Determination of Disulfide Bonds in Highly Bridged Disulfide-Linked

Jan 1, 1998 - A Program for the Identification of Disulfide Bonds in Proteins from Mass Spectra. Stephan Wefing, Volker Schnaible, and Daniel Hoffmann...
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Anal. Chem. 1998, 70, 136-143

Determination of Disulfide Bonds in Highly Bridged Disulfide-Linked Peptides by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry with Postsource Decay Michael D. Jones, Scott D. Patterson, and Hsieng S. Lu*

Department of Protein Structure, Amgen Inc., Amgen Center, Thousand Oaks, California 91320-1789

Matrix-assisted laser desorption/ionization mass spectrometry with postsource decay was used to generate fragment ions from peptide fragments containing heteropeptides linked together by two disulfide bonds. Postsource decay analysis of these peptide samples generates a series of singly charged fragment ions that, in addition to the peptide sequence ions, provide useful information for assigning disulfide arrangement in highly bridged disulfide-linked peptides. The assignment was made possible by fragmentation at peptide bonds between two Cys residues in a peptide that constitutes the highly bridged fragment, while retaining the disulfide linkage to the other peptide. Fragmentation using other types of instruments, such as quadrupole ion-trap mass spectrometry with collision-induced dissociation, usually did not generate such fragment ions. The data obtained from postsource decay also provide fragment ions derived from both symmetric and nonsymmetric cleavages of disulfide bonds. The present method is a highly sensitive technique which requires no further sample handling and should be complementary to other classical chemical methods. The method proved useful in facilitating the assignment of disulfide structure in tumor necrosis factor binding protein (TNFbp), which contains 162 amino acids and 13 disulfide bonds (Jones, M.; et al. Biochemistry, in press). Postsource decay analysis of large disulfidecontaining peptides usually produces no fragmentation but generates a series of high-intensity ions derived from both symmetric and nonsymmetric cleavages of disulfide bonds. Disulfide bond formation between Cys residues is a posttranslational event that commonly occurs in proteins synthesized in the endoplasmic reticulum. As the cysteine residues linked by disulfide bonds are usually apart in the primary structure, the intermolecular or intramolecular disulfide formation between them is associated with three-dimensional folding of the polypeptide * Address correspondence to this author at Amgen Inc., M/S 14-2-E, Amgen Center, Thousand Oaks, CA 91320-1789. Telephone: 805-447-3092. Fax: 805499-7464.

136 Analytical Chemistry, Vol. 70, No. 1, January 1, 1998

chain.1 In many occasions, disulfide bond formation contributes to the stability of the tertiary structure of folded protein molecules.2 In the cell, the formation of disulfide linkages and proper protein folding is facilitated by protein disulfide isomerase and various chaperones.1 In contrast, recombinant proteins expressed in bacteria usually require an in vitro oxidative refolding procedure to facilitate formation of disulfide bonds.3,4 Determination of disulfide linkages has been a challenging task since the original use of the elegant diagonal electrophoresis method developed by Brown and Hartley.5 Similar approaches used in recent years are still applied, except that electrophoresis is now replaced by various methods with increased sensitivity. Under appropriate conditions, a protein that contains disulfides can be enzymatically or chemically cleaved into peptides, and the peptides that contain disulfides or are linked by them can be identified. Generally, the disulfide-containing peptides are detected as peptides whose separation character or other molecular property is changed by cleavage of disulfide bonds. Recently, mass measurement of the disulfide-linked peptides before and after chemical reduction of disulfides has allowed rapid and direct assignment of the contributing peptides with no further separation. Mass spectrometric analyses by fast atom bombardment (FABMS),6 matrix-assisted laser desorption/ionization (MALDI-MS), and electrospray ionization (ESI-MS)7 have provided powerful techniques for such types of studies. In several cases, chemical reduction to break the disulfide bonds is unnecessary since the disulfide bonds can be fragmented in situ during the analysis. This has been seen as a fragmentation event at the source, or prompt fragmentation, in MALDI-MS8,9 or as a postsource decay (PSD) (1) Creighton, T. E. Proteins: Structure and Molecular Properties, 2nd ed.; W. H. Freeman and Co.: New York, 1993. (2) Creighton, T. E. BioEssays 1988, 8, 57-63. (3) Jones, M. D.; Narhi, L. O.; Chang, W. C.; Lu, H. S. J. Biol. Chem. 1996, 271, 11301-8. (4) Lu, H. S.; Hara, S.; Wong, L. W.; Jones, M. D.; Katta, V.; Trail, G.; Zou, A.; Brankow, D.; Cole, S.; Hu, S.; Wen, D. J. Biol. Chem. 1995, 270, 4775-83. (5) Brown, J. R.; Hartley, B. S. Biochem. J. 1966, 101, 214-28. (6) Yazdanparast, R.; Andrews, P. C.; Smith, D. L.; Dixon, J. E. J. Biol. Chem. 1987, 262, 2507-13. (7) Stewart, A. E.; Raffioni, S.; Chaudhary, T.; Chait, B. T.; Luporini, P.; Bradshaw, R. A. Protein Sci. 1992, 1, 777-85. (8) Patterson, S. D.; Katta, V. Anal. Chem. 1994, 66, 3727-32. (9) Crimmins, D. L.; Saylor, M.; Rush, J.; Thoma, R. S. Anal. Biochem. 1995, 1, 355-61. S0003-2700(97)00769-5 CCC: $14.00

