1-5 and protein ions, as in top-down protein identification via tandem mass spectrometry.6-10 Fragmentation reactions provide primary structure information that can be essential in identifying a gene from which a gene product is derived and in characterizing the gene product. Of particular importance in characterizing proteins is the analysis of post-translational modifications. To characterize a post-translationally modified protein via tandem mass spectrometry, therefore, it is important to understand how such modifications affect the dissociation chemistries of peptide * To whom correspondence should be addressed. Phone: (765) 494-5270. Fax: (765) 494-0239. E-mail: [email protected]. 10.1021/pr025561z CCC: $22.00

 2002 American Chemical Society

and/or protein ions. We have been studying the dissociation behavior of disulfide-linked polypeptides and proteins under ion trap collisional activation conditions and have found that multiply charged polypeptide/protein anions undergo specific fragmentation reactions at disulfide linkages. We report here the processes involved and illustrate how the reaction phenomenology might be used in protein identification/characterization scenarios. Disulfide linkages play an important role in stabilizing protein structures so that they can fulfill their biological functions. Techniques have therefore been developed to map disulfide bonds within proteins. From the standpoint of identifying proteins via tandem mass spectrometry (MS/MS) of whole protein ions, however, disulfide bonds can present complications. For example, in top-down6-8 approaches to proteomics, disulfide bonds often limit the range of product ions observed to portions of the protein that are not contained within disulfide linkages, greatly limiting the amount of information that can be derived from these methods.9 This situation tends to prevail for multiply protonated proteins subjected to collisional activation. One approach to circumvent this problem is to reduce the disulfide bonds prior to analysis.9 The resulting protein can then be analyzed using collisional activation and MS/MS as with any other nondisulfide-linked protein. Another approach is to use an alternative ion dissociation method. McLafferty and co-workers have shown that the electron Journal of Proteome Research 2002, 1, 549-557

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research articles capture dissociation (ECD) of multiply charged disulfide-linked protein cations preferentially cleaves disulfide bonds.10 They have used ECD to map the disulfide bonds of viral prolyl 4-hydroxylase.11 They were able to determine the location of the 2 disulfide bonds, despite 10 potential S-S pairs, by measuring the masses of the fragments produced by ECD and identifying fragments containing cysteines whose measured masses differed from those expected by 1 or 2 Da. Most mass spectrometric analysis of proteins and peptides is done in the positive ion mode. One reason for this is the fact that it is generally straightforward to ionize proteins and peptides as positive ions via electrospray. Negative electrospray ionization of proteins and peptides is less facile, and complicated by the possibility of electrical discharge. Another reason is that the dissociation of positive peptides is more widely understood and generally easier to interpret than those of negative peptide ions. Bowie and co-workers, however, have studied negative peptide dissociation extensively and have characterized various major fragmentation mechanisms.12-16 In many cases, as much or more sequence information has been derived from negative peptide dissociation as that from positive peptide dissociation.12-18 The dissociation behavior of multiply charged protein anions has apparently not been studied systematically. We report here the results of ion trap collisional activation of intact multiply charged protein anions that contain disulfide linkages. We observe the facile cleavage of disulfide bonds and, in some cases, a subsequent cleavage of the peptide chain N-terminal to the cysteine residues involved in the disulfide bonds. Previously, selective cleavage at disulfide bonding sites has been reported for singly charged positive ions formed through either fast atom bombardment19,20 or MALDI.21-26 Observation of the cleavage N-terminal to cysteines involved in disulfide bonds has also been reported previously as a product of the dissociation of (M + H)+ of bovine insulin in an ion trap.27 We show here that an analogous process appears to be general for negative disulfide-linked protein ions.

