Anal. Chem. 1996, 68, 3422-3430
Characterization of Unstable Intermediates and Oxidized Products Formed during Cyanogen Bromide Cleavage of Peptides and Proteins by Electrospray Mass Spectrometry Xinyi Zhang,† Lieve Dillen,† Koen Vanhoutte,‡ Walter Van Dongen,‡ Eddy Esmans,‡ and Magda Claeys*,†
Departments of Pharmaceutical Sciences and Chemistry, University of Antwerp (UIA and RUCA), Universiteitsplein 1, B-2610 Antwerp, Belgium
Products formed during cyanogen bromide (CNBr) digestion of r-endorphin, β-endorphin, and horse heart myoglobin are examined using reversed-phase high-performance liquid chromatography and electrospray mass spectrometry. It is demonstrated that unstable intermediate reaction products may be formed, as well as oxidized products when the CNBr reaction is performed in 0.1% TFA in water/acetonitrile (6:4 v/v) and that, under other conditions commonly employed for the CNBr cleavage reaction, unstable intermediate products are also generated. The formation of the expected cleavage products is found to be improved by adjusting the hydrolysis conditions. The structure of the intermediate formed from r-endorphin is examined using electrospray mass spectrometry in combination with low-energy collision-induced dissociation and tandem mass spectrometry and is shown to have a cyclic hydrated homoserine iminolactone part. The results obtained in this study explain the formation of partially cleaved proteins in the case of Met-Thrcontaining sequences, which likely have a cyclic hydrated homoserine iminolactone part instead of the putative homoserine residue. Cyanogen bromide (CNBr) cleavage is widely used in protein chemistry because it generally results in a limited number of smaller protein fragments, which are more amenable to sequencing or peptide mapping. CNBr selectively attacks methionine residues and yields peptidyl homoserine lactone and aminoacyl peptide fragments.1 Depending on the reaction conditions, the homoserine lactone part may be hydrolyzed and converted to a homoserine residue. Since Gross and Witkop2 have shown that CNBr reacts with methionine under mild conditions and that the reagent is very suitable for nonenzymatic cleavage of the methionyl peptide bond, this chemical digestion method has been successfully applied for protein identification.3-8 †
Department of Pharmaceutical Sciences. Department of Chemistry. (1) Gross, E. Methods Enzymol. 1967, 11, 238-255. (2) Gross, E.; Witkop, B. J. Biol. Chem. 1962, 237, 1856-1860. (3) Iadarola, P.; Zapponi, M. C.; Stoppini, M.; Meloni, M. L.; Minchiotti, L.; Galliano, M.; Ferri, G. J. Chromatogr. 1988, 443, 317-328. (4) Edelbaum, O.; Ilan, N.; Grafi, G.; Sher, N.; Stram, Y.; Novick, D.; Tal, N.; Sela, I.; Rubinstein, M. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 588-592. (5) Potter, S. M.; Johnson, B. A.; Henschen, A.; Aswad, D. W. Biochemistry 1992, 31, 6339-6347. ‡
3422 Analytical Chemistry, Vol. 68, No. 19, October 1, 1996
In most cases, CNBr cleavage reactions have been performed in 70% formic acid or 70% trifluoroacetic acid (TFA). A solution of 0.1% TFA in water/acetonitrile (6:4 v/v) has also been used as a suitable solvent for CNBr digestion.9 Allmaier et al.10 presented evidence for the formation of formylated peptides when 70% formic acid is used. Goodlett et al.11 demonstrated that the use of 70% TFA instead of 70% formic acid during CNBr digestion eliminates formylation of free amine or hydroxyl groups on reactive amino acid residues (e.g., serine, threonine, and lysine). Morrison et al.12 stated that there is a need for efficient, reproducible methods for obtaining CNBr fragments in preparative amounts while avoiding damage to amino acid residues. These authors showed that the use of 70% TFA results in less damage to tyrosine and tryptophan residues compared to the use of 70% formic acid and has the added benefit of increasing the extent of cleavage of the Met-Ser bond by 50% for human AI apolipoprotein. It has also been demonstrated that, if a methionine residue of a protein is oxidized and converted to a methionine sulfoxide residue, CNBr cleavage cannot occur.13 Using matrix-assisted laser desorption/ionization (MALDI) mass spectometry, Bai et al.14 have examined fragments formed by reaction of CNBr with electroblotted proteins on nitrocellulose membranes. It is clear from their study that there are discrepancies between predicted and measured masses of CNBr fragments of β-casein, ribonuclease A, and alkylated ribonuclease A, indicating that there is a need to re-evaluate the CNBr digestion method using mass spectrometric techniques. (6) Bouchon, B.; Jaquinod, M.; Klarskov, K.; Trottein, F.; Klein, M.; Dorsselaer, A. V.; Bischoff, R.; Roitsch, C. J. Chromatogr. B 1994, 662, 279-290. (7) Pittenauer, E.; Ferna´ndez, C. Q.; Schmid, E. R.; Allmaier, G. J. Am. Soc. Mass Spectrom. 