Identification of disulfide-linked peptides from recombinant human growth hormone (Nutropin)
Human growth hormone has been used to treat children with hypopituitarism or growth hormone deficiency.32
Native human growth hormone derived from the pituitary gland consists a single polypeptide (monomer) with two intra-disulfide linkages.33
Recombinant human growth hormone (Nutropin) was expressed in an E.coli cell line with the identical gene and recovered from the down-stream purification process.34
Since the correct disulfide linkages are critical to assess the recombinant DNA process, the 4 cysteines linked together as 2 disulfide bonds of Nutropin are the focus of this work.
Nutropin was digested with trypsin without reduction and then analyzed, as described in . As shown in , a disulfide-linked peptide ion (m/z 468.0, 3+), was selected from the MS scan for CID-MS2 (), and ETD-MS2 (), and one of the highest intensity ions (m/z 785.0) from the ETD-MS2 spectrum was automatically selected for CID-MS3 ().
CID, ETD, and MS3 analysis of the disulfide-linked peptide (Cys182 – Cys189, T20-T21) from the tryptic digest of human growth hormone (hGH). A
Using CID (), the disulfide bond was not broken, and only a few characteristic b and y fragmentation ions were observed. On the other hand, with ETD (), the disulfide bond was found to dissociate into two separate peptide ions (P1 and P2), along with a typical ETD fragmentation pattern of the backbone cleavages (c and z ions) with several high intensity ions consisting of charge-reduced species of the precursor ion ([M+3H]2+•, [M-NH3
+3H]2+•, [M+3H]+••, [M-NH3
+3H]+••, and [M-2NH3
+3H]+••). The loss of NH3
(17 Da) from the N-terminus, common in ETD fragmentation, is due to the NH-Cα bond at the N-terminus (see ).24, 25, 29
Thus, the loss of 2 NH3
(34 Da) could result from the disulfide-linked precursor ion, which contained both peptides (P1 and P2). The P1 and P2 ions were observed as the highest intensity ions in the ETD spectrum, indicative of preferred cleavage. One of these peptide ions, P2 (circled in ) was automatically isolated for further fragmentation in the MS3
step (). This peptide ion was found to be fragmented into b and y ions, along with characteristic side-chain losses of amino acid residues, such as loss of 18 (water), 34 (SH2
) or 46 Da (SCH2
) from the cysteine residue. These losses could be explained by this peptide containing a mixed population, as P2-SH and P2-S•, with the protonated form generating b and y ions and the odd electron (electron-transferred) form generating characteristic side chain losses of SH2
, along with c and z ions. Similar observations of the side chain losses have been described by others.25
Cysteine-containing peptides can often undergo these types of side chain losses when they are ionized in the gas phase over a longer period of time, as evident by the observation of increased side chain losses in the cysteine-containing product ions (e.g. [b6-SCH2
] and [b7-SCH2
]) in the MS3 spectrum of .
The sequence information of the P2 peptide generated in provided the identification of this peptide without the assumption of a molecular weight modification on the cysteine residue (see the Experimental Section). In a similar manner, the P1 peptide, which was another high abundant ion in , was next selected for MS3
and backbone cleavages with a similar fragmentation pattern was generated (see Figure S1, Supplementary Material
). In summary, from and S1
, both the disulfide-linked and disulfide-dissociated peptides were obtained and simultaneously characterized, in contrast to the widely used two step protocol to obtain the same information (i.e. with and without chemical reduction).
The second disulfide-linked peptide in growth hormone was next examined, as shown in . The CID-MS2 spectrum () of the precursor ion, m/z 941.8 (4+), was observed with a few characteristic fragmentation ions (e.g. y6 and b17 ions at proline residues). The ETD-MS2 spectrum () of the same precursor ion showed a typical ETD fragmentation (c and z ions), along with several high intensity ions, including charge-reduced species (labeled as [M+4H]3+• and [M+4H]2+••), and side-chain loss ions ([M+4H-H2O]2+••). For clarity, several characteristic side chain losses are not labeled in . One of the high intensity charge-reduced species, [M+4H]2+••, m/z 1881.8, was automatically isolated for further fragmentation in the MS3 step (). The disulfide-dissociated P2 peptide ion along with the backbone cleavage ions (c and z ions) were observed. The P2 ion was further fragmented (MS4) in an additional LC-MS run to obtain the backbone sequence information (data not shown).
