Therapeutic Protein and Glycoprotein Production, Optimization, and Analysis Methods

Proteins and glycoproteins have the potential to improve health when used as therapeutic products for prevention or treatment of disease, but methods are needed for solving some remaining challenges to production, optimization, and analysis. In this dissertation, novel glycosylation engineering strategies are used to solve challenges in conventional protein production and are used to improve therapeutic protein stability by blocking asparagine deamidation, a ubiquitous cause of protein degradation. Glycan structures on therapeutic glycoproteins must be optimized to avoid negative impacts on pharmacological properties. Methods to optimize glycans are described within, using novel, extracellular glycan trimming reactions performed with glycosidases that can be implemented without harm to protein activity or stability. Finally, analysis of proteins with mass spectrometric peptide mass fingerprinting techniques can be complex, so mass defect filters used for data analysis were improved by determining the correct filter size, using experimental human protein data

Improving mass defect filters for human proteins

The mass defect of a substance can be used in mass spectral analysis to identify peaks as likely belonging to a compound class, such as peptides, if the mass defect is within the known range for that compound class. For peptides, a range of possible mass defects was calculated previously, using a set of theoretical peptides, where all possible amino acid combinations were considered (Mann, M. Abstract from the 43rd Annual Conference on Mass Spectrometry and Allied Topics; 1995, ASMS). We compare that range of theoretical peptide mass defects to new values obtained from in silico tryptic digests of proteins that are abundant in human serum and human seminal fluid. The range of mass defect values encompassing 95% of peptides for the human protein data sets was found to be up to 50% smaller than the previously reported mass defect range for the theoretical peptides. The smaller range established for human tryptic peptides can be used to improve peptide mass defect filters by excluding more species that are not likely to be peptides, thus improving filter selectivity for peptides during proteomic data analysis

Collision Induced Dissociation Products of Disulfide-bonded Peptides: Ions Result from the Cleavage of More than One Bond

This document is the Accepted Manuscript version of a Published Work that appeared in final form in the Journal of the American Chemical Society, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see http://doi.org/10.1007/s13361-010-0064-x.Disulfide bonds are a posttranslational modification (PTM) that can be scrambled or shuffled to non-native bonds during recombinant expression, sample handling, or sample purification. Currently, mapping of disulfide bonds is difficult due, to various sample requirements and data analysis difficulties. One step towards facilitating this difficult work is developing a better understanding of how disulfide-bonded peptides fragment during Collision Induced Dissociation (CID). Most automated analysis algorithms function based on the assumption that the preponderance of product ions observed during the dissociation of disulfide-bonded peptides result from the cleavage of just one peptide bond, and in this report we tested that assumption by extensively analyzing the product ions generated when several disulfide-bonded peptides are subjected to CID on a QTOF instrument. We found that one of the most common types of product ions generated resulted from two peptide bond cleavages, or a double cleavage. We found that for several of the disulfide-bonded peptides analyzed, the number of double cleavage product ions outnumbered those of single cleavages. The influence of charge state and precursor ion size was investigated, to determine if those parameters dictated the amount of double cleavage product ions formed. It was found in this sample set that no strong correlation existed between the charge state or peptide size and the portion of product ions assigned as double cleavages. This data shows that these ions could account for many of the product ions detected in CID data of disulfide bonded peptides. We also showed the utility of double cleavage product ions on a peptide with multiple cysteines present. Double cleavage products were able to fully characterize the bonding pattern of each cysteine where typical single b/y cleavage products could not