Ideication of disulfide-linked peptides by isotope profiles produced by peptic digestion of proteins in 50% ¹⁸O water

Abstract

Determination of the disulfide-bond arrangement of a protein by characterization of disulfide-linked peptides in proteolytic digests may be complicated by resistance of the protein to specific proteases, disulfide interchange, and/or production of extremely complex mixtures by less specific proteolysis. In this study, mass spectrometry has been used to show that incorporation of ¹⁸O into peptides during peptic digestion of disulfide-linked proteins in 50% ¹⁸O water resulted in isotope patterns and increases in average masses that facilitated ideication and characterization of disulfide-linked peptides even in complex mixtures, without the need for reference digests in 100% ¹⁶O water. This is exemplified by analysis of peptic digests of model proteins lysozyme and ribonuclease A (RNaseA) by matrix-assisted laser desorption/ionization–time of flight (MALDI-TOF) and electrospray ionization (ESI) mass spectrometry (MS). Distinct isotope profiles were evident when two peptide chains were linked by disulfide bonds, provided one of the chains did not contain the C terminus of the protein. This latter class of peptide, and single-chain peptides containing an intrachain disulfide bond, could be ideied and characterized by mass shifts produced by reduction. Reduction also served to confirm other assignments. Isotope profiling of peptic digests showed that disulfide-linked peptides were often enriched in the high molecular weight fractions produced by size exclusion chromatography (SEC) of the digests. Applicability of these procedures to analysis of a more complex disulfide-bond arrangement was shown with the hemagglutinin/neuraminidase of Newcastle disease virus.

Disulfide linkages can be crucial to the structure and function of a protein (Wedemeyer et al. 2000), and determination of these linkages can provide insights into the domain structure and folding of the protein (Wedemeyer et al. 2000). Knowledge of disulfide-bond connectivity can also facilitate X-ray crystallography (Jones and Kjeldgaard 1977), NMR (Klaus et al. 1993), and molecular modeling studies (Pitt et al. 2000). Disulfide linkages of a protein may be determined by ideication and characterization of disulfide-linked peptides (DLPs) from proteolytic or chemical digests of the protein.

If the sequence of a protein is known, then specific proteolysis with proteases such as trypsin can yield DLPs whose chromatographic behavior and/or mass are affected by reduction. Masses of resultant cysteinyl peptides may be used to idey the sequences containing cysteines involved in disulfide linkages (Morris and Pucci 1985; Yazdanparast et al. 1986 1987). However, extensively disulfide-linked proteins may be resistant to specific proteolysis (Brown and Hartley 1966; Smyth 1967). This may be particularly relevant to viral surface proteins, many of which are resistant to specific proteases, and which may yield very few peptides of interest (Boosman 1978; Moore and Cowan 1978; Kowalski et al. 1989; Zhou et al. 1992). In such cases, more aggressive proteolysis with combinations of specific proteases or nonspecific proteases such as pepsin or proteinase K may be required to generate peptides (Brown and Hartley 1966; Smyth 1967), with the drawback of generating complex proteolytic mixtures that can be significantly more difficult to interpret (Brown and Hartley 1966; Pitt et al. 2000).

DLPs may be indirectly ideied in peptide mixtures by comparison of chromatographic, usually reverse phase HPLC (RP-HPLC), behavior following reduction to highlight DLPs against a background of peptides whose behavior remains unchanged (Pitt et al. 2000). An alteration of the behavior of chromatographic peaks indicates the presence of DLPs that are thus collected for further analysis. However, complex mixtures of peptides, such as those produced by nonspecific proteases, may have so many overlapping chromatographic peaks that such chromatographic shifts may be masked. In such cases, further fractionation of complex mixtures into simpler mixtures may be required to yield peaks, which show movement after reduction.

Strong cation exchange HPLC (SCX-HPLC) has been described for the purification of DLPs from proteolytic peptide mixtures based on the greater positive charge of the multiple basic residues on the DLP N termini compared to single-chain peptides (Crimmins 1997). SCX-HPLC has been used to enrich DLPs and very basic single-chain peptides but by its nature may not be reliably used for purification of highly acidic DLPs and peptides with intramolecular disulfide bonds. Alternative methods may still be required to detect the full complement of DLPs in SCX-HPLC procedures.

Electrochemical detection (Sun et al. 1991) allows detection of DLPs following HPLC, but is impractical for routine disulfide analysis. DLPs cannot be readily labeled in a way that distinguishes them chromatographically while retaining their disulfide linkages. Fluorescent alkylating agents can be useful for the labeling of cysteinyl peptides (Gorman et al. 1987) and free cysteines (Yen et al. 2000); however, labeling generally requires a reduction step, which necessarily destroys information regarding disulfide connectivity.

Liquid chromatography (LC) coupled to electrospray ionization (ESI) mass spectrometry (LC-MS) is a powerful technique for analysis of peptides in chromatographic eluates (Bruins et al. 1987). Tandem mass spectrometry (MS/MS) in appropriately equipped ESI instruments offers the ability to derive sequence information from peptides (Hunt et al. 1983; Wilm 2000), but interpretation of often complicated MS/MS data requires knowledge of the disulfide linkage status of the peptide. Variation of ion-source parameters and/or collision-induced dissociation (CID) may be used to produce fragment ions characteristic of cotranslational and posttranslational modifications such as glycosylation and phosphorylation (Annan and Carr 1997). However, no such comparable method has been described to detect DLP ions by LC-MS. Such an on-line mass spectrometric system would be invaluable for rapid detection of DLPs of interest, which could then be analyzed by MS/MS or collected for further analysis. An ideal disulfide detection system would alter the mass spectrometric behavior of DLPs in a consistent manner, while retaining the disulfide linkage and preserving the chemical properties of the peptides so that downstream analysis could be carried out by conventional methods.

Enzymatic incorporation of ¹⁸O into peptides has been shown by mass spectrometry during proteolysis (Rose et al. 1983, 1988; Schnolzer et al. 1996) and PNGaseF deglycosylation of N-linked glycans from asparagine residues (Kuster and Mann 1999). Such incorporation has facilitated ideication of sites of N-linked glycosylation (Kuster and Mann 1999) and C-terminal peptides (Rose et al. 1983, 1988), peptide quantitation by matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry (MALDI-TOF-MS) (Mirgorodskaya et al. 2000), and differentiation of b and y ions for de novo peptide sequencing by MS/MS (Schnolzer et al. 1996; Shevchenko et al. 1997). ¹⁸O incorporation changes the mass of peptides without affecting their chemical properties and subsequent analysis. Isotope patterns of such peptides are also altered if digestions are performed in media containing <100% ¹⁸O water (Rose et al. 1983, 1988). These altered patterns aid in ideying C-terminal fragment ions resulting from MS/MS of labeled peptides (Schnolzer et al. 1996; Shevchenko et al. 1997). A strategy has been described for the ideication of DLPs from proteins digested in trypsin in 100% ¹⁸O water based on their mass increase (ABRF Electronic Discussion Group 1999). However, this strategy is hampered by the need for reference digests in 100% ¹⁶O water, the potential resistance of highly disulfide-linked proteins to trypsin digestion (Smyth 1967), the potential for peptide disulfide interchange at the alkaline pH optimum of trypsin (Sanger 1953; Ryle and Sanger 1955; Spackman et al. 1960; Brown and Hartley 1966; Morris and Pucci 1985), and the inconsistent incorporation of multiple ¹⁸O atoms into single-chain tryptic peptides (Rose et al. 1988; Schnolzer et al. 1996).