1995, 6, 892-905. (8) Carr, S. A.; Bean, M. F.; Hemling, M. E.; Roberts, G. D. In Biological Mass Spectrometry; Burlingame, A. L., McCloskey, J. A., Eds.; Elsevier: Amsterdam, 1990; pp 621-652. (9) Glocker, M. O.; Arbogast, B.; Milley, R.; Cowgill, C.; Deinzer, M. L. Proc. Natl. Acad. Sci. U.S.A. 1994, 91,5868-5872. (10) Allmaier, G.; Chao, B. H.; Khorana, H. G.; Biemann, K. Proceedings of the 34th Annual Conference on Mass Spectrometry and Allied Topics, Cincinnati, OH, June 8-13, 1986; pp 308-309. (11) Goodlett, D. R.; Armstrong, F. B.; Creech, R. J.; van Breemen, R. B. Anal. Biochem. 1990, 186,116-120. (12) Morrison, J. R.; Fidge, N. H.; Grego, B. Anal. Biochem. 1990, 186, 145152. (13) Banerjee, S. K.; Mudd, J. B. Arch. Biochem. Biophys. 1992, 295, 84-89. (14) Bai, J.; Qian, M. G.; Liu, Y. H.; Liang, X.; Lubman, D. M. Anal. Chem. 1995, 67, 1705-1710. S0003-2700(96)00222-3 CCC: $12.00
© 1996 American Chemical Society
Chart 1. Amino Acid Sequencesa
a (a) R-Endorphin (M = 1745.9). The expected fragments of r CNBr cleavage are R-endorphin1-5 (Mr = 525.2) and R-endorphin6-16 (Mr = 1190.3). (b) β-Endorphin (Mr = 3438.0). The expected fragments of CNBr cleavage are β-endorphin1-5 (Mr = 525.2) and β-endorphin6-31 (Mr = 2882.4). (c) Apomyoglobin (Mr = 16 951.5). The expected fragments of CNBr cleavage are apomyoglobin1-55 (Mr = 6216.0), apomyoglobin56-131 (Mr = 8161.5), and apomyoglobin132-153 (Mr = 2512.9).
Problems experienced in our laboratory with the application of the CNBr reaction to cleave R-endorphin, which only contains one methionine residue, has prompted us to examine this reaction in more detail. To accurately determine the masses of the reaction products, electrospray (ES) mass spectrometry15,16 was used after isolating the products by reversed-phase high-performance liquid chromatography (RP-HPLC). ES in combination with collisioninduced dissociation (CID) and tandem mass spectrometry was applied in order to support the structure proposed for the unstable intermediate formed from R-endorphin. Our results indicate that an unstable intermediate is formed not only in the case of R-endorphin but also for larger peptides and proteins, such as β-endorphin and horse heart myoglobin. EXPERIMENTAL SECTION Materials. The peptides, R- and β-endorphin, and the protein horse heart myoglobin (Chart 1) were purchased from Sigma (St. Louis, MO). Cyanogen bromide (CNBr) solution (5 M) was obtained from Fluka (Buchs, Switzerland). Cyanogen bromide (97%) and trifluoroacetic acid (TFA) were from Janssen Chimica (Beerse, Belgium). Distilled water was prepared with a Milli-Q distillation apparatus from Millipore-Waters (Marlboroughs, MA). All solvents used were HPLC-grade. CNBr Cleavage. Method i: 6-7 nmol of R-endorphin, β-endorphin, or horse heart myoglobin was dissolved in 100 µL of 0.1% TFA in water/acetonitrile (6:4 v/v) and mixed with 1 µL of 5 M CNBr.9 The samples were reacted at room temperature in the dark for 16 h. Afterward, 1 mL of water was added, and the samples were incubated at different temperatures for different time periods. Following hydrolysis, the mixtures were evaporated with a Speed Vac centrifuge and analyzed by HPLC. Method ii: 30 nmol of R-endorphin was dissolved in 100 µL of 70% formic acid or 70% TFA, and CNBr (100-fold molar excess of the methionine present; 14 µmol) dissolved in the same solvent was added. The mixture was allowed to react in the dark for 25 h at 4 °C or at room temperature and then was diluted with 1 mL of water, cooled with liquid nitrogen, and freeze-dried. When 70% (15) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Science 1989, 246, 64-71. (16) Chait, B. T.; Kent, S. B. H. Science 1992, 257, 1885-1894.
