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Abstract
The purpose of this research is to utilize liquid chromatography-mass spectrometry (LC-MS) to investigate significant questions concerning hydrophilic post translational modifications (PTMs). PTMs are modifications which take place following protein biosynthesis, such as glycosylation and the deamidation of asparagine. There are hundreds of PTMs which have been discovered and possibly many more which have yet to be discovered. The many possible combinations of proteins and PTMs create daunting challenges in proteomic analysis compared to the analysis of the 20 unmodified amino acid residues, and LC-MS is a powerful analytical tool capable of aiding in overcoming these challenges and performing significant analysis of hydrophilic PTMs.The most diverse PTM, glycosylation, is of great importance leading to possible bio-markers for investigation and the production of possible biotherapeutics. In 2016, a new protocol was published describing a new technique for releasing Asparagine (Asn)-linked glycans from a peptide backbone for the large scale preparation of Asn-linked glycans which is much cheaper and faster than standard protocols. The release of Asn-linked glycans from their peptide backbone is essential in the characterization of the structure of these glycans. Here, the validityand practicality of this protocol was compared to an established technique, the use of peptide: N-glycosidase F (PNGase F), for the release of Asn-linked glycans on much smaller scale.Another well-studied modification is the deamidation of Asparagine (Asn) residues to form aspartic acid (Asp) and iso-aspartic acid (i-Asp). The biological consequences of Asn forming i-Asp include the mutation of the secondary structure and conformation of a protein. The biological consequences demonstrate the importance of identifying the formation of deamidation products, especially the formation of i-Asp, accurately. The deamidation of asparagine residues is often detected after trypsin digestion, and when products from this reaction are seen they are often presumed to have been present in the native protein. Here, the possibility of the conditions of trypsin digestion playing a role in causing the deamidation of immunoglobulin G (IgG) peptides was investigated.