© 1997 American Chemical Society Published on Web 01/01/1998

event observed with a scanning reflector in negative ion mode.10 The PSD fragmentation event was observed to be due to fragmentation of the disulfide bond. Nonsymmetric cleavage with retention of the sulfurs was found to be enthalpically favored over symmetric fragmentation or complete release of sulfurs.10 Collision-induced dissociation (CID) performed with FAB-MS has also been shown to fragment disulfide bonds.11 However, the above mass spectrometric methods are limited to the assignment of disulfide linkages in peptides linked by a single disulfide bond. Therefore, in a single peptide with two intrachain disulfide bonds or a peptide fragment in which more than two peptides are disulfide-linked and at least one peptide contains two or more Cys residues in its sequence, the challenge to disulfide characterization arrives when there are no suitable enzymatic or chemical cleavage sites in the peptide to identify subfragments containing only a single disulfide. In some cases, direct sequence analysis can be used to identify the linkages among these peptides.12,13 Partial reduction in conditions that resist disulfide exchange, followed by isolation and characterization of the partially reduced peptide, has also been used successfully to elucidate the disulfide linkages of some highly bridged peptides.13-15 However, it can become very difficult to isolate the partially reduced peptides if all disulfides exhibit similar reduction kinetics.13 Therefore, alternative and sensitive methods are needed for disulfide bonds that are difficult to assign. One of the approaches applied successfully in this report is MALDI-PSD, a method routinely used in the fragmentation of peptides for sequence determination either by scanning the reflectron16 or by using a curved field reflectron.17 By MALDI-PSD, we deduced disulfide linkages for some of the peptide fragments generated from an engineered recombinant human tumor necrosis factor binding protein (TNFbp), a monomeric molecule that has 162 amino acids with 13 disulfide bonds and plays a role in antagonizing the binding of TNF to its receptor.13 When PSD fragmentation of the peptide backbone between two Cys residues precedes cleavage of disulfide bonds, the identification of unique fragment ions obtained provides unequivocal evidence in making disulfide assignment for peptides containing two disulfide bonds. Fragmentation of sample induced by CID in an electrospray quadrupole ion-trap mass spectrometry (ESI-QIT-MS) analysis did not generate similar fragment ions. MATERIALS AND METHODS Materials. Premade matrix solution, HCCA (R-cyano-4-hydroxycinnamic acid; 33 mM in acetonitrile/methanol/water, 5:3:2 v/v), was purchased from Hewlett Packard (Palo Alto, CA). All other reagents were of the highest quality available. Escherichia (10) Zhou, J. E., W.; Poppe-Schriemer, N.; Standing, K. G.; Westmore, J. B. Int. J. Mass Spectrom. Ion Processes 1993, 126, 115-22. (11) Bean, M. F.; Carr, S. A. Anal. Biochem. 1992, 201, 216-26. (12) Haniu, M.; Acklin, C.; Kenney, W. C.; Rohde, M. F. Int. J. Pept. Protein Res. 1994, 43, 81-6. (13) Jones, M.; Hunt, J.; Liu, J.; Patterson, S.; Kohno, T.; Lu, H. Biochemistry, in press. (14) Gray, W. R. Protein Sci. 1993, 2, 1732-48. (15) Haniu, M.; Hui, J.; Young, Y.; Le, J.; Katta, V.; Lee, R.; Shimamoto, G.; Rohde, M. F. Biochemistry 1996, 35, 16799-805. (16) Kaufmann, R.; Spengler, B.; Luetzenkirchen, F. Rapid Commun. Mass Spectrom. 1993, 7, 902-10. (17) Cordero, M. M.; Cornish, T. J.; Cotter, R. J.; Lys, I. A. Rapid Commun. Mass Spectrom. 1995, 7, 1356-61.