Figure 1. HPLC separation of CNBr cleavage products obtained by reacting R-endorphin in a solution of 0.1% TFA in water/acetonitrile (6:4 v/v)). The reaction mixture was diluted with 1 mL of H2O and analyzed (a) directly without incubation or (b) after incubation at 60 °C for 3 h. (c) HPLC separation of products obtained by further incubation of peak 3 (Mr ) 1715.8) in water at room temperature for 16 h. For the designation of the peaks, see Table 1.
formic acid was used, the freeze-dried residue was redissolved in water, cooled, and freeze-dried again using a Speed Vac centrifuge.17 HPLC Separation of CNBr Cleavage Products. The CNBr cleavage products of R- and β-endorphin and horse heart myoglobin were subjected to HPLC on a RP column (Hipore C4; 5 µm, 4.6 mm × 250 mm, 30 nm pore size; Bio-Rad, Richmond, CA) using a Waters 600 MS gradient controller. The elution solvents were 0.1% TFA in water (A) and 0.1% TFA in acetonitrile (B). Chromatography was performed using a linear gradient from 100% solvent A either to 40% solvent B during 45 min for R- and (17) Compagnini, A.; Fichera, M.; Fisichella, S.; Foti, S.; Saletti, R. J. Chromatogr. 1993, 639, 341-345.
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Table 1. ES/MS Data of r-Endorphin Products Formed during CNBr Cleavagea or during Reaction of Component 3 HPLC peak 1 2 3
4
1′ 2′ 3′ 4′
a
measd Mr (RA,b %)
calcd Mr
R-Endorphin Products Formed during CNBr Cleavage 1190.2 1190.3 525.2 525.2 1715.8 (100) 1745.9c - 30 ) 1715.9 1737.8 (9) 1715.9 + 22 ) 1737.9 1753.6 (10) 1715.9 + 38 ) 1753.9 1697.9 (10) 1715.9-18 ) 1697.9 1785.0 (11) 1745.9 + 16 + 22 ) 1783.9 1761.8 (100) 1745.9 + 16 ) 1761.9 1784.2 (20) 1761.9 + 22 ) 1783.9 1799.9 (39) 1761.9 + 38 ) 1799.9 1715.8 (28) 1745.9-30 ) 1715.9 1738.2 (6) 1715.9 + 22 ) 1737.9 1753.8 (14) 1715.9 + 38 ) 1753.9
assignment R-endorphin6-16 R-endorphin1-5 intermediated of R-endorphin intermediate-Na+ salt intermediate-K+ salt dehydrated intermediate oxidized R-endorphin-Na+ salt oxidized R-endorphin oxidized R-endorphin-Na+ salt oxidized R-endorphin-K+ salt intermediate intermediate-Na+ salt intermediate-K+ salt
Reaction Products Formed from Component 3 during Incubation in H2O at Room Temperature for 16 h 1189.9 1190.3 R-endorphin6-16 525.3 525.2 R-endorphin1-5 1715.8 1745.9-30 ) 1715.9 intermediate of R-endorphin 1715.7 (100) 1745.9-30 ) 1715.9 intermediate of R-endorphin 1738.2 (63) 1715.9 + 22 ) 1737.9 intermediate-Na+ salt 1753.9 (58) 1715.9 + 38 ) 1753.9 intermediate-K+ salt
Solvent: 0.1% TFA in H2O/CH3CN (6:4 v/v). b Relative abundance. c Mr of R-endorphin. d The intermediate will be assigned later.
eV. Mass analyzer resolutions of MS1 and MS2 were approximately 200 and 350, respectively.
Figure 2. ES mass spectrum obtained for peak 3 (Figure 1a), corresponding to an unstable intermediate of R-endorphin. The inset shows the transformed spectrum.