The impact of modulating the isotope profiles of DLPs by conducting digestion in <100% ¹⁸O water with less specific proteases has not been investigated. Accordingly, we have investigated pepsin-mediated incorporation of ¹⁸O into peptides following digestion of nonreduced disulfide-linked proteins in 50% ¹⁸O water, as a means of detecting DLPs mass spectrometrically via their isotope profiles. Size exclusion chromatography (SEC) was also investigated as a means of reducing the complexity of the peptic digests. The results of this study indicated that isotope profiling of peptides produced by peptic digestion in 50% ¹⁸O water fulfills most of the criteria of an ideal DLP detection method.

Results

Comparison of theoretical isotope profiles of peptides expected from proteolysis in ¹⁶O water and 50% ¹⁸O water

Proteolytic cleavage of peptide bonds involves the transfer of an hydroxyl group from water to the C-terminal carboxyl group produced by cleavage. The mass spectra of single-chain and disulfide-linked peptides from a digest in 100% ¹⁶O water consist of a series of peaks separated by 1 Da, predominantly owing to different proportions of ¹²C and ¹³C in the peptides (Fig. 1A ▶). The patterns of the relative intensities or ratios of these isotopic peaks vary with the mass of the peptides according to the corresponding variation in their ¹³C content. If the digestion buffer contains 50% ¹⁸O water, and the only transfer to any newly generated C terminus is via enzymatic cleavage of the peptide bond, then statistically 50% of any newly generated C terminus should incorporate the heavier oxygen isotope (Rose et al. 1988). The mass spectrum of a single-chain peptide from a digest in 50% ¹⁸O would appear as the superposition of two identical series of ¹⁶O-only isotope patterns with a 2 Da offset (Fig. 1B ▶).

Fig. 1.

Theoretical isotope patterns of peptides of the same mass. Theoretical isotope patterns were generated for singly charged peptides of varying disulfide bond configurations by the processes described in the text. Possible disulfide bond configurations that would give rise to each pattern are shown, with polypeptide chains represented by solid lines and disulfide bonds represented by dashed lines. Oxygen isotopes at the C terminus of each peptide are shown by ¹⁶O only (no ¹⁸O incorporation) and ¹⁶O/¹⁸O (statistical incorporation of equal proportions of ¹⁶O and ¹⁸O). Divisions of the m/z axis are 1 Da. (A) No ¹⁸O incorporation. (B–D) Statistical incorporation of ¹⁸O for one (B), two (C), and three (D) primary protease-mediated incorporation events.

A DLP consisting of two peptide chains, where one is the C terminus of the protein, only has one terminus labeled during ¹⁸O incorporation and would have an isotope pattern identical to that of a single-chain peptide, which also has only one terminus subjected to ¹⁸O incorporation (Fig. 1B ▶). Therefore, peptides that appear as unlinked by ¹⁸O mass spectrometry but that dissociate into two peptides after reduction probably contain the C terminus of the protein.

Similarly, a single-chain peptide with an intramolecular disulfide bond would have incorporated ¹⁸O at only one terminus and would have an isotope pattern identical to that of an unlinked peptide of the same mass (Fig. 1B ▶). Such a looped peptide would, however, display a mass shift of +2 Da following reduction of the internal disulfide bond.

A single-chain peptide derived from the C terminus of the protein, regardless of whether it contains an intramolecular disulfide bond, has no termini available for ¹⁸O incorporation and therefore would have the same isotope pattern as an ¹⁶O peptide (Fig. 1A ▶), which would be readily distinguished from those of nonterminal peptides and DLPs (Rose et al. 1983, 1988).

A peptic cleavage product consisting of two peptide chains linked by a disulfide bond, neither of which is the C terminus of the protein, would have two termini, each of which would have had a 50% chance of incorporating the heavier oxygen isotope: 25% of the DLPs would have incorporated only ¹⁶O at both termini and would have the same mass as the peptide from a 100% ¹⁶O digest; 25% would have incorporated ¹⁸O at only one terminus and would be 2 Da heavier; another 25% would have incorporated ¹⁸O only at the other terminus and would also be 2 Da heavier; and the remaining 25% would have incorporated ¹⁸O at both termini and would be 4 Da heavier. The isotope peak pattern of such a peptide would therefore consist of a superposition of identical series of ¹⁶O isotopic peaks displaced by 0, 2, and 4 Da, in the ratio of 1:2:1, respectively (Fig. 1C ▶). A peptide containing three chains, one of which is the C terminus of the protein, would also show this isotope pattern. Such isotope patterns would be readily differentiated from that of a single-chain peptide. Taking the sequence further, a DLP consisting of three chains with three termini, none of them the protein C terminus, would be expected to yield superimposed ¹⁶O patterns displaced by 0, 2, 4, and 6 Da, in the ratio of 1:3:3:1, respectively (Fig. 1D ▶). This isotope pattern would be distinguishable from that of peptides incorporating two or fewer ¹⁸O atoms.

The differences in isotope patterns between the peptides produced by proteolysis in ¹⁶O water and protease-mediated ¹⁸O incorporation in 50% ¹⁸O water, as discussed above, would be accompanied by average mass differences. The centroids of isotope envelopes of peptides containing ¹⁸O would be higher than those with only ¹⁶O, by 1 Da per ¹⁸O incorporation event. There would therefore be two criteria, isotope patterns and average masses, for prediction of the number of ¹⁸O incorporation events. In combination these properties define the profiles of isotope envelopes of peptide ions. Comparison of these profiles forms the basis for DLP ideication by ¹⁸O incorporation during peptic digestion in 50% ¹⁸O water.

Pepsin- and trypsin-mediated incorporation of ¹⁸O into single-chain peptides

The theoretical isotope profiles detailed above are those produced under ideal conditions in which no other mechanisms for ¹⁸O incorporation are taken into account. However, in addition to the primary event of proteolytic incorporation of ¹⁸O into peptides, serine proteases such as trypsin and other proteases have previously been reported to catalyze secondary ¹⁸O incorporation events (Rose et al. 1988; Schnolzer et al. 1996). This apparently occurs by transfer of ¹⁸O from water in the medium to the C terminus of peptides during incubation, via multiple cycles of transient covalent interaction between the peptide and protease (Rose et al. 1988; Schnolzer et al. 1996). In addition, secondary ¹⁸O incorporation events can occur via nonenzymatic mechanisms. To determine whether these phenomena could also affect isotope profiles produced by peptic cleavage, reduced and alkylated lysozyme was digested in 50% ¹⁸O water using trypsin or pepsin. The resulting ¹⁸O isotope profiles were compared with those predicted for each peptide resulting from a single ¹⁸O incorporation event.

