Hybrid structural modeling of alloantibody binding to human leukocyte antigen with rapid and reproducible cross-linking mass spectrometry

Protocol for structural modeling of antibody to human leukocyte antigen interaction using discovery and targeted cross-linking mass spectrometry

“… 3 Here, we describe a technique that uses discovery and targeted XL-MS acquisition of purified antibody (Ab) and purified human leukocyte antigen (HLA). 1 The cross-link data can be used as a distance restraint for molecular docking of Ab to HLA, together with protein structures of Ab, HLA and predicted interacting surfaces of Ab and HLA. The obtained structural model can be used to understand the molecular basis of antibody to HLA interaction.…”

“…The current protocol was performed on three different antibodies that bind to HLA-A∗11:01. 1 After cross-linking, a representative stained protein gel should indicate that antibody is cross-linked to HLA ( Figure 4 A). After performing cross-linking and discovery XL-MS, 4 inter-links between Ab to HLA were identified ( Figure 4 B).…”

“…The antibody-HLA pair that was characterized by Ser et al. 1 demonstrated a 4-6 Å difference between the molecular docking model and the crystal structure for hydrogen bonding residues. However, this discrepancy is likely to vary depending on antibody-HLA pair used, quality of cross-linking data and predicted inputs.…”