Microstructure Characterization of Polymers and Polymer-protein Bioconjugates by Hyphenated Mass Spectrometry

Download or read book Microstructure Characterization of Polymers and Polymer-protein Bioconjugates by Hyphenated Mass Spectrometry written by Selim Gerislioglu and published by . This book was released on 2018 with total page 183 pages. Available in PDF, EPUB and Kindle. Book excerpt: Mass spectrometry (MS) has been a powerful technique for the characterization of synthetic polymers especially after the introduction of soft ionization methods such as electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI). One-dimensional MS analysis provides molecular weight information by detection of the mass-to-charge (m/z) ratio of gas-phase analyte ions. However, tandem MS (MS/MS) capabilities, which involve separation and subsequent fragmentation of a selected analyte ion, are needed to obtain more detailed structural information. MS/MS can provide essential information on the backbone connectivity for synthetic polymers. For the sequence analysis of polymers having hydrocarbon backbones, multistage fragmentation (MSn) that combines different ion activation methods may be necessary. In addition to MS or MS/MS analysis, additional dimensions of separation can be achieved by online hyphenation of various dispersion techniques which allows for the investigation of more complex materials and mixtures. While online combination of liquid chromatography (LC) with MS has been the most common hyphenated technique, ion mobility (IM) spectrometry has been emerging as a much more rapid alternative method for the separation of gas-phase analyte ions based on their charge state, size, and architectural differences. These two separation techniques can be combined in a high-throughput 3D LC-IM-MS method or utilized separately depending on the sample complexity. In this dissertation, applications of MSn and hyphenated MS techniques are illustrated for the analysis of synthetic polymers and complex synthetic polymer-protein conjugates. In Chapter IV, various MSn approaches were investigated for the in-depth structural characterization of poly(N-isopropylacrylamide)s (pNIPAM). Different ion activation methods including collisionally activated dissociation (CAD) and electron transfer dissociation (ETD) provided structural information on end group and side chain functionalities. Sequential application of these ion activation methods in an MS3 fashion was necessary to obtain -C-C- bond cleavages and elucidate the backbone connectivity. In Chapter V, various hyphenated techniques that involve IM and LC were examined for the qualitative structural characterization of synthetic polymer-protein conjugates that were prepared by covalent attachment of poly(ethylene glycol) (PEG) to different proteins (PEGylation). The 3D LC-IM-MS technique was combined with in-source dissociation (ISD) for the site-specific characterization of PEGylated insulin with or without pretreatment of the sample.Finally, in Chapter VI, a methodology for occupancy analysis of PEGylated proteins was investigated on a model PEGylated myoglobin conjugate. Contrary to the qualitative approach discussed in Chapter V, the method presented in this final chapter involved a subtractive relative quantification by LC-MS which provided complete occupancy information on each possible PEG attachment site.