Table 1. Sequences of TNFbp Peptide Fragments and the Assigned Sequence Positions peptides fragment 1 A1 B1 C1 fragment 2 A2 B2 C2 fragment 3 A3 B3 C3 fragment 4 A4 B4 C4

sequence

sequence calcd calcd parent position MH+ (Da) ion mass (Da) 1418.5

LRENEC VCTC LC

134-139 125-128 108-109

763.8 425.5 235.3

FCCS LSCQEK C

104-107 116-121

459.5 707.8 122.1

LCLPQ VSCSNCKK SLEC

154-158 140-146 148-151

573.3 868.3 451.2

43-57 58-64 68-81

1667.7 777.9 1554.7

1283.4

1890.1

3994.3 ECESGSFTASENHLR HCLSCSK EMGQVEISSCTVDR

coli-derived recombinant human TNFbp was expressed and purified according to methods described previously.13 The recombinant molecule contains 162 amino acids, including an N-terminal methionine at position 1 and 13 disulfide bonds. Table 1 shows four disulfide-containing peptide fragments derived from recombinant human TNFbp. Each fragment contains three peptides linked by two disulfide bonds. Fragment 1 was an HPLC fraction obtained from HPLC separation of a thermolysin digest. Fragments 2 and 3 were obtained from thermolysin digestion of a large TNFbp tryptic fragment Z (referred to as peptides Z11 and Z13, respectively, in ref 13). Fragment 4 is a tryptic fragment obtained from tryptic digestion of the whole TNFbp molecule. The specific sequence position in the TNFbp sequence and the respective theoretical mass (MH+) for each cysteinyl peptide in a fragment are also indicated in Table 1. The separation of these peptide fragments was performed by reversed-phase HPLC using procedures identical to those reported elsewhere.13 Mass Spectrometry. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) of E. coli-derived TNFbp peptides was performed on a Kompact MALDI IV mass spectrometer (Kratos Analytical, Ramsey, NJ) equipped with a curved field reflectron and a precursor ion gate. Spectra were obtained in either the linear mode, at an acceleration voltage of 20 kV, or the reflectron mode, with the linear voltage at 20 kV and the ion mirror set at 21 kV. The MALDI IV laser power can be adjusted from a relative scale (from 0 to 180) set by the manufacturer, in which the threshold of detection ranged from 45 to 65 in the linear mode and from 85 to 105 in the reflectron mode for postsource decay. Aliquots of sample (0.7 µL) and matrix solution (0.4 µL) were properly mixed and spotted onto the probe slide. The amount of samples loaded on the probe slide ranged from 1 to 10 pmol. Following an air-dry step, the sample was ready for analysis. In the linear mode (referred to as MALDI-MS), some prompt fragmentation of disulfide bonds in disulfide-linked peptide samples occurs at low laser power, but it can be enhanced by increasing the laser power during analysis.8 In the reflectron mode (referred to as MALDI-PSD), specific parent ions can be selected by the precursor ion gate and fragmented at a higher laser power (see above). For linear mode analysis, the instrument was calibrated externally with oxidized insulin B-chain (m/z 3497) Analytical Chemistry, Vol. 70, No. 1, January 1, 1998