CID, ETD, and MS3 analysis of the disulfide-linked peptide (Cys53 – Cys165, T6–T16) from the tryptic digest of hGH. A
As recently described,29,37, 38
charge-reduced species become dominant product ions in the ETD spectrum for precursor ions with m/z >900, as evident for the two disulfide-linked peptides (compare to ). The generation of several charge-reduced species with high intensities in the ETD spectrum allowed the determination of the charge state of the precursor ion (4+). As noted, the disulfide-dissociated peptides became the major ions in the MS3
step. It has been suggested that the two disulfide-linked peptides, even with the disulfide bond dissociated, are still held together by non-covalent forces in the charge-reduced species.24,25
As a consequence, the charge-reduced species could be a mixture of two populations, one peptide species that is backbone-dissociated (with the electron already transferred but the disulfide is still intact) and the other a disulfide-dissociated peptide species (either P1-SH or P1-S•, or both). With additional kinetic energy in the MS3
step, the peptide backbone-dissociated species (with the disulfide still linked) would yield c and z ions, and the disulfide-dissociated peptide (held together by non-covalent forces) would result in two separated polypeptides (i.e. P1 and P2). The P2 ion is seen in the MS3
spectrum () while the other peptide ion P1 (m/z 2617 with 1+ charge), not seen in , could appear beyond the mass detection window of this experiment.
The charge-reduced species can also be dissociated using supplemental activation without isolation.38, 39
In this case, we would anticipate that the two fragmentation steps, ETD-MS2
(CRCID), merge to a single step with product ions from both ETD-MS2
and CRCID and with minimal charge-reduced species. While supplemental activation may reduce the instrument cycle time, the observation of product ions in two separate spectra can be useful for simpler interpretation of complicated product ions. Moreover, the MS3
step is still required for the fragmentation of P1 or P2 peptides in the analysis of disulfides.
Since the generally larger Lys-C fragments, relative to tryptic fragments, provide more choices of multiple higher charge states for improved ETD/CRCID fragmentation,29
growth hormone was next digested with Lys-C to examine the influence of the size and charge states of the disulfide-linked peptides on ETD fragmentation. Using the strategy in , a large disulfide-linked Lys-C peptides was analyzed, and the spectra are shown in Figure S2
). In this example, the precursor ion of the disulfide-linked peptide (m/z 773.6, 6+) was isolated and dissociated as high abundant P1 and P2 peptide ions by ETD-MS2
). Compared to the corresponding tryptic peptide in , the advantages of using this Lys-C peptide are readily seen. While only the charge-reduced species were observed with high abundance for the tryptic peptide, the high abundant P1/P2 ions from the Lys-C fragment were automatically selected for the subsequent MS3
step to obtain the backbone sequence information (P1 fragmentation is shown in Figure S2C
). In contrast, the backbone of P2 ion would be fragmented at MS4
in an additional LC-MS run and the P1 ion was not seen at MS3
(beyond the mass detection window) for the corresponding tryptic peptide. The strategy to produce enzymatic fragments with high charge and low m/z is preferred for ETD fragmentation on disulfide-linked peptides in this LC-MS method as well.29
There are only two disulfide bonds in rhGH, CID fragmentation alone could be sufficient to identify the disulfide linkages even though an incomplete ion series is produced.40
Nevertheless, rhGH is a good model to illustrate the determination of the linkages by the fragmentation strategy of . With this determination strategy in mind, a more complicated example, a recombinant monoclonal antibody (Herceptin) with multiple disulfide bonds was next selected for analysis.
Identification of disulfide-linked peptides from a monoclonal antibody (Herceptin)
Herceptin has been used to treat woman with metastatic breast cancer, particularly with Her2 positive gene.41
The protein, a typical therapeutic monoclonal antibody, has constant (Fc) and variable (Fab) domains with inter- and intra-disulfide bonds between the heavy and light chains, as illustrated in . The identification of the peptide sequences with disulfide bonds in the Fc region are the focus in this paper.
Structural diagram of a monoclonal antibody with disulfide bonds
Herceptin was first digested with Lys-C without reduction. The precursor ion of the disulfide-linked peptide (m/z 907.8, 6+), which was associated through two inter-disulfide bonds between two heavy chains in the hinge region of the Fc domain (Cys229 with Cys229 and Cys232 with Cys232, see ), was selected for analysis, and the LC-MS results are shown in . As expected, the disulfide bonds were not dissociated by the CID-MS2
fragmentation (), and the characteristic CID cleavages at proline residues were observed with high abundance (i.e. y25, y19, and y5). It should be noted that the two disulfides are very close to each other, and no cleavages were observed for the peptide residues inside these two disulfides even with the presence of proline residues. Similar observations were reported by others as well.42, 43
CID, ETD, MS3, and MS4 analysis of the disulfide-linked peptide (Cys229 - Cys229 and Cys232– Cys232 in the hinge region of the Fc) from the Lys-C digest of Herceptin. A
The same precursor ion produced a typical ETD spectrum (c and z ions), along with several high intensity ions, consisting mainly of charge-reduced species of the precursor ion, see . The highest intensity ion ([M+6H]3+•••, m/z 1814.5) was automatically fragmented in the MS3 step (). Significantly, while the disulfide-dissociated peptide ions were not detected in the ETD spectrum (), they were the dominant ions in the MS3 spectrum (). The lack of disulfide-dissociated peptide ions (P1 or P2) in the ETD spectrum could be due to the structure of this peptide (two disulfide bonds) with a high m/z (>900). However, fragmentation of the charge-reduced species (CRCID) produced the P1 or P2 peptide ion. To obtain the sequence information, the P1/P2 peptide ion was further fragmented in a CID-MS4 step (an additional LC-MS run) to confirm the correct assignment (). The multi-fragmentation strategy provided good complementary information for this disulfide-linked peptide.
