Photolytic Cross-Linking to Probe Protein鈥揚rotein and Protein鈥揗atrix Interactions in Lyophilized Powders
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- 作者：Lavanya K. Iyer ; Balakrishnan S. Moorthy ; Elizabeth M. Topp
- 刊名：Molecular Pharmaceutics
- 出版年：2015
- 出版时间：September 8, 2015
- 年：2015
- 卷：12
- 期：9
- 页码：3237-3249
- 全文大小：705K
- ISSN：1543-8392
文摘
Protein structure and local environment in lyophilized formulations were probed using high-resolution solid-state photolytic cross-linking with mass spectrometric analysis (ssPC鈥揗S). In order to characterize structure and microenvironment, protein鈥損rotein, protein鈥揺xcipient, and protein鈥搘ater interactions in lyophilized powders were identified. Myoglobin (Mb) was derivatized in solution with the heterobifunctional probe succinimidyl 4,4鈥?azipentanoate (SDA) and the structural integrity of the labeled protein (Mb-SDA) confirmed using CD spectroscopy and liquid chromatography/mass spectrometry (LC鈥揗S). Mb-SDA was then formulated with and without excipients (raffinose, guanidine hydrochloride (Gdn HCl)) and lyophilized. The freeze-dried powder was irradiated with ultraviolet light at 365 nm for 30 min to produce cross-linked adducts that were analyzed at the intact protein level and after trypsin digestion. SDA-labeling produced Mb carrying up to five labels, as detected by LC鈥揗S. Following lyophilization and irradiation, cross-linked peptide鈥損eptide, peptide鈥搘ater, and peptide鈥搑affinose adducts were detected. The exposure of Mb side chains to the matrix was quantified based on the number of different peptide鈥損eptide, peptide鈥搘ater, and peptide鈥揺xcipient adducts detected. In the absence of excipients, peptide鈥損eptide adducts involving the CD, DE, and EF loops and helix H were common. In the raffinose formulation, peptide鈥損eptide adducts were more distributed throughout the molecule. The Gdn HCl formulation showed more protein鈥損rotein and protein鈥搘ater adducts than the other formulations, consistent with protein unfolding and increased matrix interactions. The results demonstrate that ssPC鈥揗S can be used to distinguish excipient effects and characterize the local protein environment in lyophilized formulations with high resolution.