An investigation into the use of cyclic ion mobility for the separation of biopharmaceutical peptide and protein modifications
James Langridge, Dale Cooper-Shepherd, Martin Palmer, Henry Shion, Weibin Chen
Aims /Introduction
As biotherapeutics become more complex, ever more sophisticated tools are being investigated to provide in-depth characterisation. Mass spectrometry (MS) is a central technique in the biopharmaceutical industry due to its ability to report on such a wide range of attributes. However, the presence of isobaric PTMs with differing biological properties can often be refractory to traditional LC-MS workflows even when chromatographically separated. In this work we investigate cyclic ion mobility technology as a means to distinguish isomeric PTMs to improve biotherapeutic characterisation.
Experimental and Results
Studies were performed on a Select Series Cyclic IMS mass spectrometer. The native and iso-aspartic variants of the synthetic peptide WGGDGFYAMDYWGQGTLVTVSSASTK (T12-D and T12-isoD, respectively) were used in this study to mimic isomerisation products from protein biotherapeutics. The peptides were analysed by reversed-phase separation prior to cyclic ion mobility-mass spectrometry. When electrosprayed the two isomeric peptides T12-D and T12-isoD formed primarily the [M+3H]3+ ion at 928 m/z. After a single pass of the cIM device the peptides were baseline separated from each other. Next we selected the T12 peptides in the resolving quadrupole and subjected them to collision-induced dissociation in the trap ion guide situated prior to the cIM device. This enables structural analysis of the resulting product ions by ion mobility separation. The peptide product ions were separated over three cIM passes. Upon comparing T12-D and T12-isoD product ions, we found several b-type ions, those that contain the isomeric aspartic acid residue, which showed differences in their arrival time distributions, indicating different structures. Interestingly, the y-type ions which do not contain the isomeric aspartic acid residue looked highly similar between the two model peptides.
Conclusions
These preliminary data suggest that the mobilities of peptide product ions can report on the sequence location of isomeric amino acid residues. The combination of collision-induced dissociation and multi-pass cyclic ion mobility-mass spectrometry has the potential to provide additional sequence information on peptides that have identical mass and product ion spectra. Further isomer examples should be investigated to generalise these findings.