Experimental Section Protein samples were obtained from Sigma (St. Louis, MO). Benzoquinoline was purchased from Aldrich (Milwaukee, WI). All of the samples were used without further purification. Protein and peptide solutions for negative electrospray were prepared by diluting aqueous stock solutions (1-5 mg/mL) to 0.1 mg/mL in 2% NH4OH. The benzoquinoline positive electrospray solution consisted of 0.1 mg/mL benzoquinoline in aqueous 1% acetic acid. Ionization was accomplished with nano-electrospray. Nanoelectrospray emitters were pulled from borosilicate glass capillaries with a 1.5-mm o.d. and a 0.86-mm i.d. using a Sutter Instruments micropipet puller, model P-87 (Sutter Instruments, Novato, CA). The nano-electrospray assembly consists of an electrode holder (Warner Instruments, P/N ESW-MISP, Hamden, CT) with a stainless steel wire that is inserted into the capillary.28,29 The voltage applied to the wire for negative nanoelectrospray was typically about -1.35 kV. To simplify the analysis of the product ions formed in an ion trap, we utilize ion/ion reactions in a manner analogous to that which has been reported previously for positive ions, i.e., proton transfer reactions to produce predominantly singly charged products.30 The instrument used for experiments in which ion/ion reactions were employed was a Finnigan-MAT (San Jose, CA) ion trap mass spectrometer, which had been 550

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Chrisman and McLuckey

modified to allow for the injection of ions of opposite polarity, generated by two different electrospray sources, through a hole in the endcap electrode, and has been described previously.31 Helium (1 mTorr) was used as a target gas for collisional activation. Typically, experiments consisted of a protein anion accumulation time (100-300 ms), followed by isolation of the ions of interest (200 ms), collisional activation of the isolated ions (300 ms), cation accumulation (200-400 ms), mutual storage time to allow for ion/ion reactions (400-800 ms), and finally mass analysis. Ion/ion reactions were used to simplify spectral interpretation and consisted of proton transfer between protonated benzoquinoline and the multiply deprotonated proteins. The cation accumulation and mutual storage times were adjusted so that the anions would be predominantly in the -1 charge state. Collisional activation was accomplished with single frequency resonant excitation. The mass analysis was performed using resonance ejection to extend the mass range of the ion trap.32 The product ion spectra shown are typically the average of 200 to 500 scans. The calibration of post-ion/ion product ion spectra was accomplished using anion charge states of either bovine insulin or bovine cytochrome c that were charge-reduced to cover the mass-to-charge ratio range desired. Selected experiments were also performed on a PE Sciex QSTAR Pulsar i (Toronto, Ontario, Canada) to take advantage of the higher mass accuracy and resolving power offered by this instrument. On the ion trap used, determination of ( 1 Da mass differences is difficult for the relatively high-mass species involved in this study. The time-of-flight mass analyzer of the QSTAR, on the other hand, easily provides the necessary resolution and accuracy. Two different forms of collisional activation are accessible on this instrument. Beam-type collisional activation can be accomplished by the acceleration of ions of interest into the collisional cell, where they collide with the target gas (nitrogen) and fragment, and are then pulsed into the TOF analyzer for mass analysis. The energy is controlled by the potential difference between Q0, an RF-only quadrupole used for collisionally cooling and focusing the ions, and Q2, the collision cell. The pressure in the collision cell is in the range of 2-10 mTorr, and the collision energies ranged from 30 to 40 eV. The instrument has been modified so that the collision cell can be operated as a linear ion trap, enabling the second type of activation, ion trap collisional activation. The activation is accomplished by the application of a dipolar excitation frequency that is resonant with the ion of interest, and is analogous to single frequency resonant excitation in a 3-D trap.33-35 The ions are accumulated and stored in the collision cell, an excitation waveform generated by an arbitrary waveform generator (Agilent Technologies, Loveland, CO) is triggered resulting in the acceleration and consequent collisioninduced dissociation of the ions, and the ions are then allowed to travel into the TOF mass analyzer for mass analysis.