β-endorphin or to 50% solvent B during 50 min for myoglobin. The flow rate was 0.8 mL/min, and the reaction products were monitored at a UV wavelength of 214 nm. Fractions were collected manually. After evaporation of the solvents with a Speed Vac centrifuge, the fractions were subjected to ES/MS or ES/ MS/MS analysis. Mass Spectrometry. Electrospray (ES) spectra were obtained with a VG Quattro-II tandem quadrupole mass spectrometer (Fisons, VG Biotech, Manchester, UK). The electrospray carrier solvent was a water/methanol mixture (50:50 v/v) containing 0.1% formic acid and was applied at a flow rate of 20 µL/min, with a split ratio of 1/20. The sample was introduced by loop injection. The capillary voltage was approximately 3600 V, the cone voltage was 25 V, and the nebulizer N2 gas flow was 15 L/h. Data were acquired in the multichannel mode by averaging 5-7 scans and scanning the mass range from 500 to 1500 Da at a rate of 6 s/scan. The mass resolution was 1000. Data processing was performed with the Masslynx (VG Biotech) software. The source temperature was 80 °C. Product ion spectra were obtained using collisional activation with argon in the hexapole gas cell. The gas cell pressure was approximately 2.7 × 10-3 mbar, and the collision energy was 39 3424
Analytical Chemistry, Vol. 68, No. 19, October 1, 1996
RESULTS AND DISCUSSION Reaction of R-endorphin, which only contains one methionine residue, with CNBr results not only in the two expected fragments (R-endorphin1-5 and R-endorphin6-16) but also in two additional products (Chart 1a). Figure 1a illustrates the HPLC chromatogram for the reaction mixture, which was obtained by reacting R-endorphin with CNBr. The first and second peaks showing Mr values of 1190.2 and 525.2 (Table 1), correspond to the expected CNBr fragments of R-endorphin. The fourth peak (Mr ) 1761.8) could be characterized as oxidized R-endorphin (1746.0 + 16 ) 1762.0). It is worth mentioning that oxidation of methionine to methionine sulfoxide residues in TFA/methanol or TFA/acetonitrile mixture has been noticed during solid-phase extraction for insulin II, glucagon, pancreatic polypeptide, and vasostatin I.18,19 The third peak (Mr ) 1715.8) could not be explained on the basis of what is normally expected for CNBr cleavage. Since this peak corresponds to the major peak in the chromatogram, we decided to characterize it and hypothesized that it could correspond to a reaction intermediate. Figure 2 shows the ES mass spectrum obtained for peak 3. In addition to the triply and doubly protonated molecules at m/z 572.9 and 858.5, respectively, ions are also detected at m/z 566.9, 580.2, and 585.5. The ion at m/z 566.9 indicates that the compound easily loses H2O, whereas the ions at m/z 580.2 and 585.5 correspond to the sodium and potassium adducts, respectively. To support the hypothesis of an unstable intermediate formed during the CNBr reaction, the reaction conditions were changed, and, more specifically, the reaction mixture was further incubated after dilution with water for different time periods and at different temperatures. Table 2 shows that the peak height ratios (RH) of peaks 1, 2, and 4 versus peak 3 are affected by varying the incubation temperature and the incubation time. When the (18) Linde, S.; Nielsen, J. H.; Hansen, B.; Welinder, B. S. J. Chromatogr. 1990, 530, 29-37. (19) Dillen, L.; Zhang, X. Y.; Claeys, M.; Liang, F.; De Potter, W. P.; Van Dongen, W.; Esmans, E. L. J. Mass Spectrom. 1995, 30, 1599-1604.
Table 2. Formation of CNBr Reactions Products of r-Endorphin under Different Experimental Conditionsa
a
incubation conditions used after CNBr cleavage and dilution with 1 mL of water
peak 1, R-endorphin6-16
peak 2, R-endorphin1-5
peak 4, oxidized R-endorphin
no incubation 3 h, room temperature 6 h, room temperature 24 h, room temperature 3 h, 40 °C 3 h, 60 °C
0.29 0.37 0.49 1.50 1.07 3.28
0.29 0.39 0.52 1.50 1.16 3.09
0.51 0.57 0.67 1.58 1.03 1.41
Peak height ratios (RH) of peak n versus peak 3.
Figure 3. ES mass spectra obtained for (a) peak 4′ (Figure 1c), corresponding to the R-endorphin intermediate and its Na+ and K+ salts, and (b) peak 4 (Figure 1a), containing mainly oxidized R-endorphin and its Na+ and K+ salts. The insets show the transformed spectra.