Turning to a second disulfide-linked peptide, connected through an intra-disulfide bond (Cys264 and Cys324) in the first loop of the Fc domain (see ), a precursor ion of (m/z 960.7, 5+) was detected in the same LC-MS run as in . Again, the disulfide bond was not dissociated by CID-MS2 fragmentation, and only a few high abundant ions with characteristic CID cleavages were observed (). For ETD-MS2, the same precursor ion yielded a typical ETD fragmentation pattern (c and z ions), along with several high intensity ions, of the charge-reduced species. One of the charge-reduced species ([M+5H]3+••, m/z 1599.4) was automatically fragmented in the MS3 step. The disulfide-dissociated peptide ions (P1) were not observed in the ETD spectrum (), but they became the high abundant ions in the MS3 fragmentation (), similar to that shown in . The lack of any observable P1 or P2 peptide ions in the ETD spectrum could again be due to the sequence structure of this peptide ion with a high m/z (>900). The very small P2 peptide (less than 250 Da) was not in the mass detection window, and the large P1 peptide ion (4,547 Da) may have existed as 2+ or 1+ charge state (m/z >2000) which was beyond the mass detection window.
CID, ETD, and MS3 analysis of the disulfide-linked peptide (Cys264 - Cys324 in the first loop of the Fc) from the Lys-C digest of Herceptin. A
For the remaining disulfide-linked peptide (labeled as Cys370 and Cys428 in the second loop of the Fc domain), NQVSLTC
LVK (P1) attached to SRWQEGNVFSC
SVMHEALHNHYTQK (P2), a precursor ion with m/z 682.8 (6+), was detected and analyzed as above. Given the high charge and low m/z, the disulfide-dissociated peptides were first identified with high abundance by ETD-MS2
(P1 and P2), and the sequence information of the dissociated P2 peptide was obtained in the subsequent MS3
step (see Figure S3 in Supplementary Material
). Finally, the Fab domain of the antibody (after Lys-C digestion) produced a similar number of the disulfide-linked peptides (2 intra and 1 inter disulfide for each light and heavy chain), as for the Fc domain (see ). Only one disulfide bond was linked the two peptides, and thus, the assignment was straightforward (data not shown). In summary, the results for the monoclonal antibody further demonstrate that multiple disulfide-linked peptides can be readily characterized by the multi-fragmentation steps shown in .
Identification of disulfide-linked peptides from recombinant tissue plasminogen activator (Activase)
We next turn to examine another therapeutic protein (for acute ischemic stroke) with the complicated disulfides, recombinant tissue plasminogen activator (rt-PA), with 17 potential intertwined disulfide bonds that are quite difficult to assign.7–9
These disulfide bonds are distributed in 7 tryptic peptides, with 4 of the 7 peptides glycosylated (with N-linked and O-linked glycosylation).35, 36
The remaining three non-glycosylated peptides are examined in this paper (see Figure S4 in Supplementary Material
). Before proceeding, it should be noted that we used Lys-C plus trypsin digestion for this analysis since the Lys-C digestion alone would produce only 2 peptide fragments, one with 2 disulfide bonds and the other with 15 disulfide bonds (>50 kDa) which would be too large to analyze. The use of trypsin alone could not efficiently digest rt-PA since a domain of the protein is known to be resistant to tryptic digestion.44
We first examine peptide A (Figure S4
) in which there are 2 disulfide linkages intertwined between three peptide backbones. This peptide with the precursor ion of 610.9 (5+) was fragmented by CID-MS2
() and ETD-MS2
(), and one of the high abundant product ions from ETD-MS2
was isolated and further fragmented by CID-MS3
(). Since there are three pe ptides linked by disulfide bonds, the assignments are difficult to determine from the CID-MS2
spectrum. On the other hand, the high abundant product ions can be assigned in the ETD-MS2
spectrum since the disulfide bond cleavages are the dominant ions (i.e. P1, P2, P3, P1-P2, and P2-P3 ions in ). From these partially disulfide-dissociated peptide ions, one can readily assign the linked peptides as P1 with P2, and P2 with P3, but not P1 with P3, see . The exact linkages (i.e. Cys6 with Cys36, and Cys34 with Cys43) required MS3
determination on the partially disulfide-dissociated peptide. In this case, the disulfide bond linked to P3 was broken, but the bond between P1 and P2 remained intact (circled as P1-P2 2+
in ). The isolation of the P1-P2 2+
ion (m/z 1041.6 in ) for further CID fragmentation generated the information that Cys6 was indeed linked to Cys36, and Cys34 was in the free form (see ). This information was critical since the ETD-MS2