Results and Discussion Reaction Phenomenology. To illustrate the dissociation phenomenology associated with collisional activation of disulfide-linked polypeptide/protein anions, it is useful to describe data obtained largely with ions comprising two polypeptide chains bound by one or more disulfide linkages. We therefore present data for partially reduced somatostatin and bovine insulin. Somatostatin is a small 14 amino acid peptide that contains one disulfide bond. It can be partially digested with trypsin, resulting in cleavage between Lys-9 and Thr-10. This

Disulfide-Linked Polypeptide/Protein Anions

Figure 1. Product ion spectrum for the collisional activation of the (M - 2H)2- ion of partially digested somatostatin on the QSTAR using a collision energy of 30 eV. Scheme 1. Diagram of the Products that Result from Cleavage of the Disulfide Bond at Each of the Three Possible Locations

produces a peptide consisting of two separate amino acid chains linked by a disulfide bond. Figure 1 shows the results of beam-type collisional activation of the (M - 2H)2- charge state of this peptide acquired with the QSTAR instrument (see the Experimental Section). Beam-type collisional activation refers to the injection of a beam of parent ions into a collision region containing a target gas.36 Most beam-type collisional activation experiments, including that of the QSTAR, select from fragmentation reactions with rates greater than about 104 s-1. The main product ions, in this case, are the two separate peptide chains, with 0, 1, or 2 sulfurs, such that a set of three products is observed for each chain. This is a result of the breaking of either the S-S bond or either of the two C-S bonds (as illustrated by Scheme 1). An examination of the masses of the products shows that this cleavage is heterolytic. In conjunction with the bond breaking is a hydrogen-atom transfer between the two chains. When cleavage occurs so that one chain contains both sulfurs, a hydrogen atom is transferred from the sulfur-less chain. Further, when cleavage occurs between the sulfurs, chain 2 (TFTSC) receives a hydrogen atom from chain 1 (AGCKNFFWK). The isotopic ratios of the products (shown in Figure 2) show that this process is essentially quantitative in this case. There is no evidence that the products are mixtures of different atomic compositions. The preference for hydrogen transfer from chain 1 to chain 2 upon cleavage between sulfurs may be an exceptional case with this system (see below). Perhaps the fact that the cysteine of chain 2 is

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Figure 2. Selected regions of the product ion spectrum for collisional activation of the (M - 2H)2- ion of partially digested somatostatin on the QSTAR. The atomic composition for each product is shown. The atomic composition listed as “expected” would be that predicted from a homolytic cleavage between the two sulfur atoms of the disulfide bond.

Figure 3. Product ion spectrum for the collisional activation of the (M - 4H)4- ion of bovine insulin obtained on the QSTAR with a collision energy of 40 eV.

next to the C-terminus, which is expected to be deprotonated in the doubly charged anion, plays a role in inhibiting hydrogen transfer from chain 2 to chain 1. A more complicated case is presented by bovine insulin, which consists of two peptide chains bound together by two disulfide bonds, in addition to an internal disulfide bond on the A chain. Figure 3 shows the results for beam-type collisional activation of (M - 4H)4- bovine insulin, also obtained with the QSTAR instrument. The results are very similar to those seen for the partially digested somatostatin, with the main products being the peptide chains. Due to the presence of multiple disulfide bonds, each chain can be composed of a wider range of sulfurs than is possible in the somatostatin case. The B chain, which is involved in two disulfide bonds, can have from 0 to 4 sulfurs, whereas chain A has, in addition, an internal disulfide bond and, thus, an additional two sulfurs. Examination of the masses of the products reveals that hydrogen atoms are transferred from one chain to the other during fragmentation. Examination of the abundance ratios of the isotopes of the product ions (Figure 4) indicates that, in this case, the products are mixtures of several different atomic compositions. This is expected because of the different routes by which a given number of sulfurs can result. For example, chain B could have Journal of Proteome Research • Vol. 1, No. 6, 2002 551

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Figure 4. Isotopic distributions of the -2 B chain products produced from the collision-induced dissociation of the (M - 4H)4- ion of bovine insulin containing (a) 0 sulfur atoms, (b) 1 sulfur atom, (c) 2 sulfur atoms, and (d) 3 sulfur atoms. Expected isotopic distributions for the product containing the given number of sulfur atoms and 226, 228, or 230 hydrogen atoms are shown.