reaction mixture, after dilution with water, was incubated at room temperature for 3, 6, and 24 h or at 40 and 60 °C for 3 h, the height of peak 3 (Mr ) 1715.8) was decreased, and those of peaks 1, 2, and 4 were increased, especially for incubation at 60 °C for 3 h (Figure 1b). These results clearly indicate that peak 3 corresponds to an unstable reaction intermediate. In a following series of experiments, peak 3 was collected, freeze-dried immediately, and incubated at room temperature for 16 h, and the incubation products were analyzed by HPLC. It was not anticipated that four peaks would still appear in the HPLC chromatogram (Figure 1c). The ES/MS results are summarized in Table 1. The molecular weights of peaks 1′, 2′, and 3′ are 1189.9, 525.3, and 1715.8, respectively, which are the same as those found previously for products formed during CNBr digestion of R-endorphin. These results indicate that the expected fragments
resulting from cleavage of R-endorphin with CNBr can also be produced from the unknown component with a Mr of 1715.8. When the incubation temperature and time were increased, the amount of the component with a Mr of 1715.8 was decreased. These observations support our hypothesis that the component with a Mr of 1715.8 is an unstable intermediate. The ES spectrum of peak 4′ is clearly different from that of peak 4 (Figure 3), although their retention times are the same. The ES spectrum reveals the presence of the intermediate in the protonated form (Mr ) 1715.8) (Figure 3a). Peak 4′ was found to contain two other components, with Mr values of 1738.2 and 1753.9, differing from those of the intermediate by 22 and 38 u, respectively, thus corresponding to the sodium and potassium salts of the intermediate. Besides the intermediate and its salt forms (Mr ) 1715.8, 1738.2, and 1753.8), peak 4 mainly contains oxidized R-endorphin (Mr ) 1761.9) and its sodium and potassium salts (Mr ) 1784.2 and 1799.9) (Figure 3b). Comparison of the results obtained with and without further incubation with water indicates that Na+ and K+ salt formation of the intermediate increases following incubation with water. Scheme 1 gives details on the mechanism of the CNBr reaction and a possible rationalization for the formation of an unstable intermediate with a Mr of 1715.9. The scheme also rationalizes how the unstable intermediate, containing a hydrated homoserine iminolactone part, can be dehydrated or give rise to Na+ or K+ salt formation. The dehydrated intermediate is likely formed during the CNBr cleavage but may also be generated during ES ionization, for which the applied source temperature was 80 °C. To further support the proposed structure of the intermediate, ES in combination with CID and tandem mass spectrometry was performed. Product ion spectra were obtained for the MH22+ ion of R-endorphin (m/z 873.6) and the intermediate product (m/z 858.6) formed during CNBr reaction (Figure 4). The amino acid sequences and observed bn, yn, and an ion series of R-endorphin, its intermediate, and the dehydrated intermediate are given in Figure 5 (ion nomenclature according to Roepstorff and Fohlman20 and Biemann21). Most of the expected singly charged bn product ions are observed in the MH22+ spectrum of R-endorphin. In addition, doubly charged b122+ (m/z 658.7), b132+ (m/z 707.2), b142+ (m/z 763.9), and b152+ (m/z 813.7) and singly charged a4+ (m/z 396.9) and a5+ (m/z 528.1) ions are detected (Figure 4a). As expected, the m/z values of b2+, b3+, and b4+ in the MH22+ spectrum of the intermediate are identical to those of R-endorphin. The singly charged product ions from b5+ to b15+ obtained for the intermediate are shifted by 30 u, whereas the doubly charged product ions (20) Roepstorff, P.; Fohlman, J. Biomed. Mass Spectrom. 1984, 11, 601. (21) Biemann, K. Biomed. Environ. Mass Spectrom. 1988, 16, 99-111.
Analytical Chemistry, Vol. 68, No. 19, October 1, 1996
3425
Scheme 1. Reaction Mechanism for CNBr Cleavage of r-Endorphina
a
The expected fragments of R-endorphin are formed via route 1 but can also be obtained from the intermediate (I) via route 2.
from b52+ to b152+ are shifted by 15 u. The low-energy product ion spectrum (Figure 4b) shows b2+ (m/z 220.9), b3+ (m/z 277.7), b4+ (m/z 424.4), b5+ (m/z 525.9), b9+ (m/z 970.6), b12+ (m/z 1287.3), b14+ (m/z 1496.2), b102+ (m/z 529.1), b122+ (m/z 644.0), b132+ (m/z 692.7), b142+ (m/z 749.1), and b152+ (m/z 798.5) ions, which are in agreement with the proposed structure (I) of the intermediate. An interesting feature of the CID spectrum is the base peak corresponding to product ion b5+ (m/z 525.9), which has a low relative abundance in the case of R-endorphin. This result indicates that cleavage between the pentacyclic ring and the N-terminus of threonine in the intermediate occurs more readily than that for the normal amide bond between methionine and threonine in R-endorphin. It is also worth noting that the a5 ion, which is observed in the MH22+ spectrum of R-endorphin but which is not expected on the basis of the proposed structure of the intermediate, is absent from the MH22+ spectrum of the intermediate. The absence of the a5 ion further substantiates that the intermediate contains a hydrated homoserine iminolactone part instead of an uncleaved methionine residue converted to homoserine.14 The latter modification was proposed for CNBr reaction products of β-casein and alkylated ribonuclease A, which showed mass differences of -26, -29, and -34 u from predicted masses by MALDI time-of-flight (TOF) mass spectrometry. In our opinion, these reaction products probably have -30 u mass differences because of the limited mass resolution of the TOF analyzer employed in this study. Furthermore, they more likely 3426
Analytical Chemistry, Vol. 68, No. 19, October 1, 1996
correspond to unstable intermediates formed during CNBr digestion as found in the present study. The formation of partially cleaved proteins containing a homoserine residue has been reported to occur by Schroeder et al.22 and Han et al.23 for proteins containing Met-Ser and Met-Thr sequences. It is pointed out that, also in the case of R-endorphin, β-endorphin (vide infra), and horse heart myoglobin (vide infra), the unstable intermediate is found to be formed for Met-Thrcontaining sequences. We hypothesize that the hydroxyl group of the threonine residue provides stabilization of the intermediate by intramolecular hydrogen bonding. A homoserine residue has been positively identified at the relevant cycle during automated Edman sequencing of partially cleaved proteins containing MetThr sequences.22 However, on reaction mechanistic grounds, one would also expect the formation of a phenylhydantoin derivative of homoserine for the unstable intermediate containing the proposed cyclic hydrated homoserine iminolactone structure. Moreover, it is difficult to rationalize why a homoserine-Thr or homoserine-Ser peptide bond would be more unstable than any other peptide bond under the conditions applied for the CNBr reaction. It is worth mentioning that Pappin and Findlay24 reported that the putative ester bonds involving the homoserine (22) Schroeder, W. A.; Shelton, J. B.; Shelton, J. R. Arch. Biochem. Biophys. 1969, 130, 551-556. (23) Han, K.-K.; Richard, C.; Biserte, G. Int. J. Biochem. 1983, 7, 875-884. (24) Pappin, D. J. C.; Findlay, J. B. C. Biochem. J. 1984, 217, 605-603.