spectrum did not allow determination of the specific linkages. Similarly, the isolation of the other partially disulfide-dissociated peptide (labeled as P2-P3 2+
in ) generated additional evidence that Cys34 was linked to Cys43, and Cys36 was in the free form (see Figure S5 in Supplementary Material
CID, ETD, and MS3 analysis of the disulfide-linked peptide (Cys6 - Cys36 and Cys34 - Cys43) from the Lys-C plus trypsin digest of Activase. A
Multiple charge states of high intensity were observed for this disulfide intertwined peptide A in the MS spectrum, and each of these ions were automatically selected for fragmentation in the data-dependent mode. As an illustration of the complementary fragmentation information produced, the different charge states for Peptide A are presented in Figure S5
. The ion with a lower charge state and thus higher m/z ion generated more CID fragmentation (3+ with m/z 1017.3 in Figure S5A vs. 5+
with m/z 682.6 in ), but less ETD fragmentation (compare and Figure S5B
), in agreement with our previous observations.29
The information obtained from and Figure S5
confirmed the disulfide linkages of the three peptides. To our knowledge, this is the first direct evidence to assign these linkages of tissue plasminogen activator by LC-MS, and the result is consistent with the prediction from homology.44, 45
As can be seen from the above, the isolation of the partially dissociated peptide ions, such as the P1-P2 or P2-P3 peptide ions, followed by CID fragmentation (MS3) was essential to determine the linkage sites. The backbone sequence information generated separately from the P1, P2 or P3 ions (or from the charge-reduced species) was not sufficient to determine the intertwined disulfide linkages. Importantly, the partially dissociated peptides would be difficult to obtain by a chemical approach (e.g. partial reduction by DTT) but are readily generated with high abundance in the ETD spectrum.
Interestingly, an extra shoulder with a different precursor mass was found on the main chromatographic peak of peptide A. However, the ETD spectrum of this precursor was similar to that of the main peak shown in . For this ETD spectrum, as seen in , the high abundant ions could be partially assigned to P2 and P3. Based on the theoretical molecular weight of the tryptic peptide fragments of Activase, the linkage with a Cys457 containing tryptic peptide (T43 peptide) was assigned as P1, replacing the T1 peptide containing Cys6. The rest of the fragment ions were consistent with this assignment (Cys457 linked to Cys36). In this example, the initial CID-MS2 spectrum could not be analyzed; however, based on the results derived from the ETD-MS2 spectrum, the CID-MS2 as well as the MS3 spectra could then be interpreted, reinforcing the assignment made from the ETD spectrum.
CID, ETD, and MS3 analysis of the disulfide-linked peptide (Cys457 - Cys36 and Cys34 - Cys43) from the Lys-C plus trypsin digest of Activase. A
The above disulfide scrambling (i.e. Cys457 linked to Cys36 instead of Cys6 to Cys36) is most likely a consequence of the digestion conditions (see Experimental Section), since higher amounts of the scrambled peptide were found with longer digestion times. In rt-PA, there are 35 cysteines, an odd cysteine without a pairing disulfide (likely at the Cys83 position based on homology), which could facilitate the disulfide scrambling under the digestion conditions.46, 47
The influence of digestion conditions on disulfide scrambling is under further study. Nevertheless, the multi-fragmentation strategy proved successful in revealing the scrambling and identifying the disulfide linkages in this example.
The assignments of the other two non-glycosylated disulfide-linked peptides were achieved using the same strategy as for . The multi-fragmentation results for Peptide C, containing 4 cysteines, Cys307 linked to Cys323 and Cys315 connected to Cys384 are shown in Figure S6
). As seen in the figure, the lack of significant fragmentation between C307 and C325 (in side the circle) indirectly established the disulfide linkages between these two residues. The other potential linkage (i.e. C307 to C315 and C325 to C384) would produce the fragmentation between C315 and C325 in the CID or ETD (or MS3
) spectrum. Peptide B (Figure S4
) with a simple disulfide-linked peptide, Cys201 connected to Cys243, could be analyzed in a straightforward manner (data not shown). The other disulfide-linked peptides with multiple glycosylation forms are currently being investigated and will be reported later. The assignments of fragment ions with multiple glycosylation and disulfide linkage forms are time-consuming and difficult to ascertain using the LTQ with ETD system. We anticipate that the newly developed ETD with Orbitrap mass spectrometer may facilitate the analysis.37