Anal. Chem. 1995, 67, 2498-2509
Characterization of Cytochrome c Variants with High=ResolutionFTlCR Mass Spectrometry: Correlation of Fragmentation and Structure QinyuanWu, Steven Van Olclen, Xueheng Cheng, Ray Bakhtiar, and Richard D. Smith*
Chemical Sciences Department and Environmental Molecular Sciences Laboratory, Pacific Northwest Laboratory, Richland, Washington 99352
The dissociation of cytochrome c ions (15+ charge state) generated by electrosprayionization has been studied by Fourier transform ion cyclotron resonance mass spectrometry (FI’ICR)using a sustained off-resonanceh d i a tiodcollision-induceddissociation (SORI-CID)technique. Over 95%of the fragmentions can be accurately assigned (to better than 10 ppm), yielding information on the primary sequences of the various proteins. Up to four stages of mass spectrometry (MS4) have been achieved without the need for quadrupole excitatiodcollisional cooling of the product ions. The subtle structural differences among the cytochrome c variants (from bovine, tuna, rabbit, and horse) are clearly reflected in their fragmentation patterns: replacing 3 out of 104 residues of the cytochrome c is shown to dramatidly change the dissociation pattern. Of particular importance are a variety of results indicating that the dissociation of the cytochrome c’s is influenced by higher-order structure and charge location, in addition to the primary structure (Le., sequence). No m e n t a t i o n is observed in the region between residues 10-20 and little dissociation between residues 70-90. This is most likely due to the interactions of the heme group with the polypeptide chain, and such a heme “footprinting” pattern is analogous to the protein conformation in solution. These studies demonstrate that electrospray ionization-FI‘ICR using SORI-CIDcan be a useful tool to probe not only the small differences in the primary sequences of proteins but also suggest the potential for probing their higher-order structures and yielding information not readily available from H/D exchange or circular dichoism studies. Characterizationof proteins of similar structures is important to understanding the biological function of the proteins and the processes with which they are involved. In a living organism, proteins are synthesized on the basis of their cDNA sequence. However, many proteins undergo various degrees of posttranslational modifications after synthesis to generate a class (or classes) of proteins of slightly different primary structures. It is therefore necessary to determine the identity and location of these small structural variations in order to understand their biological functions. Similarly,it is common for different organisms to have proteins of high homology (i.e., sequence similarity), often differing by only a few amino acid residues. One such protein is cytochrome c, a peripheral protein bound to the mitochondrial membrane, which is involved in the respiratory chain of all aerobic 2498 Analytical Chemistry, Vol. 67, No. 14, July 75, 1995
organisms. In general, cytochrome c has an iron-containig heme group covalently bound to two cysteines in a polypeptide chain of -100 residues. Cytochrome c variants typically have similar conformations in solution and exhibit almost identical absorption spectra and redox potentials.’ These variants were also chosen as a model system in the development of electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry @SI-FTICR) for the structural analysis of large biomolecules due to their previous broad use in ESI-MS and their ready availability in high purity. There have been extensive studies of various cytochrome c The primary sequences of many variants have been well determined, and three-dimensional structures of horse heart and tuna heart cytochrome c have been elucidated using X-ray diffraction: Circular dichoism (CD) , magnetic circular dichoism (MCD) and nuclear magnetic resonance (NMN have been applied to study their tertiary structures in s ~ l u t i o n . ~Mass ~ ~ ~spectrometry * provides molecular weight information with high sensitivity and is particularly useful for the analysis of mixtures (or impure samples). The electrospray ionization process has served to extend mass spectrometry into the realm of large molecules, including proteins, large oligonucleotides, and other large biological and synthetic p01ymers.l~Charge state distributions in mass spectra of electrospray-generated cytochrome c ions have been shown to be related to conformations in s o l ~ t i o n .Hydrogen/ ~~~~ deuterium exchange has been combined with mass spectrometry (MS) to explore the conformational relationshipbetween solution and gas An attractive feature of the ESI process is that it is amenable to a wide range of solution conditions, potentially allowing a wide range of solution-derived structural (1) Stryer, L., Ed. Biochemisty, W. H. Freeman and Co.: New York, 1988. (2) Theorell, H.; Akesson, A]. Am. Chem. SOC.1941,63, 1804-1820. (3) Knapp, J. A; Pace, C. N. Biochemisty 1974, 13, 1289-1294. (4) Takano, T.; Kallai, 0. B.; Swanson, R; Dickerson, R E.]. Biol. Chem. 1973, 218, 5234-5255. (5) Goto, Y.; Takahashi, N.; Fink, A L. Biochemistry 1990, 29, 3480-3488. (6) Dyson, H. L.; Beattie, J. K.J. Bid. Chem. 1982, 257, 2267. (7) Hawkins, B. IC;Hilgen-Willis, S.; Pielak, G. J.; Dawson, J. H. J. Am. Chem. SOC. 1994, 116, 3111-3112. (8) Mcdonald, C. C.; Phillips, W. D. Biochemisty 1973, 12, 3170-3186. (9) Chowdhury, S. K; Katta, V.: Chait, B. T. J. Am. Chem. SOC.1990, 112, 9012-9013. (10) Smith, R. D.; Loo, J. A; Barinaga, C. J.; Edmonds, C. G.; Udseth, H. R. J. Am. SOC.Mass Spectrom. 1990, 1 , 53-65. (11) Suckau, D.; Shi, Y.; Beu, S. C.; Senko, M. W.; Quinn, J. P.; Wampler. F. M.; McLafferty, F. W. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 790-793. (12) Wagner, D. S.: Anderegg, R J. Anal. Chem. 1994,66, 706-711. (13) Winger, B. E.; Light-Wahl, K J.; Smith, R. D. J. Am. SOC.Mass Spectrom. 1992,3, 624-630. (14) Smith, R D.: Loo, J. A; Loo, R. R. 0.; Busman, M.; Udseth, H. R Mass Spectrom. Rev. 1991, 10. 359-451. 0003-2700/95/0367-2498$9.00/0 0 1995 American Chemical Society
effects to be examined by mass spectrometry. Retention of some elements of higher-order structure in the gas phase has been indicated by the detection of noncovalent complexes by mass spectrometry when electrosprayed from solution in which they are known to exist.'5 It has also been found that structural d ~ e r e n c e sin solution can be manifested by differences in the charge state distributions for the resulting mass spectragJ6and in the levels of H/D exchange.11J7J8 However, there is yet no clear evidence regarding the similarity of the gas and solution phase three-dimensional molecular structures from these studies (e.g., the interactions between the subunits of a protein). Primary structural information of the gas phase ions may be obtained by using tandem MS methods. One of the major attributes of the ESI process for large molecules is that their proportionally high charge states make dissociation reactions more facile. Multiply charged biomolecular ions can be effectively fragmented by adjusting voltage gradient, capillary temperature, and pressure at the atmosphere/vacuum interface14 or by collisions with gaseous targets in the mass spectrometer (collisioninduced dissociation, CID) . Our laboratoryfirst demonstrated that large molecules could be efficiently dissociated by the use of high electric fields in the ESI interface,lga process driven by the very large number of low-energy collisions in this region. Our initial reports of tandem mass spectrometric studies for large multiply charged molecules showed that these large ions could be effectively dissociatedz0and that an abundance of structurally related information was ~ b t a i n a b l e . ~ O *The ~ ~ cross - ~ ~ sections for collision-induceddissociation for cytochrome c was shown to be quite large (>lo3 Az) compared to singly charged species of comparable mass to charge ratios ( ~ / Z ) , ~ Oand dissociation in triple quadrupole mass spectrometers was shown to be the result of a large number of low-energy collisions ('5 eV center of mass frame). The CID efficiency for such large multiply charged ions was also shown to be much higher than the singly charged ions10~20~z1 and dependent upon the ESI interface condition^.