Figure 4. Low-energy CID spectra obtained for the MH22+ precursor ions of (a) R-endorphin (m/z 873.6) and (b) the R-endorphin intermediate (m/z 858.6). Product ions corresponding to dehydrated forms are marked with an asterisk.
Figure 5. Product ions observed in the MH22+ spectra of R-endorphin and the intermediate of R-endorphin.
carbonyl and the side chains of homoserine and Thr residues are clearly unstable both in 70% TFA and under the conditions employed in automated sequencing, suggesting that they were
Figure 6. HPLC separation of CNBr cleavage products formed from R-endorphin in 70% formic acid. After dilution with 1 mL of H2O, the reaction mixture was analyzed directly. For the designation of the peaks, see Table 3.
probably dealing with the same unstable intermediates found in the present study. The ES/MS analysis of the intermediate reveals the presence of a second component with a Mr of 1697.9 (Table 1), correspondAnalytical Chemistry, Vol. 68, No. 19, October 1, 1996
3427
Table 3. ES/MS Data of CNBr Cleavage Products Formed from r-Endorphin during Reaction in 70% HCOOH HPLC peak
measd Mr (RA, %)
calcd Mr
assignment
1 2 3
1189.9 525.4 1715.5 (100) 1698.0 (12) 1743.8 (100) 1771.8 (100) 1799.5 (17)
1190.3 525.2 1745.9 - 30 ) 1715.9 1715.9 - 18 ) 1697.9 1715.9 + 28 ) 1743.9 1715.9 + (28 × 2) ) 1771.9 1715.9 + (28 × 3) ) 1799.9
R-endorphin6-16 R-endorphin1-5 intermediate of R-endorphin dehydrated intermediate singly formylated intermediate doubly formylated intermediate triply formylated intermediate
4 5
Table 4. ES/MS Data of CNBr Cleavage Products Formed from β-Endorphina HPLC peak
measd Mr
calcd Mr
assignment
1 2 3 4
525.4 2882.5 3408.1 3454.0
525.2 2882.4 3438.0b - 30 ) 3408.0 3438.0 + 16 ) 3454.0
β-endorphin1-5 β-endorphin6-31 intermediate oxidized
a
Solvent: 0.1% TFA in H2O/CH3CN (6:4 v/v). b Mr of β-endorphin.
Figure 7. HPLC separation of CNBr cleavage products formed from β-endorphin in a solution of 0.1% TFA in water/acetonitrile (6:4 v/v). The reaction mixture was diluted with 1 mL of H2O and analyzed (a) directly without incubation or (b) after incubation at 60 °C for 3 h. For the designation of the peaks, see Table 4.
ing to a dehydrated form. Product ions corresponding to the dehydrated intermediate and dehydrated R-endorphin are detected in the MH22+ spectra, but there are some differences between the two spectra (Figure 4). Ions derived from dehydrated R-endorphin include b10+ (m/z 1070.7), b11+ (m/z 1198.1), b12+ (m/z 1299.7), b122+ (m/z 649.6), b132+ (m/z 698.6), b142+ (m/z 754.6), and b152+ (m/z 804.5). Since no b2, b3, b4, b5 and b9 ions can be detected, it is suggested that the dehydration occurs between the Ser(10) and Thr(15) residues of R-endorphin, probably in Ser(10) or Thr(12) (Figure 5). It also should be noted that the abundances of the product ions formed from dehydrated R-endorphin MH22+ ions are lower than those formed from R-endorphin. The MH22+ spectrum of the R-endorphin intermediate (Figure 4b) shows ions at m/z 953.5, 1268.7, and 1478.9, which have m/z values 18 u lower than those of the singly charged bn+ ions, b9+ (m/z 970.6), b12+ (m/z 1287.3), and b14+ (m/z 1496.2), 3428 Analytical Chemistry, Vol. 68, No. 19, October 1, 1996
Figure 8. HPLC separation of CNBr cleavage products formed from horse heart myoglobin in a solution of 0.1% TFA in water/acetonitrile (6:4 v/v). The reaction mixture was diluted with 1 mL of H2O and analyzed (a) directly without incubation or (b) after incubation at 60 °C for 3 h.