Although reduced and alkylated lysozyme digests did not contain DLPs, both tryptic and peptic digests of this protein were observed to contain peptides with isotope profiles indicative of more than one ¹⁸O incorporation event per peptide when the digestions were performed in 50% ¹⁸O water (Tables 1 and 2). Based on average mass increases, 65% of the observed peptic peptides showed isotope ratios and average mass increases consistent with 0.9–1.3 ¹⁸O incorporation events (Table 1), which probably reflects primary proteolytic incorporation only when experimental errors are taken into account. Fewer peptic peptides, 35%, evidently had between 1.3 and 2 incorporation events, but peptides with more than 2 incorporation events were never apparent in this peptic digest.

Table 1.

Isotope profiles of single chain peptides from peptic digestion of reduced and alkylated lysozyme in 50% ¹⁸O water analyzed by MALDI-TOF-MS

^a Monoisotopic m/z observed in ¹⁸O digest.

^b Isotopic peak intensity indicated graphically. First bar represents the intensity of the monoisotopic MH⁺peak, with no ¹⁸O incorporation.

^{c 16}O isotope pattern based on elemental composition of peptide.

^{d 18}O isotope pattern based solely on hydrolytic incorporation of ¹⁸O, with no other incorporation events. For single peptides not derived from protein C terminus ¹⁸O patterns are those generated by statistical incorporation into one site. For single chain peptides derived from the protein C terminus, ¹⁸O patternsare equivalent to ¹⁶O patterns.

^e Difference in average masses of ¹⁸O patterns from calculated ¹⁶O patterns indicated under each displayed pattern.

^f Peptide derived from the lysozyme C terminus.

Table 2.

Isotope profiles of single chain peptides from tryptic digestion of reducedand alkylated lysozyme in 50% ¹⁸O water analzyed by MALDI-TOF-MS

^a Monoisotopic m/z observed in ¹⁸O digest.

^b Isotopic peak intensity indicated graphically. First bar represents the intensity of the monoisotopic MH⁺ peak, with no ¹⁸O incorporation.

^{c 18}O isotope pattern based solely on hydrolytic incorporation of ¹⁸O, with no other incorporation events. For peptides not derived from protein C terminus ¹⁸O patterns are those generated by statistical incorporation into one site.

^d Difference in average masses of ¹⁸O patterns from calculated 16O patterns indicated under each displayed pattern.

In contrast, all of the single-chain tryptic peptides were observed with isotope patterns and average mass increases indicative of at least 1.2 ¹⁸O incorporation events (Table 2). Furthermore, the tryptic peptides of m/z = 1675.8 and m/z = 1753.9 displayed average mass increases of 2.9 Da (Fig. 2D ▶) and 2.2 Da, respectively. These isotope profiles are inconsistent with one ¹⁸O incorporation event into single-chain peptides and indicate the potential for trypsin digestion to lead to false ideication of peptides as being disulfide-linked. Sequence analysis of the peptide of m/z = 1678.5 by MS/MS showed that it was assigned with the correct identity and also provided an explanation for the observed isotope profile. A series of y-type ions was generated by peptide-bond fragmentation during MS/MS, in agreement with the sequence assignment of this peptide as I₉₈VSDGNGMNAWVAWR₁₁₂ (Fig. 3 ▶). Ions that corresponded to cleavage events N-terminal to the asparagine at residue 103 all displayed isotope patterns and average mass increases indicative of more than two ¹⁸O incorporation events. In contrast, y ions of cleavage products C-terminal to asparagine 103 all displayed isotope patterns and average mass increases consistent with less than two ¹⁸O incorporation events (Fig. 3 ▶). These results indicated that in addition to trypsin-catalyzed ¹⁸O incorporation at the C terminus of this peptide, ¹⁸O was also nonenzymatically incorporated into the internal asparagine residue at position 103. The presence of an adjacent glycine residue at position 104 indicated that deamidation via cyclic imide formation and subsequent hydrolytic ring opening were responsible for the additional mass, partly from an additional proton from deamidation and partly from ¹⁸O incorporation during ring opening (Tam et al. 1988). In support of this observation, this peptide has also been observed to show altered isotope patterns in ¹⁶O digests consistent with a 1 Da mass increase caused by deamidation (data not shown). The lysozyme tryptic peptide of m/z = 1753.9 contains an asparagine–serine m that also has the potential to be subject to deamidation. This process would be expected to be more pronounced at the alkaline pH of trypsin digestion, and was therefore not observed for peptides with the corresponding asparagine residues produced by peptic digestion of lysozyme under acidic conditions (Table 1).

Fig. 2.

MALDI-TOF-MS isotope profiles of single-chain peptides produced from reduced and alkylated lysozyme by digestion in 50% ¹⁸O water. (A) Isotope profile of peptic peptide of m/z = 1362.9, representing one ¹⁸O incorporation event. (B) Isotope profile of peptic peptide of m/z = 1047.6, representing 1.7 ¹⁸O incorporation events. (C) Isotope profile of tryptic peptide of m/z = 1325.6, representing 1.6 ¹⁸O incorporation events. (D) Isotope profile of tryptic peptide of m/z = 1675.8, representing 2.9 ¹⁸O incorporation events. (a.i.) Absolute intensity.

Fig. 3.

MS/MS of single-chain tryptic peptide with isotope profile indicative of multiple ¹⁸O incorporation events. Masses and sequence of y ion series produced by MS/MS of the peptide of m/z = 1675.8 from reduced and alkylated lysozyme tryptic digest in 50% ¹⁸O water are shown. Insets show expanded views of isotope patterns of representative ions. ΔM_av deviations from theoretical ¹⁶O average masses are shown in bold above the m/z values or below the insets for all y ions. Differences in ΔM_av values of this peptide (2.32) and that shown in Table 2 (2.98) reflect the fact that the peptides were analyzed from different tryptic digests. (cps) Counts per second.

Two peptic peptides corresponding to the C terminus of lysozyme, which would not be expected to incorporate ¹⁸O by proteolysis, were observed with isotope patterns and average mass changes indicating an incomplete ¹⁸O incorporation event. A possible explanation for this observation is that the C-terminal leucine was susceptible to secondary enzymatic incorporation and/or secondary chemical incorporation had occurred. Regardless of this secondary incorporation, the isotope patterns of these C-terminal peptic peptides were not consistent with proteolytic incorporation of ¹⁸O.