^^ Early work examined the 15+ states of nine cytochrome c variants using CID in a triple quadrupole instrument.'O It was demonstrated that these variants had distinctive, sometimes subtle, CID fragmentation patterns. However, the fragment ion peaks in the lowresolution spectra were complex and the spectra could not be interpreted in terms of amino acid sequence. While molecules as large as albumin (-66 kDa) have now been shown to yield significant "sequencerelated" informati0n?~95%in oxidized form, purchased from Sigma, St. Louis, MO) were dissolved in 5%HOAc solutionsto concentrations of 1.5 mg/mL. The solution were infused into a modiiied Analytica (Branford, CT) ESI source, which included a stainless steel desolvation capillary and a skimmer cone.33 Typically, the infusion rate was 0.5 pL/min as controlled by a Harvard Apparatus (South Natick, MA) syringe pump. The electrospray source voltage was 3.5 kV. The capillary was biased at 180 V and resistively heated with -18 W power. Ions were transported to the FTICR cell through the magnetic field gradient using two sets of rf-only quadrupoles. After exiting the ESI source, the ion beam passed through a mechanical shutter assembly into the first section of the rf-only quadrupole ion guide. The first section of quadrupole guide was 15 cm long and terminated at a second shutter assembly, When in their “open” positions, each shutter constituted a 3 mm conductance limit. The second section of the rfonly quadrupole guide was 100 cm in length and extended from the second shutter to the trapped ion cell, which was positioned in the center of a 7 T superconducting magnet. The trapped ion cell used in these studies measured 5 cm x 5 cm x 7.6 cm in length and had holes in the front and rear trap plates of approximately 3 and 6 mm diameter, respectively. A typical experimental sequence is illustrated in Figure 1. Ions were injected for 200 ms and trapped in the cell using pulses of dry nitrogen (peak pressure -10-5 Torr) (introduced through a piezoelectric valve) and differential trapping voltages selected to enhance ion accumulation. Pressure in the trapped ion cell was 2500
Analytical Chemistry, Vol. 67,No. 14, July 75, 1995
allowed to return to base pressure Torr) by a 3 s delay before detection. The ions of interest were then isolated using stored waveform inverse Fourier transform technique (SWIFT)43,44 to radially eject unwanted ions. In SORI-CID experiments, a single-frequency excitation of low amplitude (typically, 2) experiments, the ion isolation and dissociation procedure (indicated by and U*n, respectively, in Figure 1) was repeated at each stage for the selected precursor ion with a 10 s interval between offresonance excitation and SWIFT isolation for the next stage of dissociation. Twenty seconds after the last SORI excitation step, the product ions were excited and detected using 500 kHz broadband digitization (512K data points). For signal averaging, the mass spectrometer was set to repeat the ion injection, isolation, dissociation, and detection procedure (see Figure 1) and spectra were co-added before Fourier transformation. The spectra reported here were subjected to a “Blackman-Harris, 3 term” apodization. “@,?
RESULTS AND DISCUSSION SORI-CID of Cytochrome c Variants. The ESI charge state
distributions of cytochrome c from bovine, tuna, rabbit, and horse are very similar. Figure 2A shows a typical electrospray MS spectrum for tuna cytochrome c, indicating a charge state distribution of 10+ to 16+, with 14+ as the most intense peak. In general, the protein is positively charged by proton attachment.14 There are 21 readily protonated groups (Le., the total number of Arg, His, and Lys basic residues plus the terminal amine for tuna heart cytochrome c. However, some of these basic residues are adjacent and would most likely provide only one charge site due to Coulombicrepulsion. (llree consecutive basic residues might, however, yield two charge sites.)I4 Therefore, 16 charge sites exist for tuna heart cytochrome c, in approximate agreement with the observed charge state distribution. Similarly, the charge sites for cytochrome c from bovine, rabbit, and horse are 18, 18, and 19, respectively. Previous studies have indicated that under the solution conditions (PH = -2.5) used in this work, cytochrome c is in the denatured ~ t a t e , j ,in~ which % ~ ~ the spacing between charge sites may be maximized, consistent with the extensive charging observed in ESI.9J2 Figure 2B shows a spectrum obtained after SWIFT isolation of the 15+ state of tuna heart cytochrome c illustrating the routinely attainable resolving power of -80 000. The inset of Figure 2B indicates that the monoisotopic peak is at 802.39 m / z and the most abundant peak (43) Marshall, A. G.: Wang, T.-C. L.; Ricca. T. L.J. Am. Chenz. SOC.1985, 107, 7893-7897. (44) Marshall, A. G.; Grosshans, P. B. Anal. Chem. 1991,63, A2155A229. (45) Marzluff, E. M.; Campbell, S.; Rodgers, M. R; Beauchamp, J. L.J Am. Chem. SOC.,in press. (46) Feng, M.-F.; Englander, S. W. J. Mol. Biol. 1991,221, 1045-1061.
15+ '6+
I
I
I
B
,15+
p
M'5-2H20
I
j
';*
I
1
11+
I
1
1o+ I
II,
Y "
800.0
900.0
1Ooo.o
1100.0
1200.0
mh Figure 2. Series of spectra obtained by SORI-CID of tuna cytochrome c (A) Charge state distribution generated by electrospray ionization; (B) SWIFT-isolated 15+ charge state; (C) product ion spectrum for CID at -1500 Hz; (D) product ion spectrum for CID at +1500 Hz. The insets show the isotope distributions and monoisotopic peaks (*) for the precursor and product ions.
is at 802.79 m/z. There are five isotopic peaks between the most abundant and the monoisotopic peak, consistent with the calculated molecular weight and the expected natural isotopic abundance.47 Similar isotopic distributions are also observed for the other three cytochrome c's and for other charge states (spectra not shown). The calculated monoisotopic peak for the 15+ charge state of reduced cytochrome c (Le., where the heme iron is in f 2 oxidation state and 15 protons are attached) is at 802.41m/z, while that of the oxidized form (with iron in its +3 state) should be at 802.34 m/z since it has only 14 protons (the heme group may be considered to have one positive charge). Therefore, the cytochrome c's studied here are in their reduced form in gas phase. (The 0.02 m/z difference between the observed was used to correct for all other reported mass measurements-i.e., for internal calibration.) These results indicate that the reduced form of cytochrome c is more stable in gas phase, in agreement with previous €31-MS~tudies.9J~l~ Since the cytochrome c's are known to be more stable in the oxidized form in solution, reduction must have occurred either in the electrospray or in the ion transfer processes. However, the X-ray structures of the oxidized and the reduced form of cytochrome c are very similar,49and the change (47) Rockwood, A L.; Van Orden, S.; Smith, R D. 42nd ASMS Conference on Mass Spectrometty and Allied Topics, Chicago, IL, May 28-June 3, 1994. (48) Henry, K. D.; Quinn, J. P.; McLafferty, F. W. J,Am. Chem. Soc. 1991, 113, 5447-5449. (49) Takano, T.; Dickerson, R E. J Mol. Biol. 1981,153, 95-115.
