Featuring the ZenoTOF 7600 system and Biologics Explorer software from SCIEX
Haichuan Liu1, Xuezhi Bi2 and Zoe Zhang1
1SCIEX, USA
2Bioprocessing Technology Institute (BTI), Singapore
This technical note highlights the power of electron activated dissociation (EAD) for confident identification and unambiguous localization of O-linked glycosylation in etanercept. The unique ability of EAD to pinpoint the positions of glycosylation further enabled differentiation of positional isomers of O-glycopeptides.
Glycosylation is a common post-translational modification (PTM) that plays a critical role in antibody effector functions.1 Comprehensive characterization of N- and O-linked glycosylation in protein therapeutics is essential for ensuring drug safety and efficacy.1 When applied to glycopeptides, traditional collision-based MS/MS approaches, such as collision induced dissociation (CID), result in the loss of labile glycan moieties. Hence, accurate determination of glycosylation sites using CID is extremely challenging, particularly for O-glycosylation without a consensus sequence. Compared to CID, EAD is superior in glycopeptide analysis given its ability to preserve the glycan structures in the fragments.2–4
Etanercept is a dimeric fusion protein consisting of two tumor necrosis factor receptor (TNFR)-Fc chains with 3 N-glycosylation and 13 O-glycosylation sites on each chain. Glycosylation of etanercept was characterized on the subunit levels in previous technical notes.5 In this work, EAD was utilized to elucidate the complex O-glycosylation profile on the peptide level.
Sample preparation: Etanercept (25 μg/μL) was denatured by guanidine hydrocholoride (7 M), reduced with dithiothreitol (10 mM) and alkylated using iodoacetamide (25 mM). The sample solution was buffer exchanged into 50 mM Tris-HCl (pH = 7.4) using Bio-Spin columns (Bio-Rad), followed by enzymatic digestion at 37ºC for 2 h using trypsin (Promega). The resulting solution was incubated with SialEXO from Genovis at 37ºC for 4 h to remove sialic acids. A total of 10–20 μL of the final solution (~5–10 μg) was injected for LC-MS analysis.
Chromatography: The peptides were separated with an LC gradient, displayed in Table 1, using a Waters ACQUITY CSH C18 column (2.1 × 150 mm, 1.7 μm, 130 Å). A flow rate of 0.25 mL/min was used for the separation. The column was kept at 60ºC in the column oven of an ExionLC system from SCIEX. The mobile phases A and B consisted of 0.1% formic acid (FA) in water and 0.1% FA in acetonitrile, respectively.
Mass spectrometry: EAD DDA and MRMHR data were acquired in SCIEX OS software using the ZenoTOF 7600 system. EAD MRMHR was specifically applied to O-linked glycopeptides containing 6 or 7 core 1 O-glycans. The key TOF MS and EAD (DDA or MRMHR) settings are listed in Tables 2–4.
Data processing: The EAD DDA and MRMHR data were analyzed using two peptide mapping templates within the Biologics Explorer software.
Etanercept consists of 3 N-glycosylation and 13 O-glycosylation sites on each TNFR-Fc chain. Most of the O-glycosylation sites are located in the hinge region. Comprehensive characterization of glycosylation using collision-based MS/MS approaches, such as CID, is challenging because these methods cannot provide site-specific information about glycosylation due to the loss of labile glycans. By contrast, EAD has the unique ability to preserve the glycans in the fragments,2–4 allowing unambiguous localization of this labile PTM. In this technical note, the EAD DDA or MRMHR method was employed to characterize the complex O-glycosylation profile of desialylated etanercept.
Figure 2 displays EAD spectra of two tryptic O-glycopeptides (L1-R19 and T243-K268) containing a core 1 glycan structure (HexNAcHex). Both peptides contain 3 potential O-glycosylate sites (2 Thr and 1 Ser), but only one of the sites was modified with HexNAcHex. As shown in Figure 2, EAD generated excellent quality MS/MS spectra with a nearly complete series of c/y/z fragments, leading to confident identification of these two O-glycopeptides and unambiguous localization of the glycan. Specifically, the O-glycosylation site in peptide L1-R19 (Figure 2A) was determined to be T8 based on the m/z of c7/c9 and y11/z12 fragments, while T245 in peptide T243-268 was modified with HexNAcHex based on the detection of the non-glycosylated c2/y23 and glycan-containing c3/y24/z24.
Figure 3 shows an example of EAD for differentiation of positional isomers of a singly O-glycosylated peptide (S186-R201). Three isomeric species were observed in the XIC of O-glycopeptide S186-R201 (Figure 3A). The high-quality EAD data revealed that the most abundant isomer detected at RT = 22.6 min corresponded to O-glycosylation at S199 (Figure 3D) while the other two species (RT = 20.8 min and 21.6 min) were modified with 1 HexNAcHex at T200 (Figure 3B and 3C). The presence of two T200 isomers might be attributed to different linkages of the glycan. This result highlights the unique capabilities of EAD for confident identification of O-glycopeptides, accurate localization of glycosylation and isomer differentiation in one experiment.
The EAD MS/MS spectra of O-glycopeptide S186-R201 modified with 2 or 3 HexNAcHex are displayed in Figure 4. For the doubly O-glycosylated species (Figure 4A), the detection of a nearly complete series of c/z fragments with or without the glycan enabled accurate localization of 2 HexNAcHex at S199 and T200. In the case of triply glycosylated species, the m/z of c series ions confirmed that the Ser residue at N-terminus was also occupied by HexHAcHex (Figure 4B), as expected.
Trypsin digestion of etanercept generated a long tryptic peptide (S202-K238) that contains 11 potential O-glycosylation sites (6 Ser and 5 Thr). EAD data confirmed that 7 out of 11 of these sites were occupied with the O-glycans. Figure 5 displays the EAD MS/MS spectra of the species containing 6 or 7 HexNAcHex. The presence of multiple proline residues (9 in total) prevented the generation of c/z fragments that correspond to the cleavages at the N-termini of the proline. This limitation was partially overcome by the detection of additional a/y fragments at KE = 7 eV. By taking all the sequence ions (c/z/a/y) into consideration, the locations of 6 or 7 HexNAcHex can be confidently pinpointed in the sequence (Figure 5).
Two positional isomers were observed for glycopeptide S202-K238 containing 6 HexNAcHex moieties. Figure 6 shows the signature EAD fragments—i.e., doubly charged c11, c12 and c15 for differentiation of these two isomers. The m/z difference (365 Da) seen for c12 produced from EAD of two isomers indicated that T213 (T12 in the sequence) was O-glycosylated in Isomer 1 (Figure 6B) but not in Isomer 2 (Figure 6E). The fact that the m/z of c15 was the same for two isomers showed that S216 (S16 in the sequence) was modified with a HexNAcHex in Isomer 2 (Figure 6F) but non-glycosylated in Isomer 1 (Figure 6C). These results highlight the power of EAD for unambiguous differentiation of positional isomers, which is challenging for traditional collision-based MS/MS approaches, such as CID.