Isotope profiles of peptides produced by peptic digestion of nonreduced RNaseA and lysozyme in 50% ¹⁸O water

Nonreduced and nondenatured RNaseA was subjected to digestion in both 100% ¹⁶O water and 50% ¹⁸O water under otherwise identical conditions. The resulting unfractionated ¹⁶O and ¹⁸O digests contained the same peptide repertoire when examined by MALDI-TOF-MS (Table 3). Closer examination of individual ion peaks within the mass spectra of the digests (Fig. 4B,C) revealed significant differences in the isotope profiles of peptides ideied as representing the same segment of the RNaseA sequence in the ¹⁶O and 50% ¹⁸O digests. The peptide of monoisotopic m/z = 1415.7 observed in RNaseA peptic digests in ¹⁶O water (Fig. 4B ▶, upper panel) corresponded to the RNaseA peptide Y₉₇KTTQANKHIIV₁₀₈, with a calculated monoisotopic MH⁺ of 1415.79. This peptide showed an isotope pattern with the monoisotopic peak the most abundant, as expected for a peptide of this mass (Fig. 4B ▶, upper panel). The same species observed in the digest performed in 50% ¹⁸O water displayed an isotope pattern (Fig. 4B ▶, lower panel) and average mass (Table 3) consistent with the incorporation of ¹⁸O into a single C terminus, with no additional ¹⁸O incorporation.

Table 3.

Isotope profiles of peptides observed by MALDI-TOF-MS analysis of unfractionated peptic digests of RNaseA performed in ¹⁶O water and 50% ¹⁸O water

^a DLP nomenclature follows (Pitt et al. 2000).

^b Monoisotopic m/z observed in ¹⁶O digest.

^c Isotopic peak intensities indicated graphically. First bar represents the intensity of the monoisotopic MH⁺ peak, with no ¹⁸O incorporation.

^d18O isotope pattern based solely on hydrolytic incorporation of ¹⁸O, with no other incorporation events. For single-chain peptides not derived from protein C terminus ¹⁸O patterns are those generated by statistical incorporation into one site. For two-chain peptides ¹⁸O patterns are generated by statistical incorporation into two sites.

^e Difference in calculated average mass of ¹⁸O pattern from ¹⁶O pattern indicated under each displayedpattern.

Fig. 4.

MALDI-TOF-MS isotope profiles of peptic peptides of nonreduced RNaseA. (A) An unfractionated 50% ¹⁸O water digest of RNaseA analyzed by MALDI-TOF-MS. Peak labels indicate observed MH⁺ ions before (upper) and after (lower) reduction of the digest. (B,C) Detailed isotope profiles of an unlinked peptide with m/z =1415.7 (B) and a two-chain DLP of monoisotopic m/z = 2007.7 (C), observed in unfractionated 100% ¹⁶O (upper) and 50% ¹⁸O (lower) water digests. (D) An RNaseA peptic peptide of m/z = 1198.5 from the digest in 50% ¹⁸O water before (upper) and after (lower) reduction.

The isotope pattern of the RNaseA peptic peptide with m/z = 2007.7 evident in the MALDI-TOF mass spectrum from the unfractionated ¹⁶O digest (Fig. 4C ▶, upper panel) was characteristic of peptides of this mass with the MH⁺ + 1 peaks most abundant but only marginally more than the MH⁺ peak (Fig. 4C ▶). The same peptide observed in the unfractionated 50% ¹⁸O water digest displayed a significantly altered isotope profile in which the MH⁺ + 2 peak had much greater intensity than the MH⁺ peak (Fig. 4C ▶, lower panel). The overall isotope profile was also broader, with increased intensity of MH⁺ + 2, MH⁺ + 4, and MH⁺ + 6 peaks. The isotope pattern and average mass increase of 2.7 Da indicated that this peptide had been subject to more than two ¹⁸O incorporation events. The mass of the peptide at m/z = 1415.7 was not affected by reduction of the digest prior to MALDI-TOF-MS (Fig. 4A ▶), confirming it as a single-chain peptide with no intrachain disulfide bond. In contrast, the peptide at m/z = 2007.7 disappeared upon reduction, with the appearance of ions at m/z = 1261.4 and to a lesser extent m/z = 1332.5 (Fig. 4A ▶). This was consistent with the peptide C₁₁₀EGNPYVPVHF₁₂₀ (calculated MH⁺ = 1261.56) disulfide-linked to peptide C₅₈SQKNVA₆₄ (calculated MH⁺ = 749.35). Alternatively, A₁₀₉CEGNPYVPVHF₁₂₀ (calculated MH⁺ = 1332.59) disulfide-linked to peptide C₅₈SQKNV₆₃ (calculated MH⁺ = 678.32) would have given rise to a DLP with the same mass. These observations are in agreement with the known sequence and disulfide-bond arrangement of RNaseA (Hirs et al. 1960). Because ions at m/z = 749.3 and m/z = 678.3 were not observed in reduced spectra, the DLP ion at m/z = 2007.7 was subjected to MALDI-PSD analysis (Fig. 5 ▶) in an attempt to derive confirmatory sequence and disulfide-bond cleavage information. Although no MALDI-PSD disulfide-bond cleavage was observed, fragments were observed that were consistent with the C₁₁₀–F₁₂₀ or A₁₀₉–F₁₂₀ disulfide linked to C₅₈–A₆₄ or C₅₈–V₆₃. These fragments corresponded to the facilitated cleavage at two internal proline residues (Hunt et al. 1983) of C₁₁₀–F₁₂₀ to produce C-terminal fragments P₁₁₄–F₁₂₀ and P₁₁₈–F₁₂₀. Lower intensity b- and y-type ions were also observed that were consistent with this segment of the RNaseA sequence.

Fig. 5.

MALDI-PSD analysis of a putative disulfide-linked peptide of RNaseA. The putative disulfide-linked peptic peptide of RNaseA of m/z = 2007.7, in an unfractionated 100% ¹⁶O water peptic digest, was analyzed by MALDI-PSD. Peak labels indicate monoisotopic m/z values. The sequence of the peptide is indicated, with theoretical proline-directed fragmentation events most likely to produce the predominant PSD fragment ions observed. Less intense b and y ion series corresponding to the sequence of this peptide are indicated. The alanine residue that can occupy either A₅₄ or A₁₂₁ (see text for details) is indicated by parentheses.

The C₅₈–C₁₁₀ disulfide bond was also evident in other peptides at m/z = 3186.7, 3257.8, 3565.4, and 3635.8 in the unfractionated digest, representing ragged cleavage (Table 3). In addition to the interchain disulfide bond between C₅₈ and C₁₁₀, these peptides contained an intrachain bond between C₆₅ and C₇₂. These peptides had isotope profiles consistent with two-chain DLPs (Fig. 1C ▶). Upon reduction, single-chain peptides were observed at m/z values consistent with A₅₆–Y₇₃ and A₅₆–Y₇₆ containing reduced C₅₈, C₆₅, and C₇₂ and at m/z values consistent with A₁₀₉–F₁₂₀ and C₁₁₀–F₁₂₀ containing reduced C₁₁₀ (data not shown).

The RNaseA peptic peptide with m/z = 1198.5, observed in the unfractionated 50% ¹⁸O water digest (Fig. 4A ▶), displayed the isotope ratios (Fig. 4D ▶) and average mass (Table 3) expected for a single-chain peptide. However, reduction of the unfractionated 50% ¹⁸O water digest resulted in disappearance of the ion at m/z = 1198.5 and appearance of an ion with the same isotope profile at m/z = 1200.5 (Fig. 4D ▶). This 2 Da mass shift was consistent with reduction of an internal disulfide bond between the two cysteines of RNaseA peptide V₆₃ACKNGQTNCY₇₃ (calculated reduced monoisotopic MH⁺ = 1200.50), in agreement with the known disulfide-bond pattern of this protein.