two sulfurs due to cleavage between the sulfurs on both disulfide bonds, cleavage after the sulfurs on one bond, or cleavage before the sulfurs on the other bond. The isotopic distributions observed, however, cannot be accounted for if cleavages between the sulfurs always proceed with hydrogen transfer from the same chain. In this case, unlike that of partially reduced somatostatin, there appears to be no preference for which chain supplies the transferred hydrogen. Data collected using beam-type collisional activation in the QSTAR instrument is useful in drawing conclusions about cleavages along the disulfide linkage because the resolving power and the mass measurement accuracy of the time-offlight analyzer are sufficiently high to easily distinguish products differing in mass by 1 Da. The beam-type collisional activation experiments, however, did not show the prominent secondgeneration fragmentation reaction of the polypeptide backbone that was observed under ion trap collisional activation conditions. Ion trap collisional activation tends to select for dissociation rates of 10 to 100 s-1. That the low contribution from sequential fragmentation in the beam-type collisional activation experiment is due to ion activation conditions can be evaluated with the QSTAR instrument, which is modified to allow for ion storage in the collision cell and for the application of a relatively low amplitude dipolar excitation of the ions stored in the collision cell. Figure 5 compares portions of the product ion spectra (m/z 700-825) collected from the (M - 2H)2- parent ion of native somatostatin under ion trap collisional activation (Figure 5a) and beam-type collisional activation conditions (Figure 5b). By far, the major product ion observed in Figure 5a corresponds to a doubly charged z12 ion. A variety of product ions are observed in the same mass-to-charge range in the beam-type collisional activation experiment. These products, which include losses of water and acetaldehyde (from threonine) and/or carbon dioxide, have been reported previously from studies of collisional activation of polypeptide anions.13 Losses of one or more molecules of H2S2 are also observed. The z122- product, which is so dominant in Figure 5a, is barely observed in the spectrum of Figure 5b. This comparison, made with the same parent-ion population in the same apparatus, 552


Figure 5. Comparison of (a) ion trap collisional activation of the (M - 2H)2- ion of native somatostatin at 37 800 Hz and 950 mV for 300 ms and (b) beam-type collisional activation of the same ion with a collision energy of 30 eV. The loss of 44 Da can correspond to loss of CO2 from the C-terminus or loss of CH3CHO from threonine.

suggests that the process leading to the z122- product is likely to have a relatively low energy requirement but is a kinetically slow process that is discriminated against in the beam-type collisional activation experiment. The data of Figure 5a also shed light on the nature of the sequential decomposition products. The mass of the z122- ion is consistent with the process shown in Scheme 2. Note that the products are nominally c- and z-type products because of the location of the cleaved bond along the peptide chain. However, these ions are not identical to those formed from ECD. In the latter case, for example, the z-type ions are radical species. In the former case, the z-type ions are even-electron and are specific to the cysteine side chain. Figure 6 shows the results of collisionally activating the (M - 4H)4- parent ion of bovine insulin in the ion trap and reducing the product ions predominantly to the -1 charge state


Figure 6. Post-ion/ion product ion spectrum for activation of the (M - 4H)4- ion of bovine insulin in a quadrupole ion trap at a frequency of 89 100 Hz and an amplitude of 250 mV for 300 ms. Scheme 2. Formation of c- and z-S Ions N-terminal of a Cysteine Residue that Has Had the C-S Bond Cleaved