and which correspond to dehydrated forms of these ions. Similarly, ions are also noted at m/z 635.2, 683.3, 740.0 and 789.8, which have shifted by 9 u compared to the doubly charged bn2+ ions, b122+ (m/z 644.0), b132+ (m/z 692.7), b142+ (m/z 749.1), and b152+ (m/z 798.5), and which also correspond to dehydrated forms. It is worth noting that the dehydrated product ions mentioned above and designated as *bn and *bn2+ in Figure 4b all have an
Table 5. ES/MS Results Obtained for CNBr Cleavage Products of Myoglobina HPLC peak
measd Mr (RA, %)
1 2
2512.0 8161.9 (80) 10675.1 (100) 10721.4 16982.3 (100) 16937.7 (95) 14424.8 (55) 6216.0
3 4 5 1′ 2′ 3′ 4′ 5′ a
2512.3 8162.5 10721.8 16983.1 (100) 14425.0 (82) 6216.0
calcd Mr
assignment
Without Incubation in H2O 2512.9 8161.5 10704.4 - 30 ) 10674.4 10704.4 + 16 ) 10720.4 16951.5c + (16 × 2) ) 16983.5 16951.5 + 16 - 30 ) 16937.5 14407.5 + 16 ) 14423.5 6216.0 After Incubation in H2O at 60 °C for 3 h 2512.9 8161.5 10704.4 + 16 ) 10720.4 16951.5 + (16 × 2) ) 16983.5 14407.5 + 16 ) 14423.5 6126.0
myo132-153b myo56-131 intermediate56-153 oxidized myo56-153 doubly oxidized myo intermediate of singly oxidized myo oxidized myo1-131 myo1-55 myo132-153 myo56-131 oxidized myo56-153 doubly oxidized myo oxidized myo1-131 myo1-55
Solvent: 0.1% TFA in H2O/CH3CN (6:4 v/v). b Apomyoglobin. c Mr of apomyoglobin.
enhanced relative abundance compared to those of the corresponding bn+ and bn2+ ions. The finding that these ions easily lose H2O can be rationalized on the basis of the proposed structure of the intermediate. No corresponding dehydrated ions could be found for the b2+ (m/z 220.9), b3+ (m/z 277.7), and b4+ (m/z 424.4) ions. These results are consistent with the proposed structure of the intermediate, for which dehydration most likely involves the hydroxyl group of the modified methionine residue. In subsequent experiments, we also evaluated whether the intermediate was formed when performing the CNBr cleavage reaction in 70% formic acid and 70% TFA. A representive RP-HPLC chromatogram for the reaction products obtained using 70% formic acid is illustrated in Figure 6. The ES/MS results summarized in Table 3 indicate that the intermediates of singly and multiply formylated R-endorphin are detected but that no oxidized R-endorphin is formed. With respect to the products formed when 70% TFA was used, a very complex reaction mixture was obtained (results not shown), of which not all the components could be identified. However, the unstable intermediate was found to be formed. The RP-HPLC and ES results also indicated incomplete reaction and pointed to derivatization of some reaction products, so this method was abandoned for further experiments. Since we found no reports in the literature describing the formation of unstable intermediates during CNBr cleavage of peptides and proteins, we also evaluated whether one or more unstable intermediates are formed for other peptides and proteins. The results obtained for β-endorphin (Chart 1b), which was subjected to CNBr cleavage using the same reaction conditions as those employed for R-endorphin, are illustrated in Figure 7 and summarized in Table 4. Comparison of the HPLC results obtained for CNBr cleavage with and without incubation following addition of H2O reveals very clearly that, also here, an unstable intermediate is formed. The relative abundance of component 3 is drastically decreased following incubation. ES/MS analysis reveals that this component has a Mr of 3408.1, which is in agreement with the structure of the expected intermediate of β-endorphin (Table 4). It is worth noting that, as observed for R-endorphin, oxidized β-endorphin is formed during the CNBr reaction. Since the CNBr cleavage reaction is mostly applied to proteins in order to generate a limited number of smaller proteins, we also evaluated whether unstable intermediates could be detected in
the case of horse heart myoglobin. The HPLC chromatograms obtained for CNBr cleavage products of myoglobin (Chart 1c) formed under different experimental conditions are shown in Figure 8. As in the case of the peptides R- and β-endorphin, CNBr cleavage of myoglobin also resulted in the formation of unstable intermediates. Comparison of the HPLC results obtained for CNBr cleavage with and without incubation following addition of H2O shows that one of the peaks of fraction 4, which contains two unresolved peaks, disappears from the reaction mixture upon incubation. ES/MS analysis of this unstable myoglobin derivative reveals that this peak contains three components, one of them corresponding to the intermediate of singly