Disulfide Mapping of VHH Antibodies

Disulfide bond mapping of SLP_V_HH22 and SLP_V_HH50, each with four Cys residues, was performed essentially as described (Kim et al. 2012b (link); Hussack et al. 2011b (link)). Briefly, tryptic fragments for subsequent mass spectrometry (MS) analysis were prepared as described (Kim et al. 2012a (link)). Aliquots of V_HH proteolytic digests were resuspended in 0.1 % (v/v) formic acid (aq) and analyzed by nanoflow reversed-phase HPLC MS (nanoRPLC-ESI-MS) with data-dependent analysis (DDA) using collision-induced dissociation (CID) on a nanoAcquity UPLC system coupled to a Q-TOF Ultima™ hybrid quadrupole/TOF mass spectrometer (Waters, Milford, MA, USA). The peptides were first loaded onto a 300 μm I.D. × 5 mm C18 PepMap100 μ-precolumn (Thermo Fisher) and then eluted into a 100 μm I.D. × 10 cm 1.7-μm BEH130C18 column (Waters) using a linear gradient from 0 to 36 % solvent B (acetonitrile + 0.1 % formic acid) over 36 min followed by 36–90 % solvent B for 2 min. Solvent A was 0.1 % formic acid in water. The peptide MS² spectra were compared with V_HH protein sequences using the Mascot™ database searching algorithm (Matrix Science, London, UK). The MS² spectra of the disulfide-linked peptides were de-convoluted using the MaxEnt 3 program (Waters) for de novo sequencing to confirm and/or determine the exact disulfide linkage positions.