via ion/ion proton-transfer reactions. The results are similar to those observed with the beam-type collisional activation experiment conducted with the QSTAR, with the exception that products associated with backbone fragmentation, labeled as B(z12-S), B(c18), and B(z24-S), are apparent, where B indicates that the products are formed from the B-chain. These products are expected to arise from the same mechanism that gave rise to the z12 product ion from somatostatin (see above). Similar dissociation behavior has been observed in the ion trap collisional activation of the +1 charge state of bovine insulin.27 In that study, it was suggested that the z-S ions were formed from further fragmentation of the polypeptide containing a cysteine that had lost its sulfur via cleavage of the cysteine C-S bond. Interestingly, more highly charged insulin parent cations (e.g., (M + 5H)5+-(M + 3H)3+) fragment almost exclusively to yield the b-and y-type ions commonly observed in dissociation of protonated peptides and proteins. It was postulated that, in the case of the (M + H)+ parent ion, the excess proton was sequestered on the lone arginine of insulin and was therefore not available to catalyze fragmentations leading to b- and y-type cleavages.37 In such a scenario, cleavages along the disulfide linkages are favored. The sequential fragmentation products were consistent with sequential fragmentation Nterminal to the cysteine that had lost its sulfur in the course of disulfide linkage dissociation. The overall process is not expected to require charge-site involvement. Hence, a similar process is expected to be taking place with the multiply charged anions. In this case, the negative charges are expected to be

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Figure 7. Results of MS3 experiments performed on the -2 B chain products of dissociation of the (M - 4H)4- ion of bovine insulin containing (a) 1 sulfur atom, (b) 2 sulfur atoms, and (c) 3 sulfur atoms. The spectra shown are post-ion/ion spectra, i.e., the charge states of the product ions have been reduced to primarily the -1 charge state.

locked down primarily at carboxylate groups, where they can play, at most, an indirect role in influencing the disulfide linkage and subsequent cleavages. It was not possible in the (M + H)+ bovine insulin study to determine if the disulfide linkage cleavages were heterolytic or homolytic. The QSTAR data acquired here show clearly that, at least for the anion case, the cleavages are heterolytic. This process, indicated in Scheme 2, can account for all of the c- and z-nS ions observed with the (M + H)+ insulin ion and all of the parent anions described in this study. Scheme 2 shows that the first step in the process involves cleavage of the C-S bond of a cysteine, followed by formation of the complementary c- and z-S ions. (Note that in the case of a single chain disulfide linked polypeptide, like native somatostatin, the mass of the z-type ion does not show a sulfur mass decrement because the sulfur lost from the cysteine is carried away by the other cysteine of the disulfide linkage.) Although the possibility for cleavage of the backbone after cysteine bond cleavage between the sulfurs or at the far C-S cannot be categorically precluded, a series of MS3 experiments involving several of the doubly charged B-chain product ions from insulin show that the process of Scheme 2, in some cases following the loss of one, two, or three sulfurs from the B-chain ion, likely accounts for all of the major structurally informative products. Figure 7 shows the results of collisional activation of the 1S, 2S, and 3S versions of the B-chain dianion formed from dissociation of the insulin (M - 4H)4- ion. The 1S B-chain ion population is expected to be comprised of two isomers, one in which Cys-7 has one sulfur and Cys-19 has no sulfur, and one in which Cys-7 has no sulfur and Cys-19 has one sulfur. Neither a z12 nor a c18-S ion is observed in the MS3 spectrum of this ion (Figure 7a). This complementary pair would be formed from the cleavage N-terminal to Cys-19 if the cysteine retained the sulfur. This result could arise if there were none of this isomer or, more likely, that the N-terminal cysteine cleavage is favored at the cysteine without the sulfur. In the case of the 2S B-chain dianion (Figure 7b), three isomers can be present. These include one form with Cys -7 having two sulfurs and Cys-19 having none, one form with Cys19 having two sulfurs and Cys-7 having none, and one form Journal of Proteome Research • Vol. 1, No. 6, 2002 553