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Figure 1. MALDI-MS analysis of fragment 1. (A) Prompt fragmentation during MALDI-MS analysis in linear mode. Prompt fragments with calculated masses are identified by letter codes as illustrated in the inset figure (see Table 1). (B) Fragment ion spectrum generated by MALDIPSD. Fragment ions are identified in Figure 2 and Table 2. Masses (881.2 and 982.0 Da) critical for assignment of disulfide linkages are identified by an asterisk in the spectrum.

and a HCCA matrix ion (m/z 172) in a least-squares regression analysis with the Kratos Kompact software. In reflectron mode analysis, the mass spectrometer was calibrated using a calibrant described by Cordero et al.17 Briefly, the peptide Pro14Arg, containing 14 proline residues and a C-terminal arginine (average protonated mass of 1534.8 Da), was fragmented, and the obtained fragment ions were calibrated on the basis of the expected fragment ion masses. Using the manufacture’s software, an elliptical equation was generated and used to automatically calibrate the fragmented ions after identification of the parent ion. Electrospray ionization quadrupole ion-trap mass spectrometry (ESI-QIT-MS) was performed on a Finnigan MAT ion-trap mass spectrometer (San Jose, CA) according to procedures previously described.18,19 Samples collected in RP-HPLC mobile phase (25100 pmol) were analyzed directly or concentrated to dryness in a polypropylene microcentrifuge tube and reconstituted in 15% (18) Jonscher, K. R.; Yates, J. R. Anal. Biochem. 1997, 244, 1-15. (19) Schwartz, J. C.; Jardin, I. Methods Enzymol. 1996, 270, 552-86.

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acetonitrile in 0.1% TFA. Analysis was performed by flow injection at a flow rate of 20 µL/min. Mass spectra were collected in the positive ion mode, scanning in the mass range of 150-1500 Da. Individual ions were selected for fragmentation in the ion trap and fragmented with a relative collision energy of 35%. RESULTS AND DISCUSSION Analysis of Fragment 1. During MALDI analysis in linear mode at increased laser power, in-source fragmentation, or prompt fragmentation, occurs at the disulfide bonds in disulfide-containing peptide fragments.8 A similar fragmentation can also be observed in a MALDI analysis of TNFbp fragment 1, shown in Figure 1A (see Table 1 for peptide sequences). In addition to the parent ion (1417.5 Da), two major fragment ions, 764.8 and 1184.2 Da, were obtained, representing a cysteine-reduced peptide A1 and a partially reduced but disulfide-linked peptide A1-B1, respectively. Although reduced peptides B1 and C1 with smaller mass ions were not detected in this analysis, the obtained prompt fragmentation data, together with sequence analysis as described else-

Figure 2. Disulfide structure of three disulfide-linked heteropeptide fragments. The theoretical masses for various fragment ions are indicated.

