Sample complexity is one of the key challenges facing contemporary proteomic analysis. A variety of methods is commonly employed to reduce this complexity, both at an intact protein and digested peptide level. For complex lysates containing many thousands of proteins, orthogonal (mutually independent), multidimensional separation methods must be employed to provide sufficient resolution to characterize the appropriate number of different species. The most common of these methods are two dimensional gel electrophoresis (2DGE) of proteins and multidimensional liquid chromatographic separation (MuDPIT) of peptides, which rely on isoelectric focusing followed by mass separation in the former, and ion exchange followed by reversed phase separation in the latter. These methods have significant drawbacks in terms of sample bias, sample preparation and reproducibility, and therefore a new methodology that combines the positive aspects of both separation technologies in an automatable, reproducible form is highly desirable. New developments in column technology have allowed rapid improved-resolution separation of intact proteins in complex samples, coupled to improved methodology for peptide and protein identification. The separation of complex protein mixtures using both on- and off-line 2D liquid chromatography using derivitized polystyrene-divinylbenzene (PS-DVB) pellicular ion-exchange resins and PS-DVB monolithic reversed-phase columns is described. Proteolytic digestion of the fractions followed by rapid liquid chromatography - tandem mass spectrometry was used to complete the analysis. An alternative methodology, relying on direct analysis of the second dimension eluents by top-down (mass spectrometric analysis of intact proteins) methodology, using an Apex IV 12 T Fourier-transform ion cyclotron resonance mass spectrometer (Bruker Daltonics) and an HCT ion trap (Bruker Daltonics) equipped with electron transfer dissociation has allowed in-depth analysis of intact proteins. Sample types investigated to establish the utility of the methodology include bacterial lysates (Bordetella parapertusis, and Escherichia coli), a eukaryotic parasite (Leishmania donovani), and transformed human cell lines. These developments lead to a multidimensional intact protein separation methodology suitable for small-sample proteomics (minimum effective protein load of 200 μg) and good reproducibility (1.5% variation in the ion exchange dimension, 0.5% variation in the reversed phase dimension). Analysis of the digested fractions gave good coverage of the proteome as well as a high predicted dynamic range, capable of detecting proteins with codon adaptation index scores ranging from 0.22 to 0.99, out of a logarithmic scale from 0 to 1. Proteins representing low (8 kDa) and high (500 kDa) molecular mass and extremes of predicted pI were identified, as well as a number of membrane proteins. Resolution of the overall protein separation was such that single protein species often occurred in one or two fractions for both the ion-exchange and reversed phase separations, with the fractions varying in complexity. Separation of modified proteins in the ion-exchange dimension demonstrated separation of isoforms. Most analyses coupled anion-exchange chromatography in the first dimension to monolithic reversed phase separation in the second dimension. To provide alternative first dimension methodologies for specific sample types, external gradient chromatofocussing (based on separation by isoelectric point) and high-pH ion-pair reversed phase were evaluated as additional techniques. In particular the pISEP (CryoBioPhysica) technique for chromatofocussing constituted an effective orthogonal separation methodology for separation, and its use in a proteomics context was compared to that of the original anion-exchange, reversed phase technology. To rapidly analyse the large number of fractions generated by the technique, bottom-up protocols required improvements in the throughput of peptide separations. PS-DVB monoliths were employed for rapid peptide separations and conditions for analysis were evaluated and optimized. The implementation of a parallel 200 μm monolith system for tryptic peptide separations ensured minimal sample loss and improved sample throughput with little loss of sensitivity. For simple mixtures, reversed-phase separation times could be reduced to a few minutes without significantly affecting data content, although rapid scanning capability is essential due to the very narrow peak widths. Quantitation is of paramount importance to any proteomic technique, and liquid chromatographic separation of intact proteins provides flexibility for differential analysis of complex samples. Three categories of quantitative analysis were evaluated for two dimensional intact protein separation by liquid chromatography: ultraviolet-absorbance maps, isotopic labeling and label-free computational analyses. UV-absorbance maps of wild-type and pentamidine resistant Leishmania donovani were compared using standard two dimensional gel electrophoresis analysis software and resulted in the identification of a small number of quantitiative differences. Modifications to the isotope coded affinity tag (ICAT) and isotope tags for relative and absolute quantitation (ITRAQ) protocols were developed to allow protein labeling prior to separation, and the related methodologies ExacTag, isotope coded protein label (ICPL) and stable isotope labels for amino acids in culture (SILAC) were evaluated. Finally, label-free techniques have been employed for protein quantitation by liquid chromatography (LC)/Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS). A particular niche for liquid chromatographic separation of intact proteins is in top-down analysis, in which mass spectrometric analysis of intact proteins is performed. The separation technology was directly coupled to high-resolution FT-ICR MS for analysis of standards, mixtures, and cellular lysates, leading to the identification of an intact Leishmania protein and post-translational modification (PTM) mapping of histone H4. In addition, the recently developed electron transfer dissociation (ETD) fragmentation methods coupled with proton transfer reaction (PTR) for charge reduction allowed analysis of standards and isotopically labeled cellular lysates using an ion trap instrument. In summary, we have developed a general proteomic methodology with unique flexibility as well as applicability to top-down analysis. It has been applied to multiple complex samples in a variety of analysis conditions including quantitation methodologies and state-of-the-art mass spectrometric techniques. In general, the method equals other separation methods in terms of protein identification rates, is substantially more reproducible and automated, but is more time-consuming. Currently, two dimensional intact protein separation by liquid chromatography using polystyrene-divinylbenzene-based columns is particularly applicable to top-down analysis of heavily modified proteins. However, there is considerable remaining room for optimization and improvement of methodologies, and further development will enhance the technique for general use.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:495317 |
| Date | January 2008 |
| Creators | Burgess, Karl |
| Publisher | University of Glasgow |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://theses.gla.ac.uk/322/ |
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