In addition to the RNaseA peptic peptides detailed above, other RNaseA peptides derived from digestion in 50% ¹⁸O water displayed isotope patterns and average mass increases consistent with their disulfide linkage status (Table 3). Between 0.8 and 1.8 ¹⁸O incorporation events were evident for all assigned single-chain RNaseA peptides, as was the case for the single-chain peptic peptides of reduced and alkylated lysozyme. Isotope profiles of peptides assigned as DLPs all had isotope patterns and average mass increases indicating incorporation of ¹⁸O at between 2.6 and 3 sites. Ion suppression arising from the high degree of complexity of the digests probably resulted in failure to observe all single-chain peptides and DLPs of RNaseA. Nevertheless, the data obtained served to illustrate the potential of ¹⁸O isotope profiling for detection of DLPs. Analysis of digests after reduction and PSD analysis of selected peptides confirmed DLP ideication made by isotope profiling.

Peptic digestion of nonreduced and nondenatured lysozyme in 50% ¹⁸O water revealed a peptide at m/z = 1994.1 in the unfractionated digest (Fig. 6A ▶, upper panel) that displayed an ¹⁸O profile expected for a single-chain peptide of this mass with an incomplete additional ¹⁸O incorporation event (Fig. 6B ▶) but that dissociated upon reduction with concomitant appearance of two ions at m/z = 1093.3 and m/z = 903.1 (Fig 6A ▶, lower panel). Examination of the ion at m/z = 903.1 showed it to have the isotope profile (Fig. 6C ▶) with minimal ¹⁸O incorporation. The mass of this peptide was consistent with its derivation from the C terminus of the lysozyme sequence W₁₂₃IRGCRL₁₂₉ (calculated MH⁺ = 903.49). The ion at m/z = 1093.3 showed an isotope profile typical of incorporation of ¹⁸O into a single C terminus with an incomplete secondary incorporation event (Fig. 6D ▶), and was localized to the N terminus of the lysozyme sequence K₁VFGRCELAA₁₀ (calculated MH⁺ = 1093.57). This arrangement concurred with the known disulfide-bond configuration of lysozyme (Spackman et al. 1960; Canfield and Liu 1965), in which cysteine at position 6 is disulfide-linked to cysteine 127. The presence of peptide ions separated by 71 Da (Fig. 6A ▶), indicative of ragged peptic cleavage at alternate alanine residues, confirmed localization of the DLP to the sequence of lysozyme.

Fig. 6.

MALDI-TOF-MS isotope profile of an intermolecular disulfide-linked peptide containing the protein C terminus. (A) MALDI-TOF-MS spectra of an unfractionated lysozyme peptic digest before (upper) and after (lower) reduction. (B) Enlarged view of the ion at m/z = 1994.1 from panel A (nonreduced). (C,D) Enlarged views of the ions at m/z = 903.1 (C) and m/z = 1093.3 (D) from panel A (reduced). Ions corresponding to ragged peptic cleavage at alanine residues are indicated.

The lack of significant ¹⁸O incorporation observed for the above C-terminal peptide contrasted with the level of ¹⁸O incorporation for the equivalent reduced and alkylated peptic peptide observed from the digest of reduced and alkylated lysozyme. This observation indicates that secondary incorporation into the carboxamidomethylated single-chain peptide was caused substantially by chemical rather than enzymatic incorporation. However, greater accessibility of the C-terminal leucine to pepsin in the single-chain peptide than in the disulfide-linked two-chain peptide cannot be ruled out.

¹⁸O isotope profile stability during LC-MS

MALDI-TOF-MS involves rapid crystallization of peptides in microliter quantities of acidic matrix solutions with low ¹⁶O water content, which allows minimal opportunity for back exchange with ¹⁶O water. In contrast, ESI-MS, and particularly LC-MS, requires exposure of ¹⁸O-labeled peptides to larger volumes of aqueous ¹⁶O buffers for more than 1 h and a correspondingly greater opportunity for back exchange (Rose et al. 1988). The stability of isotope profiles of ¹⁸O-labeled DLPs during LC-MS of peptic digests was therefore investigated. Nonreduced RNaseA was digested with pepsin in 50% ¹⁸O water, and the digest was subjected to RP-HPLC-MS. The peptide of m/z = 2007.7, previously ideied by MALDI-TOF-MS as an RNaseA DLP, was readily observed by LC-MS (Fig. 7A ▶) and displayed the same isotope profile as seen for this peptide in unfractionated digests by MALDI-TOF-MS (Fig. 7B ▶), although at the lower resolution typical of a single-quadrupole instrument. This result indicated that the ¹⁸O label introduced into peptides by digestion in 50% ¹⁸O water is stable in ¹⁶O buffers over the duration of LC-MS analysis under the conditions used. Longer term storage of ¹⁸O-labeled peptides in ¹⁶O buffers is not recommended because of pH-dependant exchange (Rose et al. 1988). ¹⁸O-labeled peptides stored in ¹⁶O buffers at −20°C were observed to lose their ¹⁸O label over periods of several days to weeks (data not shown); however, lyophilized ¹⁸O-labeled peptides stored under dry nitrogen at −20°C retained their label indefinitely.

Fig. 7.

Stability of ¹⁸O profiles to analysis by LC-MS and MALDI-TOF-MS. Previously ideied disulfide-linked RNaseA peptide of m/z = 2007.7 from peptic digest in 50% ¹⁸O water, as visualized during LC-MS (A) and MALDI-TOF-MS (B) of an unfractionated digest.

Size exclusion chromatography (SEC)

Peptic digestion, particularly of large proteins, can yield complex mixtures that need to be simplified by fractionation before DLPs can be ideied or isolated. The use of RP-HPLC and SEC for the fractionation of peptic digests of RNaseA was compared (Fig. 8 ▶). RP-HPLC generated complex chromatograms with large numbers of peaks, each containing peptides related by hydrophobicity (Fig. 8A ▶). In contrast, SEC of the same digest generated simpler chromatograms (Fig. 8B ▶) with fewer peaks, each containing larger numbers of peptides related by size (Fig. 8C ▶). Ragged peptic cleavage peptides, which may have had quite different hydrophobicities, coeluted in SEC fractions as observed by MALDI-TOF-MS (Fig. 8C ▶). Closer examination of the peptides that coeluted with the m/z = 2007.7 DLP in SEC fraction 2 showed mass differences indicative of ragged peptic cleavage of alanine and valine residues (Fig. 9 ▶). All were consistent with the known sequence and disulfide-bond configuration of RNaseA, although the presence of multiple valine and alanine residues at the termini of this DLP resulted in several possible peptide configurations for some of the observed masses. Although the intensities of the cleavage product ions differed, they showed isotope profiles typical of DLPs when digestions were performed in 50% ¹⁸O water. The preservation of ¹⁸O isotope profiles during SEC also indicates the stability of these profiles to limited exposure to ¹⁶O buffers under the conditions used for chromatography.

Fig. 8.