in heme oxidation state should not affect the following discussion regarding fragmentation/structure correlation. The spectra in Figure 2C,D were acquired using SORI-CID pulses of 16.5 V,, to the SWIFT-isolated 15+ state ions. Since any fragment ions with cyclotron frequencies in the region of the excitation pulse are either ejected or further dissociated, full information was obtained by SORI-CIDexperiments at both lower (-1500 Hz in Figure 2C) and higher (+1500 Hz in Figure 2D) frequencies. The insets of Figure 2C,D show the high resolving power (>40 O00 at 850 m/z) obtained for measurements of the dissociation products. A feature of SORI-CID is that the extent of dissociation can be controlled by simply varying the amplitude, the duration of excitation, or the difference between the excitation frequency and the cyclotron Under our instrumental conditions, dissociation can be achieved with a frequency difference of -1000--2000 Hz and the excitation time could be varied between -50 ms to more than 5 s. However, variation in excitation for different isotopic components (as indicated by a distorted isotopic distribution) was observed experimentally when the excitation was applied too close to the cyclotron frequency. In this work, SORICID was performed at A1500 Hz for 300 ms and no significant distortion in isotopic distributions for the product ions was found. The insets of Figure 2C,D illustrate the isotopic distributions for a large and a small fragment ion, which match well with the calculated distributions. Figure 3 illustrates the dependence of ion dissociation on the excitation amplitude for -1500 Hz SORICID of 15+ charge state horse heart cytochrome c, where all other parameters were held constant. Each of the spectra represents a single experiment and is normalized to the most intense peak. At low excitation levels (Figure 3A,B), the ions primarily undergo dehydration. As the excitation amplitude increases, greater water loss is observed but other channels of dissociation gradually become dominant Figure 3C-E). Increasing the excitation amplitude beyond 21.5 V,, was found to decrease the total ion intensity. Figure 4 shows the SORI-CID (at -1500 Hz) of 15+ charge states of cytochrome c ions from bovine, tuna, rabbit, and horse. Each spectrum represents the sum of 10 experimental sequences with detection range of 700-2500 m/z (experiments were also extended to 200 m/z, but no other major fragment ions were detected). In order to compare the behavior of the variants, the excitation amplitudes were maintained at 21.5 V,, and identical ESI, ion transportation, and collisional trapping conditions were used. The experiments were also performed at +1500 Hz under otherwise identical conditions and complementary information was obtained (spectra not shown). All of the variants were effectively dissociated and produced spectra qualitatively different from one another, as was evident at low-resolution,10although most of the fragment ions fall into narrow m/z ranges. Accurate Assignment of the Fragment Ions. The general pattern for the SORI-CID spectrum of cytochrome c from tuna heart (Figure 5A) is similar to that generated earlier using a triple quadrupole mass spectrometer.1° However, the high-resolution FI'ICR results (Figure 5B) show that what appears to be a single peak in the low-resolution spectra actually includes contributions from several fragment ions. Figure 5C further expands a small region in 5B and shows it to consist of three overlapping peaks. Due to the high-resolution,-110 peaks can be identified for each Analytical Chemistry, Vol. 67,No. 14, July 15, 1995
2501
Ds3'"
I""- "
L
750.0
800.0
850.0
900.0
950.0
1000.0 750.0 800.0 850.0 900.0 950.0 1000.0 1050.0 1100.0
ln/Z
m/Z
Figure 3. SORI-CID spectra of horse cytochrome c (15+ charge state ions) at different off-resonance excitation levels. Amplitudes (Vpp) of the single frequency excitation: (A) 16.3 V; ( 6 ) 17.2 V; (C) 18.1 V; (D) 18.9 V; (E) 19.8 V; (F) 20.6 V.
spectrum in Figure 4. From these high-resolution spectra, it is clear that HzO losses from the precursor, fragment ions, or both significantly complicate the spectra. The number of peaks increases by 30-40% in the SORI-CID spectra due to the additional HzO loss channel. As a result, peak overlap possibilities increase, as is evident in Figure 5C. Another consequence of water loss is that average peak intensities decrease due to the spreading of a limited ion population over more peaks. The number of ions that can be trapped in an ICR cell is limited by space charge effects and is determined by the charges on the ion and cell dimensions, among other experimental parameters. The collision energy studies (Figure 3) demonstrated that H20 losses are lower energy processes. As will be seen from the accurate peak assignments, larger fragments generally display more HzO losses than the smaller ones. H2O loss is probably one of the common features of SORI-CID for large p r 0 t e i n s . 3 ~ ~ ~ ~ ~ ~ ~ Dehydration, signal spreading, peak overlapping, multiple charge states arising from a single cleavage site, and the competing fragmentation pathways all serve to increase the complexity of the spectra and peak assignments. For the cytochrome c variants, the possibilities of heme loss must also be considered for -50% of the peaks. In general, a b, fragment ion is different from a (c, - H20) ion by only 1 Da, as is a y, fragment from (2, - H2O). Consideringall these possibilities and ___
(50) Bakhtiar, R;Wu, Q.;Hofstadler, S. A: Smith, R 1994, 23. 707-710.
''
Figure 4. SORI-CID spectra (-1500 Hz) of the 15+ charge state ions under identical experimental conditions: (A) bovine cytochrome c; (B) tuna cytochrome c; (C) rabbit cytochrome c; (D) horse cytochrome c.
the precision of the measurements, our criterion is that an unambiguous assignment should, in general, agree to within 0.04 m/z of the calculated value. It is difiicult to completely assign these spectra manually, and there is yet no suitable commercially available software for this purpose. We therefore developed a PGbased computer program, PROFRAG, to facilitate the assignment of fragment ions of a known amino acid sequence. The program generates a list of the possible a, b, c and x, y, z fragment ions with user-selected charge states, using Biemann's n~menclature.~~ PROFRAG can also add or subtract from the sequence user-defined groups and calculate masses of the corresponding fragment ions. For example, the mass and m/z changes due to addition of a heme group, Na adduct, and water loss were calculated. The program provides the average mass, monoisotopic mass, or the most abundant mass for all of the fragments, and the masses of internal fragment ions can also be calculated. More accurate peak assignments can be achieved by using the most abundant, the monoisotopic, or both peaks identified from high-resolution spectra. As illustrated in the insets of F i r e 2C,D, the monoisotopic peak for the smaller ions (i.e., y3g5) is observed with high intensity, but the intensity decreases relative to the most abundant peak as the mass of the ions increase (i.e., b98I4). In this work, the most abundant peaks were used to identify
D.Biol. Mass Spectrom.
