mass-spectrometric-approaches-to-study-protein-structure-interactions

Mass Spectrometric Approaches to Study Protein Structure and Interactions in Lyophilized Powders

Amide hydrogen/deuterium exchange (ssHDX-MS) and side-chain photolytic labeling (ssPL-MS) followed by mass spectrometric analysis can be valuable for ...

Purdue University

Peptide and protein microarray and microneedle array technology provides direct information on protein function and potential drug targets in drug discovery and delivery. Because of this unique ability, these arrays are well suited for protein profiling, drug target identification/validation and studies of protein interaction, biochemical activity, immune responses, clinical prognosis and diagnosis and for gene, protein and drug delivery.

Microarrays and microneedle arrays for delivery of peptides, proteins, vaccines and other applications.

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.

Photolytic labeling to probe molecular interactions in lyophilized powders.

Lyophilization can induce aggregation in therapeutic proteins, but the relative importance of protein structure, formulation and processing conditions are poorly understood. To evaluate the contribution of protein structure to lyophilization-induced aggregation, fifteen proteins were co-lyophilized with each of five excipients. Extent of aggregation following lyophilization, measured using size-exclusion chromatography, was correlated with computational and biophysical protein structural descriptors via multiple linear regression. Descriptor selection was performed using exhaustive search and forward selection. The results demonstrate that, for a given excipient, extent of aggregation is highly correlated by eight to twelve structural descriptors. Leave-one-out cross validation showed that the correlations were able to successfully predict the aggregation for a protein "left out" of the data set. Selected descriptors varied with excipient, indicating both protein structure and excipient type contribute to lyophilization-induced aggregation. The results show some descriptors used to predict protein aggregation in solution are useful in predicting lyophilized protein aggregation.

Lavanya K. Iyer

Department of Industrial and Physical Pharmacy

Purdue University

Mass Spectrometric Approaches to Study Protein Structure and Interactions in Lyophilized Powders

Lavanya K. Iyer

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Industrial and Physical Pharmacy

Purdue University

Mass Spectrometric Approaches to Study Protein Structure and Interactions in Lyophilized Powders

Department of Industrial and Physical Pharmacy