where,13 indicated that A1 and C1 peptides are linked to B1 peptide by two disulfide bonds. Fragment 1 was further subjected to MALDI-PSD analysis, and the observed data are shown in Figure 1B. A number of peaks are detected at masses lower than that of the parent ion with lower intensity, and the proposed assignments for most fragment ions are indicated in Figure 2 and Table 2. Two groups of triplet ions, i.e., 1217.4, 1186.0, and 1151.7 Da, as well as 793.4, 760.7, and 727.7 Da, show a mass difference of a sulfur between each group of ions, indicating nonsymmetric cleavage of disulfide bonds. The 1305.3 Da is a fragment ion for A1-B1-C1y1. Other observed fragment ions, i.e., 270.6, 371.9, 399.8, 484.6, 512.2, 612.6, and 640.8 Da, are from fragmentation of peptide A1 and represent a and b series of ions which provide good sequence information for the peptide. These ions are assigned as fragment ions of A1b2, A1a3, A1b3, A1a4, A1b4, A1a5, and A1b5, respectively. The most important ions representing the cleavage of Cys-Thr-Cys sequence for peptide B1 are 982.0, 881.2, and 555.3 Da. The 982.0 and 881.2 Da ions represent fragments A1-B1y2 and A1-B1y1, while the 555.3 Da ion is B1b3-C1. These data, therefore, provide unequivocal evidence for the disulfide arrangement of fragment 1, as shown in Figure 2. Two prominent ions at 253.6 and 382.7 Da are assigned as the loss of water from 270.6 and 399.8 Da (fragments A1b2 and A1b3, respectively). Analysis of Fragment 2. Figure 3A shows a mass spectrum derived from prompt fragmentation of fragment 2. In addition to the major parent ion (1285.2 Da), two fragment ions with masses of 708.9 and 1166.4 Da were detected, which correlate with the expected peptides A2 and A2-B2. Ions corresponding to B2, C2, and B2-C2 peptides were not found. In the MALDI-PSD analysis shown in Figure 3B, several fragment ions were detected, and their assignment is listed in Table 2. The fragmentation of peptide A2 in the intact fragment 2 occurred such that ions of A2y5-B2C2 (1170.4 Da), A2b5-B2-C2 (1136.1 Da), and A2y4-B2-C2 (1083.5 Da) are clearly seen. Fragmentation of peptide A2 from the C-terminus generates peptides A2y3 (404.3 Da) and A2y2 (277.1 Da). As shown in Figure 3B, the detection of two important fragment ions, B2b2-C2 (370.4 Da) and A2-B2y2 (913.4 Da), clearly demonstrates the cleavage of a peptide bond between two adjacent Cys residues in peptide B2 and confirms the disulfide linkage as indicated. Triplet ions (738.2, 705.8, and 672.2 Da) are centered (20) Biemann, K. Methods Enzymol. 1990, 193, 886-7.

Table 2. Assignment of Fragment Ions Obtained from MALDI-PSD of Fragments 1 and 2 fragment 1 assigned structure A1-B1-C1 A1-B1-C1y1 A1-B1 (+S) A1-B1 A1-B1 (-S) A1-B1y2 A1-B1y1 -b A1 (+S) A1 A1 (-S) B1-C1 A1b5 A1a5 B1b3-C1 A1b4 A1a4 A1b3 A1b3 (-H2O) A1a3 A1b2 A1b2 (-H20)

fragment 2

mass (Da) obsd calcd 1418.3 1305.3 1217.4 1186.3 1151.7 982.0 881.2 834.9 793.4 760.7 727.7 683.5 658.5 640.8 612.6 555.3 512.2 484.6 416.3 399.8 382.9 371.9 270.6 253.6 83.8

1418.6 1305.4 1217.3 1186.3 1153.3 984.0 882.9 794.8 763.8 730.8 657.8 642.7 614.7 554.7 513.6 485.6

assigned structure A2-B2-C2

mass (Da) obsd calcd 1283.4

A2y5-B2-C2 A2b5-B2-C2 A2y4-B2-C2 A2-B2y2 A2-B2y2 (-NH3)

1283.1 1197.5 1170.4 1136.1 1083.5 913.4 896.0

A2-B2y2 (-2NH3) A2 (+S) A2 A2 (-S) B2-C2 A2y3 A2y3 (-H2O) B2b2-C2 A2y2

879.0 738.2 705.8 672.2 577.2 472.6 404.3 387.2 370.4 277.1

880.0 738.8 707.8 674.8 578.6

1170.2 1137.2 1083.2 914.0 897.0

404.4 386.4 369.4 276.3

399.5 381.5 371.5 270.3 252.3

a Fragmentation nomenclature for Tables 1-3: Each peptide contributing to the disulfide-linked fragments is identified by a capital letter followed by a number representing the peptide fragment number (i.e., An, Bn, or Cn). Peptide fragments obtained from mass spectrometric analysis are indicated by their associated peptide followed by the fragmentation type and number by the classical naming system.20 For example, the b fragment of peptide B2 at the third amino acid would be indicated by B2b3. If this peptide is linked to peptide C2 by a disulfide, the fragment is identified as B2b3-C2. The presence of an additional sulfur or the loss of a sulfur in fragments (nonsymmetric fragmentation of disulfide bonds) is recognized by the name of the ions followed by (+S) and (-S), while the loss of ammonia or water (i.e., neutral losses) is recognized by (-NH3) or (-H2O). b Observed ion was not assigned.