2502 Analytical Chemistry, Vol. 67,No. 74, July 75, 7995
(51) Biemann, K Biomed. Eflviron. Mass Spectrom. 1988,16,99-111
-
s
10.0
d
a
0.0
\
\
g 5
838.00
838.50 miz
839.00
839.50
/
1
B
0.0
750.0
L . 800.0
850.0 m/z
900.0
950.0
1
1OOO.O
Figure 5. Accurate peak assignments for SORI-CID product ions based upon the high-resolution capability of FTICR: (A) SORI-CID (at -1500 Hz) of tuna cytochrome c(15+ charge state); (B) expansion of a small region in A; (C) isotopic peaks in the region with three overlapping peaks with sequence assignments.
the majority (>95%) of the fragment ions. Nevertheless, there are a few cases when the most abundant peaks cannot be readily identified, i.e., where the peak intensities are too low (i.e., with S/N < -3), where there are two peaks with comparable intensities or both. The latter typically occurs when the mass of the fragment ions are close to -n x 1800 Da (where n is an integer). Isotopic peak intensity is dependent upon the natural abundance and the elemental composition of the ion because each peak can include isotopic contributions from several elements. In such cases, the approximate mass of the ions (based upon the observed charge state and m/z) was used to generate a simulated isotopic di~tribution~~ using the general formula, CnH1.6nN0.3nO0,3nSO.~~~, for cytochrome c. The result was then compared with the observed distribution to identify the monoisotopic and the most abundant peaks. We can assign the mass and sequence of ions with a signaltenoise ratio as low as 3:l with high confidence. The assignment can also be confirmed by identifying ions of the same mass but with different charge states or ions of the same charge state but with one or more HzO losses. For example, when a low-intensity peak could be assigned as either bJy8 ions or (c, - HzO)/(z, H20) ions (which differ by 1 Da), due to the uncertainty in the identification of the most abundant and monoisotopic peaks, it was assigned only if other ions of the same series were identified and had greater intensity (invariably b,/yn ions in this work). Improved methods for the identitication of large ions with very low peak intensities might be feasible with enhanced product ion collection efficiency based upon selective ion acc~mulation~~ and broad-band ion axialization techniques.28~53 (52) Bruce, J. E.; Anderson, G. A; Hofstadler, S. A; Van Orden, S. L.; Sherman, M. S.;Rockwood, A L.; Smith, R D. Rapid Commun. Mass Spectrom. 1993, 7, 914-919.
With the methods discussed above, we have successfully assigned over 95%of the peaks. The results in Table 1for tuna cytochrome c give the observed m/z, charge state, mass of the fragment, and difference between the observed and the calculated value. The majority of the assignments are within 0.01 m/z of the experimental values, and the average accuracy in mass determination is better than 10 ppm. Table 1also shows several ions without assignments. There are other peaks (not listed in Table 1)with very low intensities, where the charge states or the most abundant isotopic peak could not be positively determined. Some of these ions probably resulted from “internal” fragmentation, i.e., where a fragment ion with sufficient internal energy undergoes further dissociation. For example, some b,-type ions may undergo subsequent y-type fragmentation as revealed below using higher-order MS. MS4 of Tuna Heart Cytochrome c. To identify the possible internal fragments and to obtain more sequence information, specific product ions can be isolated and further dissociated. Higher-ordermass spectrometrictechniques should be especially useful for the structural characterization of proteins with unknown sequences. The combination of ESI interface dissociation with CID in FTICR has previously been shown to generate more extensive data reflective of the primary structure of carbonic anhydrase.% However, since the precursor ions cannot be isolated during dissociation in the ESI interface, the spectra can be dficult to interpret for unknown or impure samples. Recently, MS4 experiments have been achieved for ubiquitin using FTICR3‘jWe have also succeeded in performing multiple-stage MS experiments, as demonstrated in Figure 6. In these experiments, the 15+ charge state ions from tuna heart cytochrome c were isolated using the SWIFTtechnique and dissociated at +1500 (Figure 6B) and -1500 Hz (inset of Figure 6B). The bl02’~+ions were then chosen for further dissociation. The spectrum in Figure 6C and its inset show the sum of 25 SORI-CID experiments with 20 V,, at +1500 and -1500 Hz, respectively. It can be seen that many of the b, ions in MS3are the same as those in the MS2 experiment, probably due to similarity of the two precursor ions (only different by one terminal amino acid residue). More interestingly, a peak at 869.30 m/z (5+ charge state) in the MS2 spectrum cannot be unambiguously assigned on the basis of the primary sequence. However, this peak is also found in the MS3 spectrum from the further dissociation of b10214+ions. With the sequence of the blo214t ions, it can be accurately assigned as { ( ~ I O Z - y39)3a5+ - HzO} (see Table 1). In the MS2experiment,this peak must be the result of internal fragmentation. A { ( ~ I O Z- y39)385t - 2H20) peak can also be identified in Figure 6C. (The nomenclature for the internal fragment ions was described previo~sly.~~ In the above case, the “bloz”and “ ~ 3 9 ’ ’indicate the positions and types of the cleavages, while the “38’’ shows the number of residues retained for this 5+ state ion.) In MS4,bd3+ions were isolated and subjected to off-resonance excitation. The -1500 Hz spectrum (Figure 6D) indicates that further dissociation is favored at the b75 and bu (Le., the (b98 yZ8)23 and (b98 - y&* ions) positions, although water loss is also a major process. The +15W Hz (the inset of Figure 6D) spectrum (53) Guan, S. H.; Wahl, M. C.; Marshall, A G.I. Chem. Phys. 1994,100, 61376140. (54) Senko, M. W.; Beu, S. C.; McIafferty, F. W. Anal. Chem. 1994,66,415417. (55) Ballard, K. D.; Gaskell, S. J. Inf.I. Mass Spectrom. Ion Processes 1991,111, 173- 189.
Analytical Chemistry, Vol. 67, No. 14, July 15, 1995
2503
Table 1. List of Product ions Observed from SORI-CiD of Tuna Cytochrome c (15+) and Sequence Assignments p
m/$
A (m/z)
Md @a)
M e @a)
6 6 6 10 10 8 4 10 10 6 10 10 4 6 6 6 15 15 15 15 15 14 14 14 14 11 9 14 9 9 9 9 14 5 9 9 5 9 9 9 14 14 14 14 14 14 14 9 13 5 13 7 5 13 13
736.25 739.25 742.25 745.78 747.58 749.66 751.42 752.48 754.28 755.09 757.08 758.88 760.42 761.10 771.93 777.95 794.56 795.74 797.94 799.15 800.36 813.50 814.78 815.49 816.78 818.14 819.30 824.88 825.41 826.53 828.52 830.54 834.02 834.85 835.98 837.98 838.48 839.08 841.10 843.10 844.02 845.30 848.66 849.94 851.22 852.52 856.16 857.63 860.15 860.68 861.53 863.42 864.30 865.62 867.00
