Photolytic Labeling To Probe Molecular Interactions in Lyophilized Powders
journal contributionposted on 2013-12-02, 00:00 authored by Lavanya K. Iyer, Balakrishnan S. Moorthy, Elizabeth M. Topp
Local side-chain interactions in lyophilized protein formulations were mapped using solid-state photolytic labeling-mass spectrometry (ssPL-MS). Photoactive amino acid analogues (PAAs) were used as probes and either added to the lyophilized matrix or incorporated within the amino acid sequence of a peptide. In the first approach, apomyoglobin was lyophilized with sucrose and varying concentrations of photoleucine (l-2-amino-4,4′-azipentanoic acid; pLeu). The lyophilized solid was irradiated at 365 nm to initiate photolabeling. The rate and extent of labeling were measured using electrospray ionization/high-performance liquid chromatography/mass spectrometry (ESI-HPLC-MS), with labeling reaching a plateau at ∼30 min, forming up to six labeled populations. Bottom-up MS/MS analysis was able to provide peptide-level resolution of the location of pLeu. ssPL-MS was also able to detect differences in side-chain environment between sucrose and guanidine hydrochloride formulations. In the second approach, peptide GCG (1–8)* containing p-benzoyl-l-phenylalanine (pBpA) in the amino acid sequence was lyophilized with various excipients and irradiated. Peptide–peptide and peptide-excipient adducts were detected using MS. Top-down MS/MS on the peptide dimer provided amino acid-level resolution regarding interactions and the cross-linking partner for pBpA in the solid state. The results show that ssPL-MS can provide high-resolution information about protein interactions in the lyophilized environment.