research articles with one sulfur on each cysteine. The absence of either a z12 ion or a z12+S ion again suggests that the backbone cleavage is preferred when no sulfur is present at the cysteine site involved in the reaction. The presence of the c18 and c18-S ions could arise from cleavages at cysteines with one or two sulfur atoms, respectively. However, given that only the z12-S ion is observed and that strong signals associated with losses of one and two sulfur atoms from the 2S B-chain ion is observed, it is likely that the c18 and c18-S ions are formed after loss of one and two sulfurs, respectively. Further evidence that this is the case comes from the appearance of z24-S and z24-2S ions, which must be formed in association with losses of one and two sulfurs, respectively. Analogous observations can be made for the 3S B-chain dianion population (Figure 7c), which can be comprised of two different forms. These include one in which Cys-7 has two sulfurs and Cys-19 has one sulfur, and one in which Cys-7 has one sulfur and Cys-19 has two sulfurs. Like the 1S ion, the 3S B-chain ion shows the z12-S and c18 combination as the only complementary pair from cleavage N-terminal to Cys-19. This is most readily rationalized by the initial loss of a pair of sulfur atoms prior to backbone cleavage. The loss of two sulfur atoms is the base peak in the MS3 spectrum. Furthermore, no z24 ions are observed, despite the fact that this is the expected product if all three sulfur atoms are retained on the B-chain. Rather, z24-S and z24-2S ions, which can only arise in conjunction with multiple sulfur atom losses, are observed. This result, in conjunction with the observations made with the other B-chain ions, suggests that most, if not all, backbone cleavages arise from the process represented by Scheme 2. Application to Single-Chain Disulfide-Linked Proteins. Data collected with two polypeptide chains bound by one or more disulfide linkages are useful in determining the underlying processes giving rise to c- and (z-S)-type ions. Many disulfidelinked systems, however, are composed of a single polypeptide chain. In these cases, the masses of the complementary ions formed from the N-terminal cysteine cleavage discussed above correspond to c- and z-type ions, rather than to c- and (z-S)type ions. This is simply due to the fact no sulfur atoms are lost as a result of a cysteine bond cleavage when no separation of polypeptide chains occurs. However, on the basis of the results related above, it is likely that the cysteine at which the polypeptide chain is broken is devoid of its sulfur atom. Several single-chain disulfide protein anions, ranging in size from turkey ovomucoid third domain (5.6 kDa) to porcine trypsin (23.5 kDa), have been subjected to ion trap collisional activation. A subset of results is presented here to illustrate the general phenomenology. Bovine pancreatic trypsin inhibitor (BPTI), a 6.5 kDa protein that contains 3 internal disulfide bonds, provides a good example of typical fragmentation behavior. Figure 8 shows the results of collisional activation of the (M - 4H)4- ion of BPTI in the ion trap, and reducing the product ions to primarily the -1 charge state. The products observed are those arising from one or more neutral sulfur losses from the precursor ion, as well as c- and z-type ions with varying numbers of sulfurs. Each c and z ion has a distribution of product ions containing various numbers of sulfurs. This arises from the presence of multiple disulfide bonds and the possibility that several of them can fragment before the polypeptide chain cleaves. Cleavage is clearly observed at 3 of the 6 cysteines present in BPTI in this charge state, with evidence for cleavage at a fourth cysteine in the region expected for z54-. Too little information is in hand to draw conclusions 554



Figure 8. Post-ion/ion product ion spectrum for the activation of the (M - 4H)4- ion of bovine pancreatic trypsin inhibitor at 89 450 Hz with an amplitude of 700 mV for 300 ms.

Figure 9. Post-ion/ion product ion spectra for the activation of several charge states of bovine pancreatic trypsin inhibitor (a) (M - 5H)5- ion activated at 89 250 Hz, 450mV, 300 ms; (b) (M 3H)3- ion activated at 59 470 Hz, 1200 mV, 300 ms; and (c) (M 2H)2- ion activated at 58 950 Hz, 1280 mV, 300 ms.

about why c and z ions are observed from some, but not all, disulfide linkages. Perhaps some disulfide-linkages are particularly stabilized by intramolecular interactions and do not compete with the dissociation of other disulfide-linkages. Or, perhaps the polypeptide chain near particular cysteines is stabilized thereby inhibiting the cleavage of the backbone. Furthermore, there may be nearest neighbor effects that influence the backbone or disulfide-linkage cleavages. More experience with these systems may shed further light on these possibilities. The fragmentation of multiply charged protein cations is known to be highly dependent upon parent ion charge state.38-40 This dependence is believed to arise, in part, from the effect of total cation charge on proton mobility.37 In the case of protein anions, charge migration along the backbone is not expected to influence directly the fragmentation of disulfide linkages. However, the charge state of the anion can, nevertheless, influence at least indirectly the observed fragmentation behavior. Figure 9 shows the post-ion/ion reaction MS/MS spectra of the (M - 5H)5- (Figure 9a), (M - 3H)3(Figure 9b), and (M - 2H)2- (Figure 9c) anions derived from BPTI. The (M - 5H)5- ion clearly shows a signal corresponding