0.00 -0.01 -0.01 0.00 -0.01 -0.02 -0.02 -0.01 -0.01 -0.01 -0.01 -0.01 -0.03 -0.01 -0.01 0.00 0.00 -0.02 -0.02 -0.01 0.00 -0.01 -0.01 -0.02 -0.01 -0.01 -0.02 0.01 -0.03 0.00 -0.01 0.00
4411.45 4429.45 4447.45 7447.72 7465.72 5989.22 3001.65 7514.72 7532.72 4524.49 7560.72 7578.72 3037.65 4560.55 4625.53 4661.65 11903.28 11920.98 11953.98 11972.13 11990.28 11374.89 11392.81 11402.75 11420.81 8988.45 7364.63 11534.21 7419.62 7429.70 7447.61 7465.79 11662.17 4169.21 7514.75 7532.75 4187.36 7542.65 7560.83 7578.83 11802.17 11820.09 11867.13 11885.05 11902.97 11921.17 11972.13 7709.60 11168.85 4298.36 11186.79 6036.88 4316.46 11239.96 11257.90
0.00 -0.06 -0.06 0.00 -0.10 -0.16 -0.08 -0.10 -0.10 -0.06 -0.10 -0.10 -0.12 -0.06 -0.06 0.00 0.00 -0.30 -0.30 -0.15 0.00 -0.14 -0.14 -0.28 -0.14 -0.11 -0.18 0.14 -0.27 0.00 -0.09 0.00 0.00 -0.20 -0.09 -0.09 -0.05 -0.18 0.00 0.00 0.00 -0.28 -0.14 -0.14 -0.28 -0.14 -0.14 -0.27 0.00 -0.05 -0.13 -0.14 0.00 0.13 0.00
0.00
-0.04 -0.01 -0.01 -0.01 -0.02 0.00 0.00 0.00 -0.02 -0.01 -0.01 -0.02 -0.01 -0.01 -0.03 0.00 -0.01 -0.01 -0.02 0.00
0.01 0.00
ZQ
5 9 7 5 13 13 5 13 5 13 5 5 10 4 5 8 5 13 13 8 4 8 8 8 8 8 8 8 12 12 8 8 8
8 12 12 4 8 6 3 8 4 2 2 6 3 3 6 6 3 1 3
m/9
A(mlz)c
Md (Da)
869.30 872.00 873.58 876.05 878.16 879.54 883.30 886.84 886.90 888.25 890.50 893.90 899.86 903.02 905.46 907.24 913.10 916.64 918.04 919.34 920.78 921.60 926.22 927.46 928.45 929.72 931.98 934.22 937.66 939.16 942.62 943.98 946.10 948.35 951.25 952.75 961.55 964.72 971.16 973.12 980.88 989.60 990.50 999.40 1007.15 1011.50 1013.60 1019.02 1038.02 1049.50 1052.62 1092.20
0.00 0.00 0.00 0.00 0.00 0.00 0.00 -0.02 0.00 0.01 -0.01
4341.46 7838.93 6108.00 4375.21 11402.98 11420.92 4411.46 11515.82 4429.46 11534.15 4447.46
0.00
8988.52
0.00 0.02 -0.02 0.00 0.01 0.00
4522.26 7249.86 4560.46 11903.22 11921.42 7346.66
0.01 0.00 -0.01 -0.02 0.00 0.01 0.00 0.00 0.01 0.01 -0.01 -0.01 -0.01 0.00 0.00
7364.74 7401.70 7411.62 7419.54 7429.70 7447.78 7465.70 11239.82 11257.82 7532.90 7543.78 7560.74 7578.74 11402.90 11420.90
0.08 0.00 -0.08 -0.16 0.00 0.08 0.00 0.00 0.12 0.08 -0.08 -0.08 -0.08 0.00 0.00
-0.02 0.00 -0.02 0.01
7709.70 5820.91 2916.34 7838.98
-0.16 0.00 -0.06 0.08
B6j
-0.02 0.02 0.01 0.01 0.00 0.00 0.00 0.01
6036.85 3031.48 3037.78 6108.07 6222.07 3145.48 1051.61 3273.58
-0.12 0.06 0.03 0.06 0.00
B50
av dev
-0.01 0.01
AMe (Da) 0.00 0.00 0.00 0.00 0.00 0.00
0.00 --0.26 0.00
0.13 -.0.05
assignment
(BIOZ- y39)38 B66 B51 B35 B97 Bg7 Y39 - 2H20 Bgg Y39 Bgg Y39
HzO
Hz0 HzO
0.00
B75
0.00
B36 B61 Y40
0.16 -0.10 0.00
0.13 0.00
0.00 0.00
0.03
- HzO
Bloz Bioz
- H20
B62-H20
B48 Bz1 B66
Bzz Yzg Bjl
Bjz B24 Y10 B25
-0.06 0.10
Observed number of charges. * Observed mass-to-charge ratio for the most abundant isotope peak. The difference between the calculated values based on the sequence assignment and the observed values. Observed mass of the most abundant isotope peak [M = ( m / z ) ( z- 1.008)]. e The difference between the calculated values based on the sequence assignment and the observed values. (I
provides complementary information by showing the b75 peak, while the water loss peak is absent. Both spectra are the sum of 25 experimental sequences. Beyond this step, the total ion intensity decreases quickly and higher irradiation amplitude results in a sharp decrease in the overall signal. It must be pointed out that these MS4 experiments are feasible because of the high dissociation efficiency and the high trapping efficiency for the product ions in the earlier steps. In these experiments, over 99% of the precursor ions are dissociated. The trapping efficiencies for the MS2 to MS4 stage are w%9 and 68%frespectivelyf as calculated from the sum of the intensities for all isotopic peaks. 2504 Analytical Chemistry, Vol. 67, No. 14, July 15, 1995
In SORI-CID, ions are dissociated near the center of the ICR cell so that product ions are generated without significantmagnetron motion. Such magnetron motion ultimately causes ion ejection, results in low resolving power for the product ions, and is believed to be one of the major reasons for the relatively inefficient resonant CID for large m o l e c ~ l e s . 3Signal ~ ~ ~ ~partitioning ~~~ among the fragment ions and ion losses during various steps of trapping, dissociation, and isolation are important factors affecting the maximum order of MS attainable. (56) Senko, M. W.; Speir, J. P.; McLafferty, F. W. Anal. Chem. 1994,66,28012808.
14+
A
13+ 12+ I
C
bm13 -H,O
D
750.0
60b.O
650.0
bw’3
I
I
9do.O 950.0 1060.0 1050.0 1100.0 ml2
Figure 6. Multiple-order SORI-CID (at $1500 and -1500 Hz) spectra of tuna cytochrome c (A) MS of the electrosprayed ions; (e) MSz of the 15+ charge state ions; (C)MS3 of the isolated bloz14 product ions from MS’; (D) MS4 of the isolated b9813 product ions from MS3,The stars indicate the precursor ions for the next step, and the arrows show the positions of irradiation. The scale factors from (A) to (D)are x l , x3, ~ 1 7and , x40, respectively.
Fragmentation Patterns and Structural Correlation. The MS4 experiments show that although internal fragmentation can occur during SORI-CID,most of the cleavages occur at the amide bonds to generate b, and ynfragmentions. In addition, H20 losses are observed for the majority of the product ions, including those from internal fragmentation. This is consistent with the collision energy studies (Figure 3), which show that HzO loss as the lowest energy pathway. This, and the lack of high-energy process products, such as d,, xn, z, and w, ions, indicates that the dissociation is a result of low-energy processes, as expected for SORI-CID. Another major channel is the loss of a CO group to form an a,, product ion from a b, product ion. Although the a,, ions could also be formed from direct cleavage of a C-C bond in the peptide backbone, their C-terminal counterpart ions, i.e., the xmions, are typically not observed. On the other hand, many y, ions are detected as the counterparts of the b, products. It should be noted, however, that only -50% of these products are truly complementary ions (for which the sums of amino acid sequences (m n) and charge states are equal to those of the precursor). The term “counterpart ions” is used here to designate those ion pairs whose sum of amino acid sequences is only different from that of the precursor by a CO group or some H2O losses, while whose sum of charge number may or may not be the same as that of the precursor ions. The observed variations in mass and charge of the fragments reflect the complexity of the fragmentation processes.