at 705.8 Da for fragment A2 and, again, were derived from the nonsymmetric cleavage of a disulfide bond, as described previously. Neutral losses were also observed for fragments A2-B2y3 (913.4 Da) and A2y3. Analysis of Fragment 4. In several cases in the analysis of disulfide-bridged peptides derived from digests of TNFbp, MALDIPSD gave no more useful information than what could be obtained Analytical Chemistry, Vol. 70, No. 1, January 1, 1998

139

Figure 3. MALDI-MS analysis of fragment 2. (A) Prompt fragmentation during MALDI-MS analysis in linear mode. Prompt fragments (see Table 1) are identified by letter codes as illustrated in the figure inset. (B) Fragment ion spectrum generated by MALDI-PSD. Fragment ions are identified in Figure 2 and Table 2. Masses (370.4 and 913.4 Da) critical for assignment of disulfide linkages are identified by an asterisk in the spectrum.

by a linear mode analysis. This is especially true when the peptide fragment is larger in molecular size. Figure 4 compares the fragment ions obtained from prompt fragmentation of tryptic peptide fragment 4 of TNFbp (Figure 4A) in the linear mode to those generated during MALDI-PSD analysis (Figure 4B). With reference to the predicted masses seen in Table 1 for fragment 4, prompt fragmentation of the peptide revealed that the obtained fragment masses represent homolytic cleavages of either or both disulfide bonds (Figure 5A). In addition to the charged molecular ions (3997.5 for MH+ and 1999.2 for MH2+), fragment ions B4 (776.5 Da), C4 (1555.7 Da), A4 (1668.9 Da), B4-C4 (2331.9 Da), and A4-B4 (2445.3 Da) were, therefore, clearly detected. In the MALDI-PSD analysis (Figure 4B), the detection of 3980.4 Da represents neutral loss (-NH3) of the molecular ion from one of the two Arg residues of peptide A4 or C4. The detection of 3841.5 Da indicated the loss of an Arg residue from the parent ion following cleavage of the amide bond from either peptide A4 or peptide C4, while a prevalent neutral loss of ammonia from the 140

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remaining Arg residue generated a more intense ion at 3823.9 Da. Unlike prompt fragmentation, we observed that MALDI-PSD fragmentation of disulfide bonds produced characteristic triplets with a mass separation of 32-34 Da, corresponding to zero, one, or two sulfurs remaining on the charged fragments. Four sets of ions are clearly detected: 1522.8, 1554.8, and 1587.8 Da for peptide C5; 1635.2, 1667.3, and 1699.0 Da for peptide A4; 2295.1, 2329.8, and 2361.6 Da for peptide B4-C4; and 2408.0, 2442.1, and 2475.5 Da for peptide A4-B4. These triplet ion spectra were derived from both symmetric and nonsymmetric cleavages of the disulfide bonds. The high intensity of the 2475.5 Da ion indicated that fragmentation of the disulfide-linked heteropeptide favors nonsymmetric cleavage, with retention of the two sulfurs on fragment A4-B4. The pattern of the relative intensity in the triplet centered at 2442.1Da corresponds to that expected for the enthalpies of formation as predicted.10 In contrast, the cysteinyl peptide A5 or C5 favored loss of both sulfurs, suggesting that these peptides

Figure 4. MALDI-MS analysis of fragment 4. (A) Prompt fragmentation during MALDI-MS analysis in linear mode. Prompt fragments (see Table 1) are identified by letter codes as illustrated in the figure inset. (B) Fragment ion spectrum generated by MALDI-PSD. Each of the masses represents cleavage of the disulfide bond, and none was able to be used for determining disulfide connectivity.

