Separations coupled to mass spectrometry (MS) are widely used for large-scale protein identification in order to reduce the adverse effects of analyte ion suppression, increase the dynamic range, and as a deconvolution technique for complex datasets typical of cellular protein complements. In this work, matrix assisted laser desorption-ionization is coupled with ion mobility (IM) separation for the analysis of biological molecules. The utility of liquid-phase separations coupled to MS lies in the orthogonality of the two separation dimensions for all analytes. The data presented in this work illustrates that IM-MS relies on the correlation between separation dimensions for different classes (either structural or chemical) of analyte ions to obtain a useful separation. For example, for a series of peptide ions of increasing mass-to-charge (m/z) a plot drift time in the IM drift cell vs. m/z increases in a near-linear fashion, but DNA or lipids having similar m/z values will have very different IM drift time-m/z relationships, thus drift time vs. m/z can be used as a qualitative tool for compound class identification. In addition, IM-MS is applied to the analysis of large peptide datasets in order to determine the peak capacity of the method for bottom-up experiments in proteomics, and it is found that IM separation increases the peak capacity of an MS-only experiment by a factor of 5-10. The population density of the appearance area for peptides is further characterized in terms of the gas-phase structural propensities for tryptic peptide ions. It is found that a small percentage (~3%) of peptide sequences form extended (i.e., helical or β-sheet type) structures in the gas-phase, thus influencing the overall appearance area for peptide ions. Furthermore, the ability of IM-MS to screen for the presence of phosphopeptides is characterized, and it is found that post translationally modified peptides populate the bottom one-half to one-third of the total appearance area for peptide ions. In general, the data presented in this work indicates that IM-MS offers dynamic range and deconvolution capabilities comparable to liquid-phase separation techniques coupled to MS on a time scale (ms) that is fully compatible to current MS, including TOF-MS, technology.
| Identifer | oai:union.ndltd.org:tamu.edu/oai:repository.tamu.edu:1969.1/2203 |
| Date | 29 August 2005 |
| Creators | Ruotolo, Brandon Thomas |
| Contributors | Russell, David H. |
| Publisher | Texas A&M University |
| Source Sets | Texas A and M University |
| Language | en_US |
| Detected Language | English |
| Type | Book, Thesis, Electronic Dissertation, text |
| Format | 7518205 bytes, electronic, application/pdf, born digital |
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