+
To compare the cytochrome c variants qualitatively, the fragmentation pattern can be most simply expressed in terms of cleavages between the residues. Figure 7 summarizes the MS/ MS results F i r e 4) for the four cytochrome c’s. As indicated in Figure 7, counterpart ions are generally not observed for large b, and y,, fragment ions (i.e., those from cleavages near N- or C-terminus). These counterpart ions are expected to be small and have low charge states. Low-mass ions may be more readily lost under the high-pressure conditions used in the SORI-CID experiments57 and may be discriminated against. Ions with fewer charges generate lower signal intensities and are detected with lower sensitivity since they generate lower amplitude signals at a given cyclotron radii. Similarly, the absence of counterpart ions for some intermediate size fragments (see Figure 7) may indicate that these ions are the products of internal fragmentation, and the smaller counterpart ions were not detected. For example, the b48 ions for rabbit cytochrome c (see Figure 7) may arise from the further fragmentation of the bg (or b49 or b51) ions. The internal fragmentation of the counterpart ions, i.e., y54 ions, is also possible. Another possibility is that the intensities of such counterpart peaks are too low, since peak intensities of these intermediate size ions are typically less than 20%of those observed to have counterpart ions (e.g., the bs3 ions in the above example). Further dissociation of the fragment ions, as illustrated in the MS4 experiments, can help determine the origin of these ions. The extent of further dissociation of these intermediate size products, however, might also serve as an indication of the internal energy partition of the cytochrome c ions during the dissociation processes. As discussed earlier, each peptide cleavage shown in F i r e 7 may produce additional fragment ions with slightly different masses (e.g., due to losses of HzO, CO, or both), as well as those due to different charge states (see Table 1). Charge state variation is usually limited to one or two for the majority of the fragments, but was observed to be up to three.for larger fragments. This variation in charge state may reflect the charge site heterogeneity in the molecular ions or local proton transfer at the cleavage Figure 8 shows a linear correlation between the total number of basic residues and the charge states of tuna cytochrome c fragment ions. Similar behavior is also observed for the other three types of cytochrome c ions. A near 1:l relationship can be found between the number of charges and the number of available charge sites if charge-charge repulsion is considered (vide supra). This suggests that no significant global rearrange ment of charge carriers (neither protons nor basic residues) occurs during the dissociation processes. Structural rearrangements have been reported for some peptides during CID processes in a quadrupole mass spectr0meteI5~and for small molecules@during SORI-CID. The fact that accurate fragment ion assignments have been readily accomplished based on the primary structures similarly excludes the possibility of protein isomerization (sequence rearrangements) as a significant channel for these SORI-CID studies. (57) Miluchihin, N. V.; Miura, K; Inoue, M. Rapid Commun. Mass Spectrom. 1 9 9 3 , 7, 966-970. (58) O’Connor, P. B.; Speir, J. P.; Wood, T. D.; Senko, M. W.; Chorush, R A; McLafferty, F.W. 42nd ASMS Conference on Mass Spectrometty and Allied Topics. Chicago, IL, May 28-June 3, 1994. (59) Tang,X. J.; Thibault, P.; Boyd, R K Org. Mass Spectrom. 1 9 9 3 , 28, 10471052. (60)Bakhtiar, R; Holmagel, C. M.; Jacobson, D. B. Organometallics 1 9 9 3 , 1 2 , 621-623.
Analytical Chemistty, Vol. 67,No. 14, July 15, 1995
2505
1. Cytc.(Bovine)
1 10 NH~~-D-v-E+J-G-K~~I-F-v-Q-K-c-A-Q-c-H-T
2.Cytc.(Tuna)
NH2-G-D-V-A-K-G-K-K-T-F-V-Q-K-CA-Q-C-H-T
3.Cytc.(Rabbit)
NH2-G-~V-E~K-G-K-Ktl-F-V-Q-K-C-A-Q-C-H-T
4.Cytc.(Horse) 20
-
40
30
1.
-V-EtK-G-G-K~H-K-T-G-P-N-L-H~G-L-F-G-RkK-T~~A
2.
-v-E~N~+G-G~K~H-K-v-G-P-N-L-w-G-+F+-R-K-T-G-Q-A
-
-
~
L
3.
-E~~~~+P-K-K-Y-~-G-T-K~M-I-F-A-G-I-~+K-K-D-E-R-A-D
4.
-E+-P-K-K-Y-I+G-T-K+-I-F-A-G-+K-K-K-T-E-R-E-D
1.
100 +L-I-A~Y-L-K-K-.)T)N~E~COOH
2.
+-+A~Y~+I+s-T~+cooH
3.
-L-I-A~YTL-K-K-A-T-N+E>OOH
4.
~L-I-~Y-L-K-K-A-T-N~E~cooH
Figure 7 . Summary of the MS/MS results for the four cytochrome 6s. A cleavage symbol represents the observed N-terminal fragment ions if it points toward the N-terminal or C-terminal ions if toward the C-terminus. A two-way symbol indicates that both types of products are observed for the particular cleavage. The basic residues are indicated as boldface letters.
25 1
20
Total Basic Residues
1510 -
5-
Available Charge Sites
Number of Charges Figure 8. Correlation for tuna cytochrome c of the number charges on the product ions with the number of basic residues. Also shown is a near 1:l relationshipbetween the charge numbers and the available charge sites.
The fragmentation patterns shown in Figure 7 are clearly sequence informative,and additional insights can be obtained by consideration of the probability of cleavage at each position. To a first approximation, the number of ions detected is reflected in 2506
Analytical Chemistv, Vol. 67,No. 74, July 75, 7995
the relative peak intensities in the mass spectra for ions of the same charge state. Under the same experimental conditions (i.e., assuming the Same cyclotron radius), ions with more charges give greater peak intensities. The fragment ion distributions and cleavage probabilities can be better represented by dividing the sum of the intensities of all isotopic peaks by the number of charges. Figure 9 shows the relative probabilities of cleavages in the entire amino acid sequences for the four types of cytochrome c ions based upon the data from both +1500 and -1500 Hz SORI-CID experiments. Due to the lack of complimentary (or counterpart) ions for some fragments, the relative probability of cleavage at each position is represented by the sum of the chargecorrected peak intensities for either the C-terminal ions or the N-terminal ions. For a position where both types of fragment ions are observed, the larger sum was used. In each case, product ions from HzO and CO losses and of different charge states are included. The reconstructed spectra in Figure 9 show two primary cleavage regions for tuna cytochrome c (residues 60-67 and the C-terminus), while the other cytochrome c’s show additional
Table 2 Differences in Amino Acid Sequences between Cytochrome c from Bovine and Those from Horse, Rabbit, and Tuna
Bovine Cytochrome C
l I l d 0
10
0
1W
._ 0
20
30
40
50
60
I L , ,-, , 70 80 90 1W
,
50
C
al
c
bovine
47
S
54 5R
N T G
M)
o0
10
20
30
40
50
60
70
80
90
100
a,
.->
61
62 89
92
c
Rabbit Cytochrome C
al
U
0 0
100
cytochrome c horse rabbit
tuna
Tuna Cytochrome C
(0
-c
residue position
1
10
20
30
40
Hone Cytochrome C
50
60
70
A 80
90
95 100 103 104
E E G E I K N E
S
v
K
T
D D A
N N I)
Q
v S
S none
1W
1
Residue Number Relative probabilitiesof dissociation along the amino acid sequence for bovine, tuna, rabbit. and horse cytochrome 6s. Figure 9.