are derived from fragmentation of the parent ion but not from secondary fragmentation of other disulfide-linked fragments, such as A4-B4 or B4-C4. Fragment ions of 1667.3 and 1554.8 Da are the fragments A4 and C4 with symmetric disulfide cleavage, which exhibit the lowest peak intensity within each of the triplet ions. This observation again indicates that these triplet ions were derived solely from cleavage of the parent ion. Each ion triplet was very intense relative to the parent ion, which is consistent with disulfide fragmentation observed in CID-FAB-MS analysis.11 However, these triplet ions are usually less intense in samples where extensive fragmentation had occurred during MALDI-PSD analysis (see Figures 1B and 5C). Cleavage of the peptide bonds did not occur in the analysis of fragment 5, and no disulfide could be assigned. The above data appear to indicate that larger fragments containing two disulfide bonds tend to produce a series of highintensity ions derived from cleavage of disulfide bonds. No further fragmentation occurs during MALDI-PSD analysis. Analysis of

complex heteropeptides containing more than three disulfide bonds was also not successful in obtaining fragment ions useful for assignment (data not shown). Many of these prompt fragments generated by linear MALDI-MS analysis contain a pair of intact disulfide and an unpaired cysteine and should be good candidates for PSD analysis to obtain secondary information for disulfide bond assignment. Unfortunately, these prompt fragments did not produce additional fragment ions following MALDIPSD analysis. Analysis of Fragment 3 by MALDI-PSD and ESI-QIT-MS Analyses. As indicated in several examples described in this report, for obtaining useful data for assigning disulfide bonds by MS analysis, a peptide containing two Cys residues that are linked to two other Cys peptides must be fragmented between the Cys residues prior to fragmentation of its disulfide bonds. However, not all MS/MS analyses using different types of equipment can produce similar fragment ions. An ESI-QIT-MS/MS analysis of Analytical Chemistry, Vol. 70, No. 1, January 1, 1998

141

Figure 5. Mass spectrometric analyses of fragment 3. (A) Parent ion scan performed in an ESI-QIT-MS analysis. (B) MS/MS analysis of a triply charged ion (630.2 Da) by ESI-QIT-MS. Fragment ions are identified in Table 3. (C) Fragment ion spectrum generated by MALDI-PSD. Fragment ions are identified in Figure 2 and Table 3. Masses (825.6, 940, and 1027 Da) critical for assignment of disulfide linkages are identified by an asterisk in the spectrum.

fragment 3, whose disulfide assignment has been previously determined by MALDI-PSD analysis,13 was, therefore, performed to evaluate if the obtained fragment ions are similar to the PSDgenerated ions and could be useful for disulfide assignment. Figure 5A shows a full MS scan of the sample to indicate that the parent ion is in the doubly and triply charged states (944.3 and 630.2 Da, respectively). The triply charged ion at the highest intensity was isolated in the ion trap and subjected to CID to produce fragments during an MS/MS analysis. Figure 5B is a full MS/MS scan for the detection of fragmentation. As assigned in Table 3, fragmentation of several peptide bonds and cleavage of disulfide bonds at various charged states occurs, but with no observation of ions associated with fragmentation between the two cysteinyl residues in peptide B3. Therefore, the linkage of two disulfide bonds could not be assigned. These data indicated that the fragmentation pattern is different from what was observed in the MALDI-PSD analysis of fragment 3 shown in Figure 5C. All PSD-generated fragment ions were in a singly charged state (see Table 3 for assignment). The two disulfide bonds of fragment 3 (see Figure 3 for structure) were assigned by the detection of three fragment ions of 1027, 940, and 825.6 Da. These ions, corresponding to B3y5-C3, B3y4-C3, and B3y3-C3 ions, respectively,13 are fragments obtained from cleavage between two cysteines in peptide B3. The above data indicated that ESI-QIT-MS/MS analysis of highly disulfide-bridged peptides can generate various fragment 142

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ions, but these ions do not aid in assigning disulfide bonds. Further evaluation and optimization of CID conditions with ESIQIT-MS may lead to more productive cleavages. For example, fragmentation of singly charged ions selected for MS/MS analysis may generate fragment ion spectra that are different from those observed for the fragmentation of doubly or triply charged ions but are similar to PSD-generated ion spectra (Scott Patterson, unpublished observation). It is also unclear that other disulfidelinked peptides of different sequences may be fragmented more efficiently in ESI-QIT-MS/MS analysis. CID-generated MALDIPSD of a disulfide-linked heteropeptide from Chinese hamster ovary cell-derived TNFbp generates fragment ions that are relatively similar to those from MALDI-PSD and are useful for making disulfide assignment.13 Mass Accuracy of MALDI Analysis. Compared to other mass spectrometry methods, accuracy in mass measurement is an inherent drawback of MALDI-MS analysis. Mass deviation (>1-2 Da) in some fragment ions has made structure assignment difficult (Tables 2 and 3). Assignment confidence could be enhanced by acquiring secondary spectra of the methyl esterified peptides and observing mass shifts that would be expected from the predicted structure. Mass accuracy may be enhanced by combining MALDI-PSD with current method enhancements such as pulsed (or delayed) extraction.21 As pulsed (or delayed) extraction reduces in-source collisional activation, the loss of PSD fragment ion yield can become significant. Therefore, a balance

