Mass spectrometric analysis of protein deamidation - A focus on top-down and middle-down mass spectrometry.

Deamidation of asparagine and glutamine alters protein structures and affects the chemical and biological properties of proteins. Protein deamidation has been demonstrated to be associated with protein folding, enzymatic activity, and degradation, as well as aging, cancer, and neurodegenerative diseases. To gain a better understanding on the biological roles of protein deamidation in aging and diseases, mass spectrometry (MS) has been employed in the identification of deamidated protein species and comprehensive characterization of deamidation sites. Three main MS approaches, top-down, middle-down, and bottom-up have been applied in the study of protein deamidation with high sensitivity, throughput, and accuracy. In this review, we discuss the application of top-down and middle-down MS in the study of protein deamidation, including sample preparation methods, separation strategies, MS and MS/MS techniques and data analysis. The advantages and drawbacks of these two approaches are also discussed and compared with those of the bottom-up method. The development of top-down and middle-down MS methods provides new strategies for protein deamidation analysis and gives new insights into the biological significance of protein deamidation in diseases.

High-throughput Carbonyl Content Method of Therapeutic mAb using size-exclusion chromatography with ultraviolet and fluorescence detection.

Monoclonal antibody (mAb), one of the major types of therapeutic proteins in the pharmaceutical industry, is predominantly manufactured using mammalian cell culture [1]. Oxidative stress, potentially present during cell culture process, may increase the protein carbonyl content in the mAb product, which was reported to positively correlate with aggregate burst rate during storage [2]. In order to monitor carbonyl content during mAb process development, we developed a high-throughput screening method for therapeutic mAbs using size-exclusion chromatography followed by ultraviolet and fluorescence detection (SEC-UV/FL), optimized from a fluorescein thiosemicarbazide (FTC) semi-microplate method. The method demonstrated a good correlation with conventional ELISA assay and FTC-based fluorometric semi-microplate method with improved throughput and precision. The method was successfully applied in three case studies to improve our understanding of mAb carbonylation, including the impact of metal-catalyzed oxidation on an IgG4 mAb, comparison of carbonyl content between several mAbs expressed by CHO cell culture with human serum antibody pool, as well as the surface charge property of carbonylated mAb assessed by ion-exchange chromatography.

Characterization of sol-gel entrapped chlorophyllase.

Immobilization of membrane proteins remains a challenge compared to soluble proteins. The membrane protein-chlorophyllase was successful entrapped in tetramethoxysilane (TMOS)-based sol-gel in the presence of lipid. Activity was examined against mixing rate, incubation temperature, time, substrate, acetone, and canola oil concentration. The external mass transfer of chlorophyll is not the rate-limiting step at higher mixing rates. Stability against temperature and acetone as denaturant was enhanced. In spite of the fact that an initial reaction lag phase was observed, 20% more chlorophyll was hydrolyzed, compared to reaction with free enzyme by the end of a 12 h assay. The initial lower activity demonstrated by entrapped chlorophyllase is likely due to the diffusion resistance of chlorophyll into and within the entrapment matrix. This hypothesis was substantiated by a low diffusion coefficient on the order of 10(-14) m(2)/s obtained for chlorophyll in nanoporous sol-gel particles. Pore size distribution of nanoporous wet TMOS-based sol-gel with or without protein was determined by thermoporometry. The change in pore morphology upon doping with chlorophyllase suggests that protein acts as a template during the sol-gel process.