HPLC fractionation of an RNaseA peptic digest. RNaseA peptic digest in 50% ¹⁸O water, separated by (A) RP-HPLC or (B) SEC. Chromatograms show absorbance at 214 nm. Numbers on the SEC chromatogram indicate SEC peaks analyzed by subsequent MALDI-TOF-MS. (C) MALDI-TOF-MS spectra of an unfractionated digest (top) and indicated SEC fractions.

Fig. 9.

Demonstration of ragged peptic cleavage products. Enlarged view of the MALDI-TOF-MS spectrum of SEC fraction 2 from Figure 8C ▶. Peak labels indicate observed monoisotopic m/z values. Possible peptide sequences that would give rise to MH⁺ values corresponding to the observed m/z values are shown.

Evaluation of ¹⁸O labeling for ideication of DLPs of Newcastle disease virus (NDV) hemagglutinin/neuraminidase (HN)

Newcastle disease virus hemagglutinin/neuraminidase (NDV HN) was digested with pepsin in both 100% ¹⁶O and 50% ¹⁸O water, and the digests were separated by SEC (Fig. 10A ▶). Individual SEC fractions of peptic digests were examined by MALDI-TOF-MS, and the isotope profiles of the observed peptides were compared. Larger peptides eluted in the early SEC fractions (Table 4). Comparison of the isotope profiles of ions in these fractions from ¹⁶O and ¹⁸O digests indicated the presence of DLPs. DLP masses were consistent with the known disulfide bonds of NDV HN (Pitt et al. 2000).

Fig. 10.

Ideication of DLPs from NDV HN by isotope profiling. (A) Peptic digests of NDV HN separated by SEC after digestion in 100% ¹⁶O water (upper) and 50% ¹⁸O water (lower). Vertical lines and numbers indicate fractions collected for analysis by MALDI-TOF-MS. (B–D) Enlarged views of spectra to show the isotope profiling of DLPs of different m/z values in SEC fractions 4 (B), 7 (C), and 3 (D) of ¹⁸O (upper) and ¹⁶O digests (lower).

Table 4.

Ideication of NDV HN DLPs

^a DLP nomenclature follows (Pitt et al. 2000).

^b Assignments represent DLPs previously reported for NDV HN.

^c Unless otherwise specified monoisotopic values are presented.

^d Differences in average mass of ¹⁶O and ¹⁸O isotope patterns are presented for the most abundant representative ion for each particular assignment where sufficient isotopic resolution was obtained.

^e Predominant SEC fraction in which ions were observed.

^f Average masses due to insufficient resolution.

The DLP of m/z = 3130.7 from SEC fraction 4 of the digest in 50% ¹⁸O water showed an isotope pattern and increase in average mass indicative of more than two ¹⁸O incorporation events (Fig. 10B ▶). This peptide mass was not observed in the previous study, and differed from the previously reported mass representing the DLP of F₁₆₁–D₁₇₉/S₁₉₄–Q₂₀₄ (observed m/z = 3245.8) by the mass of an aspartate (F₁₆₁–F₁₇₈/S₁₉₄–Q₂₀₄). The m/z = 3245.8 DLP observed in the previous study was observed in this study in the same SEC fraction as the m/z = 3130.7 DLP (data not shown). These peptides had very similar isotope profiles, indicating their relationship as DLPs in agreement with the known sequence and disulfide linkage pattern of NDV HN.

In contrast to the above two-chain DLP, the peptides observed with m/z = 2004.1, 2105.0, and 2206.1 represent ragged peptic cleavage products of the same intrachain DLP of sequence T₅₂₇–A₅₄₆. ¹⁸O isotope patterns and average mass values of these peptides were consistent with incorporation of ¹⁸O into a single C terminus, with an incomplete secondary incorporation event by other mechanisms, in confirmation of this disulfide linkage arrangement (Fig. 10C ▶).

DLPs of m/z > 4000 displayed reduced MALDI-TOF-MS resolution, increased peak broadness, and an increase in average mass indicative of multiple ¹⁸O incorporation (Fig. 10D ▶). The peptide of monoisotopic m/z = 4324.3 is typical of such a peptide and corresponded to a known DLP of NDV HN (Table 4).

Except for high mass disulfide-linked glycopeptides, peptides were observed representative of all the disulfide linkages of NDV HN (Table 4). Failure to observe these high mass glycopeptides in complex mixtures was not unexpected because of the lability and heterogeneity of these peptides, which required further purification in order to be observed in the previous study. Although the exact positions of peptic cleavage varied from the original report, peptides were apparent that could be correlated to the known sequence and disulfide linkage pattern of NDV HN. In all cases, peptides ideied as DLPs had increases in the m/z of their most abundant isotope consistent with disulfide linkages when digests were performed in 50% ¹⁸O. In the case of poorly resolved isotope patterns of larger peptides, their increases in average mass were consistent with disulfide linkage.

Discussion

The readily available model proteins RNaseA and lysozyme were initially chosen for evaluation of DLP detection through isotope profiles produced by pepsin digestion in 50% ¹⁸O water. These proteins are small and have a small number of well-defined disulfide links (Hirs et al. 1960; Spackman et al. 1960; Canfield and Liu 1965). The suitability of pepsin digestion of proteins and consistent incorporation of ¹⁸O was evaluated, and differences in the isotope patterns and average masses of ¹⁸O-labeled unlinked and disulfide-linked peptic peptides were quaied. The behavior of ¹⁸O-labeled peptic peptides during both ESI-MS and MALDI-TOF-MS was examined, as were the application of SEC and RP-HPLC to isolation of DLPs. The success of the technique with these simpler proteins led to evaluation of the potential of pepsin-mediated ¹⁸O incorporation for ideying DLPs of a more complex glycoprotein. The technique was therefore applied to the 457-residue NDV HN ectodomain, which contains 13 cysteines. The complete disulfide-bond arrangement of NDV HN has been previously determined in our laboratory, largely by comparative reduction of peptic digests separated by RP-HPLC (Pitt et al. 2000). The DLP status of NDV HN peptic peptides was therefore known in some detail and formed a useful basis for evaluation of the technique.

The results obtained with all three proteins showed the feasibility of using mass spectrometric isotope ratios and average mass calculations to idey DLPs produced by peptic digestion in 50% ¹⁸O water. Pepsin-mediated ¹⁸O labeling of peptides enabled mass spectrometric detection of DLPs via isotope profiling without the need for reference ¹⁶O mass spectra.

