Introduction:

Post-translation modifications (PTMs) on monoclonal antibodies (mAb) play an important role in the safety, efficacy, and binding of a therapeutic to its target. Common examples of PTMs that seek to be identified include glycosylation and phosphorylation. One challenge in the identification of these PTMs is that dissociation techniques such as collision-induced dissociation (CID) can break apart these fragile modifications. Here, we describe the use of an electron-based dissociation technique (ExD) that supplies low-energy electrons for the fragmentation of tryptic digests that contain glycosylation at asparagine consensus sites (NXS/T). The fragmentation of trastuzumab tryptic digest on a 6545XT AdvanceBio LC/Q-TOF using CID is compared to illustrate that CID alone will fragment these labile PTMs.

Experimental:

Tryptic digests for mAbs were generated using the Agilent AssayMAP Bravo system using either an in-solution digestion or single-pot (SP3) protocol. Following digestion, peptides were reconstituted in 0.1% formic acid in water at a concentration of ~0.5 µg/µL. Peptides were added to a G7167B multisampler at 6^oC inside a 1290 Infinity II LC system. LC/MS data was collected on a 6545XT equipped with an ExD cell and searched using a pre-released version of MassHunter BioConfirm 12.1 software. For tuning, the Extended Dynamic Range (2 GHz) mode was used with the 100 – 3000 m/z range.

Preliminary data:

A) Sequence coverage of trastuzumab and NIST:

LC/MS data was acquired on the 6545XT AdvanceBio LC/Q-TOF system operated in ExD or CID mode. Following peptide mapping search in MassHunter BioConfirm software, the sequence coverage was 99% for the light- and heavy-chains of NIST and trastuzumab. Similarly, the sequence coverage of mAbs in CID mode was 99%, however this fragmentation technique resulted in glycopeptide fragmentation along the peptide backbone.

B) NIST and Trastuzumab glycosylation identification from MS/MS

The peptide TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK with G1F (NIST) and G0F (Trastuzumab) was examined because this glycopeptide had a strong MS¹ response that would provide amino-acid resolution along the peptide backbone around the glycosylation site at the N9 position. Sequence coverage of these glycopeptides were found to be 100%, providing unambiguous assignment of the glycosylation PTM. In particular, the z₂₁²⁺ monoisotopic fragment ion was identified at m/z 1930.4256 for Trastuzumab, resulting in a deisotoped mass of 3858.85 Da. Moving one amino acid to the right (toward the C-terminus), the z₂₀²⁺ monoisotopic fragment ion was identified at m/z 1150.6100, resulting in a deisotoped mass of 2299.22 Da. The deisotoped mass shift between z₂₁ and z₂₀ (1559.63 Da) is consistent with the G0F+Asparagine residue with H₂O loss. A mass shift of 1721.63 Da is observed for NIST with the G1F+Asparagine residue with H₂0 loss.