fragmentation sites. Indeed. the sequence of tuna cytochrome c is different from bovine by 16 residues, while those of horse and rabbit differ by only 3 and 4 residues, respectively (see Table 2). It has been found that SORI-CID fragmentation patterns can be quite different for protein ions of different charge states,=.:* More interestingly. even with only three different amino acid residues (at positions 47.60 and 89) between bovine and horse cytochrome c, the dissociation probabilities in the residue 40-50 and 60-70 regions are strikingly different, and the abundant fragmentation at residue 75 for bovine cytochrome c is replaced by a high intensity at the Gterminus for horse cytochrome c. Similariy. with four different residues from bovine cytochrome e, Le., at 44,62, 89. and 92, rabbit cytochrome c shows a significant fragmentation near the C-terminus. However, the fragmentation patterns from residues 50 to 90 are essentially the same for bovine and rabbit cytochrome c, although their sequences are different by two residues in this region (see Table 2). This is in sharp contrast to the large differences in fragmentation patterns between bovine and horse cytochrome c, which also different by two amino acid residues in this region. The similarity between bovine and rabbit cytochrome c may be attributed to the structural similarity between a glutamic acid residue (Le.. Glu" for bovine) and an aspartic acid residue (Le.. Asp"4 for rabbit) (see Table 2). The differencesbetween bovine and horse cytochrome c are evidently
due to the change at position 60 from a Gly (G) to a Lys 0.the later being a basic residue. Since the basic residues are generally effectively charged, as indicated by the data in Figure 8. the substitution of a Gly (G)by a Lys 0 residue would likely result in changes in charge distribution. It has been shown that, by blocking Lys charge sites through acetylation. the conformation of a cytochrome c molecule in solution can be altered from a totally unfolded structure to a nativelike structure."' In the native state of horse cytochrome c. Lysm is situated close to another charge site, Lysn, as determined by X-ray diffradon." Although the X-ray structures of native cytochrome c variants are similar,' the differences in basic residues may become important in determining the structures of the denatured states due to the electrostatic interactions. The additional repulsive forces between charged Lysmand LysNsites for horse cytochrome c in the gas phase might lead to conformationalvariations, which could iduence or change energy deposition and redistribution processes and result in changes in the fragmentation pattern. It has been shown by magnetic circular dichoism and electron paramagnetic resonance techniques' that replacing the invariant residue of Phe with a His residue at position 82 of cytochrome c dramatically changes its higher-order structure. Our results indicate that such structural differences may be manifested as differences in the SORI-CID fragmentation patterns and, therefore, may be probed by lowenergy collision methods. The effects of charge sites on dissociation seem to be general for all four cytochrome c's studied. As shown in Figure 7 and in Figure 9, cleavage appears less likely at basic residues and no charge sites are clearly evident in the regions of greatest fragmentation. This is in apparent contrast to a recent repot+' showing that fragmentation at charge sites of hipeptides was favored during SORI-CID. Chargepromoted and chargeremote (61) Golo. Y.: Nishikiori. S . J Mol. Bid. 1991.222.679-686. (62)Bushnell. G. W.: Louie. G . V.: Brayer, G.D.1. Mol. B i d 1990.214,?AT-
595. (63)Carroll. J. A Wu.J.: Do.T.: Lebrilla. C. B.42ndASMS Con/erenrr on Moa Splclromnehy and Allied To,Dirr. Chicago, IL May 23-June 3 . 1'34.
Analyiical
Chemishy. Vol. 67. No. 14, July 15, 1995 2507
dissociations have all been observed for small ions,64,65 and the heme - H20)13+ ions. Moreover, the probability of the heme effects of multiple charging on large biomolecular fragmentation detachment process was found to be very low (see Figure 9 for have been of For the proteins studied, it is possible the blol fragments of bovine cytochrome c), and no fragmentation that proton attachment may serve to stabilize the charge sites and of the heme (for instance, loss of its tail carboxyl groups) was reduce dissociation. Further experiments are underway to observed. These results suggest that the heme group is protected investigate this apparent charge suppressed dissociation. by the polypeptide backbone. This suggestion is also consistent Another general observation is that dissociation does not occur with the lack of dissociation in the region of residue 10-20, which near many of the proline or aspartic acid sites. A proline (P) supports the preservation of the strong binding of the heme to residue can be extremely important in determining protein this part of the polypeptide chain in the gas phase, thus leaving c~nformation~~ due to its nonrotatable N-C bond enclosed in a a heme “footprint” on the fragmentation pattern. (The term five-membered ring. This structural constraint has also been footprint is often used in molecular biology to represent an area suspected of hindering internal energy transfer and potentially of the substrate, such as DNA, which loses reactivity to certain making nearby peptide bonds susceptible to dissociation.68 On reagents due to its interactions with species.) The other part of the other hand, aspartic acid (D) residues have recently been the heme is most likely protected by interacting with the residues recognized as potentially labile in the gas phase and may be near MePo, which is well reflected by another footprint in the involved in a proton transfer-hydride formation m e c h a n i ~ m . ~ ~ , ~region ~ from residue 70-90 as shown in Figure 9. The minor Of the 15 aspartic acid residues in the four types of cytochrome dissociation observed near M e P (see Figures 7 and 9) indicates c (see Figure 7), only 3 at position 50 were found to undergo the nature of the weaker binding in this region and is analogous signifcant dissociation (see Figure 9). Similarly, only 3 out of 14 to the denatured structure of the protein in the solution. Further proline (Pro) sites fragment with high probabilities. The strongly experiments are needed to understand whether these effects arise favored Ile75-Pro76 dissociation observed for bovine and rabbit from some intermolecular hydrogen bonds, hydrophobic interaccytochrome c ions is not observed for horse cytochrome c, in tions, or iron coordination. which more fragmentation occurs at the Pro44residue. Residues The structures of cytochrome c variants in their denatured 60,76, and 79 have been determined by X-ray difh-action to be in states have not been as extensively studied as the native states. a triangular geometry with located at the protein surface In fact, only in recent years have the denatured states of proteins well away from the heme.49 If this structure is preserved in the been recognized as biologically important.i0 In general, cytogas phase, the observed changes in fragmentation can be chrome c has three helices: N-terminal, Ws, and C-terminal helix. qualitatively rationalized. The charge-charge repulsion between In the native state in solution, the C-terminal helix of cytochrome Lys60 and LYS’~in horse cytochrome c (vide supra) may expand c is known to fold back on the protein surface and is close to the the triangle, retracting the Pro76residue from the protein surface N-terminal These two helices are thought to play and making it less accessible to collisions. Similarly, the unfaimportant roles in the initial stages of protein f ~ l d i n g . ~All~ . ~ ~ . ~ ~ vored aspartic acid and proline residues may be buried inside the three helices are known to be well preserved between the native protein 3-D structure. and molten globular states, and the helical content decreases This potential fragmentation/conformation correlation is furquickly as the proteins are denatured.46J1,72Previous studies ther supported by the following observations. For the four suggested that such secondary structure might be transferred into variants of cytochrome c studied, there is no fragmentation in the gas phase and affect the protein’s dissociation b e h a ~ i o r . Figure ~~,~~ region of residue 10-20, as evident from Figure 9. Only minor 9 clearly shows that fragmentation is mainly observed in the dissociation is observed from residues 70 to 90, except at regions corresponding to the three major helices and from as discussed above. It has been well-known’ that, in the native residues 40 to 50. Generally, greater fragmentation is observed state of cytochrome c, the heme group is covalently bound to Cy@ for the C-terminus region than for the N-terminus. These and Cyd7and noncovalently to His@and MePo. Residues 70-80 observations appear consistent with the known features of the denatured state in solution, since it is conceivable that greater are conserved in all known cytochrome c’s and are in direct contact fragmentation may occur as the helical content is decreased. with the heme group during oxidation and reduction in respiration. Interestingly, the residue 40-50 region is expected to be exposed In the denatured state, neither Hid8 nor MePo is coordinated to on the protein surface at close to neutral pH (based upon the X-ray the heme iron, while either of these two positions can be coordinated in the intermediate state(^).^.^ Cytochrome c is in structure under crystallization conditions) .49 No dissociation is observed for the N-terminal region of tuna cytochrome c, while a the denatured state under the solution conditions used, and the variable extent of fragmentation was found in this region for other native state 3-D structures were reported to be substantially degraded.jI6 In these dissociation experiments, products due to cytochrome c’s. The primary structure in the first 20 residues of heme detachment are rarely observed. In the case of bovine the N-terminus is identical for all of the cytochrome c’s, with the cytochrome c, only the blol fragments was detected as @IO, exception of a Glu (E), which is replaced by an Ala (A) at residue 4 of tuna cytochrome c. The crystal structure of horse cytochrome (64)Fabris, D.; Kelly, M.; Murphy, C.; Wu, Z.; Fenselau. C. J. Am. Sot. Mass Spectrom. 1993,4, 652-661. c (in the native state) shows a loose N-terminal helii with the (65) Contado, M. J.; Adams, J.; Jensen, N. J.; Gross, M. L. J. Am. Sot. Muss first two residues extended, while the N-terminal helii of tuna Spectrom. 1991,2, 180-183. cytochrome c is c0mpact.~z*~9 These observations suggest that (66) Rockwood, A. L.; Busman, M.; Smith, R. D. Int. J, Mass Spectrom. Ion Processes 1991,111, 103-129. residual helical structures may exist in the denatured state and (67) MacArthur, M. W.; Thomton, J. M.J. Mol. Bid. 1991,218. 397-412. (68) Light-Wahl, K J.; Loo, J. A; Edmonds, C. G.; Smith, R D.; Witkowska, H. E.; Shackleton, C. H. L.; Wu. C. S . C.Biol. Mass Spectrom. 1993,22, 112120. (69) Yu, W.; Vath, J. E.; Huberty, M. C.; Martin, S. A.Anal. Chem. 1993,65, 3015-3023.