oxidized apomyoglobin (Mr ) 16 937.5). In addition, ES/MS analysis also shows that peak 2 contains not only one of the expected fragments (apomyoglobin56-131) but also the intermediate of apomyoglobin56-153 (Mr ) 10 704.4), which upon incubation disappears from the reaction mixture. In contrast to the results obtained for the peptides R- and β-endorphin, the intermediate products were completely absent from the CNBr reaction mixtures of myoglobin upon incubation following addition of H2O. Besides the expected fragments, apomyoglobin1-55, apomyoglobin56-131, and apomyoglobin132-153, the CNBr reaction mixture also contains oxidized products. In addition to singly and doubly oxidized apomyoglobin, oxidized apomyoglobin56-153, oxidized apomyoglobin1-131, and the intermediate of singly oxidized apomyoglobin are formed (Table 5). CNBr digestion products of myoglobin have been examined using MALDI-TOF by Bai et al.14 and Zhang et al.25 Bai et al.14 have reported the formation of partially cleaved myoglobin, apomyoglobin1-131, and apomyoglobin56-153 but did not observe modifications, possibly because of the limited mass resolution of the TOF analyzer employed in their study. Zhang et al.25 found mass discrepancies for apomyoglobin1-131 (Mr ) 14 409) and intact apomyoglobin (Mr ) 16 951), which were typically 20-50 u in excess of the calculated values, but did not determine the source of these discrepancies. In view of the results obtained in the present study, it is suggested that the CNBr digests also contained (25) Zhang, W. Z.; Czernik, A. J.; Yungwirth, T.; Aebersold, R.; Chait, B. T. Protein Sci. 1994, 3, 677-686.
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oxidized partially cleaved fragments. Baker et al.26 encountered difficulties for the isolation of CNBr fragments formed from Apolipoprotein glutamine I. These authors were able to improve the yield of isolated fragments by an additional pretreatment step before performing chromatography on Bio-Gel P-30 and have attributed their difficulties to the tendency of some CNBr fragments to form aggregates. Taking into account results obtained in this study, it is likely that the isolation problems experienced by these authors were due to the formation of unstable intermediates, which upon further treatment were converted to the expected fragments. Partially cleaved CNBr fragments containing a methionine residue converted to homoserine have also been reported by Carr et al.8 for the cleavage of interleukin-1R and of the C-terminal domain of phosphorylase b kinase, and by Buzy et al.27 for the cleavage of the Aa6 subunit of the scorpion Androctonus australis hemocyanin. These partially cleaved fragments may correspond to intermediates containing a modified methionine residue with a hydrated homoserine iminolactone structure instead of a homoserine residue. It is worth mentioning that Buzy et al.,27 who performed the CNBr reaction in 70% TFA, also found a fragment corresponding to the partially cleaved protein containing one methionine sulfoxide residue. (26) Baker, H. N.; Jackson, R. L.; Gotoo, A. M., J. Biochemistry. 1973, 12, 38663871. (27) Buzy, A.; Gagnon, J.; Lamy, J.; Thibault, P.; Forest, E.; Hudry-Clergeon, G. Eur. J. Biochem. 1995, 233, 93-101. (28) Fenselau, C., Advances in Mass Spectrometry; Cornides, I., Horva´th, Gy., Ve´key, K., Eds.; Wiley: Chichester, 1995; Vol. 13, pp 129-149.
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CONCLUSION The present study shows that CNBr cleavage of peptides and proteins may give rise to the formation of unstable intermediates as well as oxidized products and that the yield of the expected cleavage products may be enhanced by adjusting the hydrolysis conditions. Identification based on molecular masses of fragments from chemical/proteolytic digestion is already an established method in protein chemistry.28 However, this method requires mass determination to be as accurate as possible and the cleavage as specific as possible. The general utility of this approach for CNBr digestion may be increased if the possibilities of oxidation of methionine residues and formation of unstable, partially cleaved intermediate products are taken into account.
ACKNOWLEDGMENT The work was funded by the Belgian National Fund for Scientific Research (Grants 9.0035.91 and 2.0133.94 N), the EU communities (Human Capital and Mobility Project ERB 4050 PL 93-2014), and the Flemish Federal Government. M.C. is indebted to the NFSR as a research director. Received for review March 7, 1996. Accepted July 24, 1996.X AC9602229 X
Abstract published in Advance ACS Abstracts, September 1, 1996.