Detection of DLPs by isotope profiles following pepsin-mediated incorporation of ¹⁸O has advantages over related methods that use tryptic ¹⁸O incorporation to detect DLPs by mass increases (ABRF Electronic Discussion Group 1999). Although it does not generate easily predicable proteolytic fragments, pepsin is a much more aggressive protease than trypsin (Smyth 1967) and can be used to effectively digest proteins that, owing to sequence, disulfide linkages, or structure, are essentially trypsin-resistant (Sanger 1953; Ryle and Sanger 1955; Spackman et al. 1960; Canfield and Liu 1965; Brown and Hartley 1966; Smyth 1967). Pepsin has been used successfully in our laboratory in the study of viral proteins that are intractable to analysis by trypsin (Pitt et al. 2000). The lower sequence specificity of pepsin digestion often results in ragged peptides that can be used as sequence tags to aid peptide ideication (Pitt et al. 2000), particularly when coupled with SEC, which tends to copurify peptides of similar masses but potentially different hydrophobicities from complex peptic digests. Although proteins may be made susceptible to tryptic digestion by denaturation, the lower pH optimum of pepsin greatly reduces the potential problems of disulfide interchange that can occur at the alkaline pH optimum of trypsin and can complicate disulfide analysis (Sanger 1953; Ryle and Sanger 1955; Spackman et al. 1960; Canfield and Liu 1965; Brown and Hartley 1966; Smyth 1967). Lower pH also appeared to prevent the additional ¹⁸O incorporation at internal residues, such as asparagine, as was evident under the conditions of tryptic cleavage. Secondary ¹⁸O incorporation into newly generated C termini was also more apparent with trypsin compared with pepsin. This increased the confidence that altered isotope profiles of peptic peptides were actually caused by proteolytic ¹⁸O incorporation into multiple DLP termini rather than by multiple incorporation events into a single peptide. It should be noted that peptic peptides may be less amenable to MS/MS fragmentation than tryptic peptides. However, this should be assessed on a case-by-case basis. Moreover, intractable peptic peptides may still produce MALDI-PSD sequence data.

Quantitation of ¹⁸O isotope patterns and average mass values of isotope envelopes, which in combination we term isotope profiling, facilitated the ideication of DLPs. This required knowledge of the ¹⁶O isotope profile, which could be calculated if the peptide sequence was known, determined empirically if the sequence was unknown, or observed directly if reference digests had been performed in ¹⁶O water. Although reliable, the latter method has the disadvantages of consuming additional protein, which may be undesirable if the protein is in limited supply, and duplicating the digestion and purification. Furthermore, the need to use reference spectra precludes use of isotope profiling for on-line detection of DLPs. Although difficulties in assigning the monoisotopic MH⁺ peak of isotope profiles may limit the applicability of ¹⁸O isotope profile quantitation for large peptides with reduced peak resolution, aggressive proteolysis with pepsin tended to yield peptides with masses of <4000 D, which reduced this problem.

In practice, it was found that quaication of ¹⁸O isotope profiles to recognize DLPs was usually not necessary. Where resolution of isotopes permitted, peptides for which the MH⁺ + 2, MH⁺ + 4, and MH⁺ + 6 isotopic peaks were significantly more intense than the MH⁺ peak were easily distinguishable from unlinked peptides in which only the MH⁺ + 2 and MH⁺ + 4 peaks were of greater intensity than the MH⁺ peak. The isotope profiles of ¹⁸O-labeled DLPs were therefore sufficiently different from unlinked peptides to make their visual ideication straightforward at all masses, without the need for reference ¹⁶O spectra. Reference ¹⁶O spectra may be useful to rule out the possibility that complex ¹⁸O isotope profiles resulted from coincidence of single-chain peptides of similar monoisotopic mass. The present data indicate that single-chain peptic peptides with the same monoisotopic mass would produce very similar isotope profiles upon digestion in 50% ¹⁸O water, typical of single-chain peptides. However, observation of single-chain peptic peptides of slightly different monoisotopic mass could confuse DLP ideication by ¹⁸O isotope profiling alone. Other strategies, such as analysis of 50% ¹⁸O water digests after reduction, MS/MS, and/or PSD analysis, could be used to confirm assignments made by ¹⁸O profiling and obviate the need for ¹⁶O spectra.

The ability to detect many DLPs without the need for reference spectra or reduction analysis is significant because it opens up the possibility of on-line detection and collection of DLPs from peptic digests using LC-MS. Mass spectrometers use differing combinations of ionization strategies (ESI, MALDI) and ion separation/detection strategies (ion-trap, quadrupole/hexapole, TOF), which suit them to different aspects of DLP analysis (Wilm 2000). MALDI-TOF-MS offers high sensitivity, resolution, and mass accuracy, and the ability to examine the same peptide before and after reduction (Yazdanparast et al. 1987; Pitt et al. 2000). MALDI-TOF-MS spectra are usually simpler to interpret owing to the predominance of singly charged peptide ions. However, the preparation of samples for MALDI-TOF-MS, which involves crystallization of samples on targets, does not generally allow for simultaneous on-line MALDI-TOF-MS analysis of chromatographic eluates. ESI generates more complex spectra with multiple peptide ion charge states and may also have lower sensitivity, resolution, and mass accuracy, particularly on single-quadrupole instruments. However, this is offset by the ability to perform simultaneous analysis, and collection via appropriate output splitting, of chromatographic output (LC-MS). Where available, ESI-TOF and ESI-qQ-TOF mass spectrometers allow increased sensitivity, resolution, and mass accuracy, while retaining LC-MS capabilities. This study showed that even at modest resolution and mass accuracy, ¹⁸O isotope profiles indicative of DLPs were readily apparent in ESI mass spectra, and could be applied to ideication and isolation of DLPs by LC-MS. Because pepsin-mediated ¹⁸O incorporation in 50% ¹⁸O water resulted in more predictable isotope patterns and average mass increases than those obtained with trypsin, isotope profiling of peptic digests may be suitable for algorithmic analysis, with the possibility of automated detection and collection of DLPs, particularly if sequence tags are also observed. Although one limitation to LC-MS may be that peptic peptides could be less favorable to MS and MS/MS than tryptic peptides, the MS properties of peptic peptides were not a limitation to determination of the disulfide-bond arrangement of the NDV HN (Pitt et al. 2000; this study).

In summary, this study indicated that pepsin-mediated ¹⁸O incorporation produces characteristic isotope patterns and average mass increases for DLPs, with only minor alterations to existing methodologies, which can facilitate subsequent determination of the disulfide linkage pattern of proteins. Observed isotope patterns and average masses of representative unlinked, disulfide-linked single-chain and two-chain DLPs derived from model proteins RNaseA and lysozyme varied from theoretical values in a predictable manner such that they could be used to assign disulfide bonds consistent with the known disulfide linkage pattern of these proteins. A more rigorous assessment of this isotope profiling approach was made using the more experimentally taxing NDV HN.

Based on our observations we propose Scheme 1 for using ¹⁸O profiling to assist in the analysis of protein disulfide bonding. This procedure should prove to be of general applicability to facilitate the characterization of disulfide-bond configurations of proteins. It may also be applicable to the characterization of other types of natural peptide chain cross links, such as ɛ-(γ-glutamyl)-lysine (Folk and Finlayson 1977) and collagen Schiff bases (Traub and Piez 1971; Graham and Mechanic 1989), as well as chemically introduced cross links. This may be particularly useful for characterization of these additional cross links in which facile cleavage may not be an option, in contrast to reduction of disulfide bonds.

Materials and methods

Reagents

All reagents were of analytical grade. Egg white lysozyme (Sigma) and bovine pancreatic ribonuclease A (RNaseA; ICN) were desalted by extensive dialysis against water and lyophilized. Newcastle disease virus (NDV) hemagglutinin/neuraminidase (HN) was purified as described previously (Pitt et al. 2000).