2508 Analytical Chemistry, Vol. 67, No. 14, July 15, 1995
(70) Dill, K A,; Shortle, D. Annu. Rev. Biochem. 1991, 60, 795-825. (71) Jeng, M.-F.; Englander, S. W.; Blove. G. A,;Wand, A. J.; Roder, H. Biochemistry 1990,29, 10433-10437. (72) Roder. H.; Elove, G. A; Englander, S. W. Nature 1988,335, 700-704. (73) Loo, J. A,; Edmonds, C. G.; Smith, R. D. Anal. Chem. 1993,65, 425-438.
persist into the gas phase. Alternatively, the solution-denatured proteins may recover some higher-order structure as the interactions with solvent molecules are removed when they are transferred into gas phase. A direct comparison of the helical content for cytochrome c in the gas phase and in solution may be feasible by ESI-MSusing H/D exchange and would serve in differentiating these two possibilities. Dissociation in the C-terminal region is very different for cytochrome c from bovine, rabbit, and horse, even though they have the same primary sequence at residue 93104 (see Figure 9 and Table 2). While there may be some conformational differences between the cytochrome c variants, as indicated by the fragmentation patterns, subtle conformational differences in the C-terminal regions for the variants have not been well studied, especially for the denatured state of cytochrome c, and limit speculation on such effects. It must also be remembered that the conformations of proteins in solution are dependent upon their environmental conditions, such as solvent and ionic strength. Charge-charge repulsion, hydrophobic and hydrophilic interaction, and inter- and intramolecular hydrogen binding likely all contribute to determining the detailed structure. The present studies demonstrate that such higher-order structure may, under certain circumstances, be transferred into the gas phase and be reflected by the fragmentation following a large number of low-energy collisions. This is remarkable since the degree of activation necessary to result in cleavage of covalent bonds (as necessary to produce the observed fragmentation) might be expected to have also resulted in prior dissociation of the noncovalent bonds responsible for the higherorder structures. One possible component of an explanation for this behavior is that the sum of the bond strengths for the noncovalent bonds may well exceed that of a covalent bond, and kinetic constraints might apply that limit the rate at which the relevant noncovalent bonds are dissociated. Such noncovalent interactions may also involve large portions of the protein, including portions buried inside the 3-D structure of the protein. During the activation by low-energy collisions, these relatively weak bonds may be more effectively protected by the stronger covalent bonds located on the protein’s surface. The dissociation of these covalent bonds is indicated by the presence of the corresponding protein fragments. Some of the intramolecular interactions (especially those close to the surface) may be interrupted by energetic collisions but will not result in observable fragmentation unless the internal covalent bonds are also cleaved. Kinetic constraints upon gross structural changes may thus allow various aspects of protein conformation to be investigated by lowenergy CID methods. Additional benefits of low-energy CID for primary sequence analysis come from the facile production of b and y fragments, which greatly facilitates sequence analysis. Clearly, more detailed dissociation studies are needed to provide additional information on the possible correlation of fragmentation and higher-order structures of protein ions in the gas phase. CONCLUSIONS
We have shown that the subtle structural differences among the cytochrome c variants are reflected in their lowenergy fragmentation patterns. The four cytochrome c variants were
found to be effectively dissociated using the sustained offresonance irradiation/collision-induced dissociation technique. By controlling experimental conditions,high-resolutionfragment ion mass spectra can be obtained with little distortion in the isotopic distribution. About 95% of the fragment ions can be accurately assigned (to better than 10 ppm) on the basis of the primary sequences of the proteins. Up to four orders of mass spectrometry @IS4) could be achieved without using reaxialization techniques for the product ions. Strikingly, the fragmentation patterns of the cytochrome c proteins studied are determined not only by their primary structures but are also greatly influenced by the protein‘s conformations. Replacement of 3 out of 104 residues of a cytochrome c molecule was found to dramatically alter the dissociation pattern. Fragmentation generally does not occur with high probability at expected charge sites, nor near the majority of the proline and aspartic acid residues. No fragmentation is observed in the region of residue 10-20, and there is little dissociation from residue 70-90. This heme footprintingpattern suggests some residual structural similarity of the gas phase ions to that in solution. The conformations of the proteins are known to be greatly dependent upon the number of charges and their distributions in the 3-D structures. We have demonstrated that both the higherorder structure and charge location may, in addition to the primary structure, iniluence the dissociation of the cytochrome c’s. Ultimately, the internal energy distribution and, therefore, the resulting fragmentation pattern are determined by the combination of the proteins’ primary sequences, conformations, and charge densities, in addition to the CID conditions. The most probable locations of charge sites can vary between the four cytochrome c’s and may be (in part) responsible for the differences in fragmentation. Previous work has correlated differences in tertiary structure with differences in the observed charge state distrib~tion.~J~ It has also been recently observed that different charge states can have remarkably different fragmentation pattems.50356 These various results suggest signilkant potential for the use of CID methods with FTICR to obtain higher-order structural data, in addition to primary (sequence and site of modification) data. These methods can provide structurally related information not readily available from H/D exchange studies or cjrcular dichoism experiments. ACKNOWLEDGMENT
The authors thank Drs. James E. Bruce, Steven A. Hofstadler, Dale W. Mitchell, and Mr. Gordon A Anderson for helpful discussions. This research was supported by internal PNL exploratory research through the US. Department of Energy. Pacific Northwest Laboratory is operated by Battelle Memorial Institute for the US. Department of Energy, through Contract DE-ACOG76RLO 1830. Received for review October 21, 1994. Accepted May
IO,
1995.a AC941034Q @Abstractpublished in Advance ACS Abstracts, June 15, 1995.
Analytical Chemistry, Vol. 67,No. 14, July 15, 7995
2509
