title: Photolytic labeling to probe molecular interactions in lyophilized powders.
abstract: 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.
authors.affiliation: Department of Industrial and Physical Pharmacy, Purdue University , West Lafayette, Indiana 47901, United States.
authors.forename: Lavanya K
authors.initials: LK
authors.lastname: Iyer
authors.forename: Balakrishnan S
authors.initials: BS
authors.lastname: Moorthy
authors.forename: Elizabeth M
authors.initials: EM
authors.lastname: Topp
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D001059:Apoproteins / Q000737:chemistry; D001577:Benzophenones / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D005612:Freeze Drying / Q000379:methods; D019791:Guanidine / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods; D009211:Myoglobin / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D010649:Phenylalanine / Q000031:analogs & derivatives / Q000737:chemistry; D010782:Photolysis; D011208:Powders / Q000737:chemistry*; D013395:Sucrose / Q000737:chemistry
chemical_list: C488060:4-benzoylphenylalanine; D000596:Amino Acids; D001059:Apoproteins; D001577:Benzophenones; D009211:Myoglobin; D010455:Peptides; D011208:Powders; C010857:apomyoglobin; D010649:Phenylalanine; D013395:Sucrose; D019791:Guanidine