Table 3. Assignment of Fragment Ions Obtained from Quadrupole Ion-Trap MS/MS Analysis and MALDI-PSD of Fragment 3 quadrupole ion trap MS/MS

a

MALDI-PSD

assigned structurea

charged state

obsd mass (Da)

calcd mass (Da)

B3-C3 (+S) A3y3-B3-C3 A3-B3-C3y2 A3By3-B3-C3 B3-C3 (+S) A3-B3-C3 A3-B3-C3 (-NH3) A3y3-B3-C3 C3 (+S) A3y3 A3y2

+1 +2 +2 +2 +2 +3 +3 +3 +1 +1 +1

1349.3 871.0 844.0 822.5 675.4 630.7 623.9 581.1 482.9 357.1 244.1

1348.6 870.9 843.9 822.5 674.8 629.6 624.0 581.0 483.2 356.3 243.2

assigned structure

obsd mass (Da)

calcd mass (Da)

A3-B3-C3 A3-B3y6-C3y3 B3-C3 (+S) B3-C3 B3-C3 (-S) B3y5-C3 B3y3-C3 B3 (+S) B3 B3y3-C3 B3y6 B3y3 B3y3 (-NH3) C3 A3y2 A3y2 A3-B3-C3y1

1890 1618 1350 1318 1284 1027 940 897 865 826 679 492 476 453 359 243 1560

1888 1615 1350 1318 1286 1028 941 901 869 827 683 493 478 452 357 244 1558

See footnote in Table 2.

of improved mass resolution versus generation of fragmentation must be maintained. Conclusion. MALDI-PSD is a powerful and sensitive mass spectrometric method useful to generate fragment ions from peptide fragments containing heteropeptides linked together by two disulfide bonds. The present report demonstrates that PSD analysis of peptide samples generates a series of singly charged fragment ions that, in addition to the peptide sequence ions, provide useful information for assigning disulfide arrangement in highly bridged disulfide-linked peptides. The assignment was made possible by fragmentation at peptide bonds between two Cys residues in a peptide that constitutes the highly bridged fragment. The use of electrospray QIT-MS with collision-induced dissociation did not generate a similar fragmentation pattern that was useful for disulfide assignment. PSD analysis also provides useful fragment ions to identify nonsymmetric cleavage of disulfide bonds. It has been previously noted that MALDI-PSD produces a large number of fragment ions,16 and, fortunately, these fragment ions are primarily singly charged, which makes assignment of fragment ions relatively easy. The method has proved to be useful (21) Kaufmann, R.; Chaurand, P.; Kirsh, D.; Spengler, B. Rapid Commun. Mass Spectrom. 1996, 10, 1199-208. (22) Biemann, K. Methods Enzymol. 1990, 193, 455-79.

in facilitating the assignment of disulfide structure in tumor necrosis factor binding protein, which contains 162 amino acids and 13 disulfide bonds.13 MALDI-PSD analysis should be highly complementary to other classical chemical methods to characterize disulfide-linked peptides. ACKNOWLEDGMENT We are grateful to Dr. Vish Katta and Mr. David Chow for their help in training and technical support for the operation of the quadrupole ion-trap mass spectrometer. NOTE ADDED IN PROOF During the preparation of the manuscript, Gorman et al. (Protein Sci. 1997, 6, 1308-1315) reported a similar observation for assignment of two disulfide bonds in a peptide fragment during MALDI-MS analysis of peptides from a viral protein.

Received for review July 16, 1997. Accepted October 10, 1997.X AC9707693 X

Abstract published in Advance ACS Abstracts, December 1, 1997.

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