Reduction/alkylation

When required, lysozyme was reduced in 2% (w/v) sodium dodecyl sulfate/10 mM dithiothreitol, and alkylated with 50 mM iodoacetamide as described previously (Gorman 1987).

Protease digestions

Trypsin digestions were performed with 30 μg of reduced and alkylated lysozyme using 3% (w/w) trypsin (modified sequencing grade; Boehringer Mannheim) in 100 mM NH₄HCO₃ at 37°C for 5 h.

Pepsin digestions were performed with ∼100 μg of substrate using 5% (w/w) pepsin (Sigma) in 100 mM acetic/100 mM formic acid at 37°C for 3 h. Time course studies showed pepsin digestion under these conditions to be essentially complete after 3 h (data not shown). For analysis of intact disulfide links, proteins were not denatured or reduced.

When ¹⁸O incorporation was desired, sufficient ¹⁸O water (>97% ¹⁸O; Enritech) was added to the substrate solution just prior to the addition of protease, to yield a final concentration of 50% (v/v) ¹⁸O water in the digest.

Digestion was terminated by freezing or by immediate analysis.

Chromatographic separation of peptic peptides

Reverse phase high performance liquid chromatography (RP-HPLC) was performed on a Hewlett Packard HP1100 liquid chromatography system. Thirty μL (containing ∼30 μg of protein) of digest was diluted to 100 μL in 0.05% (v/v) aqueous trifluoroacetic acid (TFA) and injected onto a 1 mm × 25 cm reverse phase C18 column (Vydac). Separations were performed at 40 μL/min using a 90-min linear gradient from 0.05% (v/v) aqueous TFA to 0.045% (v/v) aqueous TFA in 80% (v/v) aqueous acetonitrile (ACN). Size exclusion chromatography (SEC) was performed on the HP1100 using a 3.2 mm × 30 cm Superdex Peptide column (Amersham Pharmacia Biotech). Thirty μL of digest was injected onto the column, and peptides were eluted with 0.05% (v/v) TFA in 6% (v/v) aqueous ACN at 30 μL/min. Peptide elution was monitored by ultraviolet absorbance at 214, 254, and 280 nm. Peptides were collected and stored at −20°C.

Matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry (MALDI-TOF-MS)

Reduced and nonreduced samples were analyzed using a Bruker Reflex MALDI-TOF-MS operated in the positive ion reflector mode. MALDI-TOF-MS data were acquired and analyzed using the Bruker XMass suite of software (Lopaticki et al. 1998; Pitt et al. 2000). A 2,6-dihydroxyacetophenone/diammonium hydrogen citrate (DHAP/DAHC; Fluka) matrix was prepared as described previously (Gorman et al. 1996). Samples were diluted 1:5 in 33% (v/v) ACN/0.1% (v/v) TFA prior to analysis. Then 1–2 μL of diluted sample was mixed with an equivalent volume of matrix, with 1 μL deposited on a Bruker Scout 26 MALDI target and allowed to air dry for 10 min before analysis. Reduction of peptides was performed using a previously described modification of the DHAP/DAHC protocol (Gorman et al 1996).

Post-source decay (PSD) analysis was performed using the Bruker Reflex mass spectrometer and samples prepared in α-cyano-4-hydroxycinnamic acid as the matrix, essentially as described previously (Gorman et al. 1997; Lopaticki et al. 1998). Variations to the previously described procedures involved use of a 100-nsec delay before extraction of ions from the source, and data acquisition using a 1-GHz digitizer.

Peptide masses were localized in RNaseA or lysozyme sequences using PAWS freeware version 8.4 (Proteometrics).

Liquid chromatography–mass spectrometry (LC-MS)

Liquid chromatography–mass spectrometry (LC-MS) was performed using a Sciex API-150EX single-quadrupole electrospray mass spectrometer operated in positive ion mode. Ions were scanned over the range 100–3000 m/z at an orifice potential of 80 V, with a dwell time of 0.4 msec and a 0.25 Da step. For LC-MS, the output from the HP1100 was fed into the ion source of the API-150EX at 40 μL/min. ESI-MS data were analyzed on-line or off-line using Biomultiview 1.4 (Sciex).

Partial amino acid sequence analysis

Peptides were subjected to partial sequence analysis by tandem mass spectrometry (MS/MS) using a Sciex QSTAR-Pulsar Quadrupole-quadrupole (Qq)-TOF-MS. Unfractionated digests were diluted 1:10 in 60% (v/v) aqueous methanol containing 0.1% (v/v) formic acid, then ∼2 μL of diluted digests was loaded into drawn capillaries coated with gold/platinum (Protana NanoES capillaries) and fitted onto a Protana NanoES electrospray ion source. Ions were sprayed with a potential of 850 V on the sample capillary. Collisionally activated decomposition was achieved with a collision energy of 44.4 eV using nitrogen as the collision gas.

Determination of isotope ratios and average masses

Isotope peak intensities of peptides produced by proteolysis in ¹⁶O and 50% ¹⁸O water were measured directly from spectra where mass measurements had been performed. For peptides produced by proteolysis in 50% ¹⁸O water only and whose sequences were assigned based on their monoisotopic masses, the ¹⁶O-only isotope peak intensities were calculated based on the precise elemental composition using IsoPro 3.0 (MS/MS Software http://members.aol.com/msmssoft/). For peptides of unassigned sequence in 50% ¹⁸O digests, ¹⁶O-only isotope peak intensities can be determined empirically by reference to a graph of the variation in isotopic intensities for known ¹⁶O-only peptides of varying mass and elemental composition as a function of mass. There is some variation in the isotopic patterns for peptides of similar mass caused by differences in elemental composition; however, these are insignificant in comparison to experimental errors involved in measurements of isotope intensities. Theoretical distributions were calculated for ¹⁸O-labeled peptides with increasing numbers of ¹⁸O labeling sites by superimposing natural ¹⁶O distributions, determined as described above, with +2-D, +4-D, +6-D, . . . , offsets and using the ratios discussed in the first Results section above.

Average masses for the unlabeled and ¹⁸O-labeled peptides were calculated using the formula below. For an isotope cluster in which each isotope peak has mass m_i and abundance A_i, the average mass (M_av) is given by

Scheme 1.

Process for ideication of disulfide-linked peptides by profiling isotope patterns and average mass changes produced during pepsin-mediated proteolysis of nonreduced protein in 50% ¹⁸O water.

Abbreviations

• DLP, disulfide-linked peptide

• HPLC, high performance liquid chromatography

• ESI, electrospray ionization

• MALDI-TOF, matrix-assisted laser desorption/ionization-time of flight

• MS, mass spectrometry

• LC-MS, liquid chromatography-mass spectrometry

• RNaseA, bovine pancreatic ribonuclease A

• SEC, size exclusion chromatography

• RP-HPLC, reverse phase HPLC

• NDV HN, Newcastle disease virus hemagglutinin/neuraminidase