Figure 10. Post-ion/ion product ion spectrum for the activation of the (M - 7H)7- ion of ribonuclease A at 59 450 Hz with an amplitude of 980 mV for 300 ms.

to the z54- ion but does not show signals associated with z29and c29- ions, as observed in the (M - 4H)4- data. The most striking trend in charge state fragmentation behavior is the dramatic decrease in contributions from backbone cleavage as charge state decreases below -4. Instead, the loss of one or more sulfurs tends to dominate the spectrum. This effect is likely due to the role that the Coulomb field plays in facilitating second-generation fragmentation. The Coulomb field can affect the extent of intramolecular hydrogen bonding by inhibiting folding. Furthermore, if charge is present on both sides of a given cysteine, cleavage of the backbone after loss of the cysteine sulfur can be facilitated by Coulomb repulsion. The predominance of the losses of relatively small neutral molecules from precursor ions of relatively low charge parents has also been noted for low charge state positive protein ions,38-40 at least under ion trap collisional activation conditions. It is clear that disulfide linkages in a protein can play a dramatic role in the appearance of the product ion spectra of the anions. It is of interest to determine how the presence of other post-translational modifications, in addition to disulfide linkages, might affect protein anion dissociation. As an initial step in addressing this question, we have examined ribonuclease A and B. Ribonuclease A is a 13.7 kDa protein that contains four internal disulfide bonds. The results of collisionally activating the (M - 7H)7- ion of ribonuclease A, and reducing the products primarily to the -1 charge state, are shown in Figure 10. At this mass-to-charge ratio range, the instrument is no longer capable of resolving the various sulfur containing ions of a given fragment type, so that relatively broad, single peaks are observed. In this case, cleavages at four of the eight cysteines are noted. Examination of other charge states yielded one more cleavage (Ala-109/Cys-110 in the (M - 5H)5- charge state, data not shown). Of the other three cysteines in ribonuclease A, the masses of the fragment ions expected for 2 of them have masses that would not be resolved from the [M-2H]2- ion, and the fragments expected for the final cysteine would not be resolved from the [M-H]- ion, so it is possible that these are occurring, and just not observable. Ribonuclease B is ribonuclease A with an N-linked sugar attached to Asn-34. Figure 11 shows the results of collisionally activating the (M - 8H)8- ion of ribonuclease B and reducing the product ions to the predominantly -1 charge state. The fragmentation shows that the sugar remains linked while the cysteine cleavages occur. The data are sufficient to locate the sugar within a 14 amino acid region, whereas CID of positively

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Figure 11. Post-ion/ion product ion spectrum for the activation of the (M - 8H)8- ion of ribonuclease B at 59 350 Hz with an amplitude of 1260 mV for 300 ms.

