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 analogs (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 photo-leucine (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 ESI-HPLC-MS, with labeling reaching a plateau at ∼ 30 min, forming up to 6 labeled populations. Bottom-up MS/MS analysis was able to provide peptidelevel 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 acidlevel 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.

Photolytic Cross-Linking to Probe Protein–Protein and Protein–Matrix Interactions in Lyophilized Powders

“…A second formulation containing Mb-SDA and Gdn was prepared with a final concentration of 1.5 M Gdn (Table 1). The formulations were lyophilized as described previously 18 . Briefly, samples were lyophilized in borosilicate clear glass vials according to the following cycle: loading samples on shelves precooled to −2 °C, freezing at −40 °C for 50 min, followed by drying under vacuum (70 mTorr) over 5 steps (−35 °C for 10 h, −20 °C for 8 h, −5 °C for 6 h, 10 °C for 6 h and 25 °C for 6 h).…”

“…Crosslinking was initiated by irradiating the freeze-dried samples at 365 nm for 30 min using a UV Stratalinker 2400 (Stratagene Corp., La Jolla, CA) as described previously 18 . The irradiated samples were reconstituted in 200 µL ammonium bicarbonate (100 mM, pH 8.0) and stored at 4 °C until further use.…”

“…Unlike ssHDX-MS, there are no constraints with respect to experimental conditions (pH, temperature) as the pLeu label is stable and does not undergo back-exchange. Using this method, we studied excipient effects on protein side-chain environment with peptide-level resolution 18 .…”

Process and Formulation Effects on Protein Structure in Lyophilized Solids Using Mass Spectrometric Methods

“…Lyophilized Mb-A and Mb-B vials containing pLeu were uncapped and irradiated at 365 nm for 40 min using Stratalinker 2400 (Stratagene Corp., La Jolla, CA) as described previously 31 . The cakes were then reconstituted with 500 μL ammonium bicarbonate (100 mM, pH 8.0), diluted to 20 pmol and analyzed at the intact protein level by LC-MS.…”

“…To identify the sites of labeling at the peptide level, the labeled Mb formulations were digested with trypsin (1:10 ratio of trypsin to protein) at 60 °C for 16 h. Labeled peptides were analyzed by LC-MS using the method described previously 31 and identified with MassHunter, using a theoretical mass list of tryptic peptides with up to 2 labels.…”