charged ribonuclease B was only able to locate the sugar to within a 23 amino acid region.41 The formation of c- and z-type ions from disulfide-linked protein anions appears to be a general phenomenon, at least when the charge states are sufficiently high to observe sequential fragmentation products, as all of the disulfide-containing proteins examined thus far have exhibited these cleavages. The fact that it is highly specific, occurring only N-terminal of cysteine residues, makes it potentially useful in proteomics applications. Furthermore, it is generally straightforward to differentiate this type of cleavage from other cleavages by the distribution of sulfur-containing products. The strong preference for disulfide cleavage and subsequent fragmentation to yield c- and z-type ions in gas-phase protein anions is somewhat analogous to the specificity of enzymatic cleavage of proteins in the condensed-phase. Hence, an approach somewhat similar to peptide mass fingerprinting42-46 can be envisioned, with the exception that, in this case, all dissociations take place within the mass spectrometer (i.e., it is a topdown approach). With peptide mass fingerprinting, a protein is enzymatically digested in the condensed-phase, and then the masses of the resulting peptides are determined mass spectrometrically. The specificity of the enzyme allows these masses to be compared to the expected products of a protein sequence contained in the database. In the approach envisioned here, fragment ions are generated from the intact protein within the mass spectrometer, and their masses can be searched against those of sequences contained in a database, restricting the search to cleavages N-terminal of cysteine. Attractive aspects of the approach derive from the fact that this is a top down methodology and include the ability, in principle, to examine complex mixtures, not having to digest the protein, and being able to determine the mass of the intact protein. In addition, when a protein is enzymatically digested, all of the peptides expected are not typically observed. This means that some parts of the sequence are not accessible. When looking for post-translational modifications, this loss of sequence coverage can be a problem, as the portion that contains the post-translational modification may not be observed. Any strategy that examines whole protein ions does not suffer from this problem, as the whole sequence is present for the analysis. As an example of how the database searching might work, the BPTI data contained in Figure 8 was used to search with Journal of Proteome Research • Vol. 1, No. 6, 2002 555

research articles an in-house database search program.47 In the case of an unknown protein, which particular product in the sulfur distribution is the c or z-S ion is not known, so all the product masses were included in the search (resulting in a total of 20 fragment masses). A mass tolerance of ( 5 Da on the product ions and ( 10 Da on the parent ion was used. Searching against the entire database (SWISS-PROT, release 40.0 modified by removing signal peptides and propeptides48) for fragments that were produced by cleavages N-terminal to cysteine, BPTI was selected as the best match, matching 5 of the products (the sixth cleavage was 6 Da off of the expected mass). The greatest number of matches that any other protein scored was 2. So even with this fairly rudimentary database search, BPTI was identified. Steps to improve the scoring of the database search can easily be envisioned. For example, in addition to searching for the c and z ions, the program could also look for matches corresponding to c- and z-type ions with up to ( 2 sulfurs. Random matches would still match some of those as well, but would be less likely to be centered on the distribution, thereby resulting in a more specific search.

Conclusions Ion trap collisional activation of multiply charged disulfidelinked protein anions results in the strongly preferred cleavage of the disulfide bonds. This cleavage is then often followed by a facile second-generation cleavage of the protein backbone N-terminal to the cysteine residues. It appears that this cleavage occurs primarily following cleavage of the C-S bond of the cysteine residue. Evidence suggests that it is a slow process, as it has so far been observed to be prominent only in trapping experiments, and not in beam experiments. It is also highly specific, in that it is almost the only backbone cleavage that occurs for these anions. The highly specific nature of the cleavage, as well as the ability to easily differentiate it from other cleavages that might occur by the presence of a distribution of sulfur peaks make it potentially very useful for protein identification with database searching. Even with a rudimentary search program and fairly wide mass tolerances, BPTI was easily identified from the data herein. The identification strategy outlined here shares commonalities with peptide mass fingerprinting, but features all the potential advantages inherent to a top-down approach, including the reduced sample processing required and the ability to determine the mass of the intact protein. Improvement of the database searching program is one area of ongoing work, as well as attempting to obtain similar data on an instrument capable of providing higher mass accuracy and resolving power.

Acknowledgment. The National Institutes of Health, Grant No. GM 45372, supported the research. The authors acknowledge Dr. J. Mitchell Wells, Dr. Gavin E. Reid, and Dr. Ethan R. Badman for helpful discussions and Jason M. Hogan for assistance in evaluating the possibility of database searches based on the chemistry outlined in this paper. The authors also acknowledge MDS-Sciex for providing use of the QSTAR Pulsar i as part of the Purdue Department of Chemistry Industrial Associates Program. References (1) Aebersold, R.; Goodlett, D. R. Chem. Rev. 2001, 101, 269-295. (2) Pandey, A.; Mann, M. Nature 2000, 405, 837-46.

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Dissociations of Disulfide-Linked Gaseous Polypeptide

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