Proteomics based on liquid chromatography coupled to mass spectrometry has developed rapidly in the last decade and become a powerful tool for protein mixtures analysis. LC-MS based proteomics involves four steps, sample preparation, liquid chromatography, mass spectrometry and bioinformatics. Improvements in each step have extended its applications to new biological research areas. This dissertation mainly focuses on method developments in both qualitative and quantitative proteomics. The first part of this dissertation focuses on qualitative analysis of T. gondii Parasitophorous Vacuole Membrane (PVM) proteins, which is very important for T. gondii’s survival. The hypothesis of this study is that proteomic approaches coupled with immunoprecipitation using polyclonal antisera as affinity reagents can successfully characterize the proteome of the T. gondii PVM. The “Three-layer Sandwich Gel Electrophoresis” (TSGE) protocol, was developed to contend with efficient salt removal and protein concentration from challenging samples. Furthermore, the TSGE coupled to 2D-LC-MS/MS was proven to be effective with the proteomic analysis of complex protein mixtures like T. gondii whole cell lysate, allowing for high-throughput protein analysis from complex samples. By using the TSGE-2D-LC-MS/MS methodology, we successfully identified 61 proteins from the PVM samples and constructed the PVM proteome. The second part of this dissertation describes a novel method for selecting an appropriate isocyanate reagent for potential quantitative proteomics application. Our hypothesis is alteration of isocyanate structure will change fragmentation pattern and ESI property of isocyanate modified peptides. The CID property of N-terminal modified peptides by phenyl isocyanate (PIC), phenethyl isocyanate (PEIC) and pyridine-3- isocyanate (PyIC) was systematically studied using LC-ESI-MS/MS. We observed that adjustment of isocyanate structure changed both ESI and fragmentation characteristic of modified peptides. We rationalized the decrease of protonation of PIC and PEIC modified peptides results from the neutral property of the both reagents. The electron withdrawing feature of PyIC leads to significant reduction of fragments during CID. Therefore, we designed a new isocyanate reagent, 3-(isocyanatomethyl) pyridine (PyMIC). The results revealed that PyMIC modified peptides had more suitable ESI properties and generated more sequence-useful fragments compared to PIC, PyIC and even unmodified peptides. PyMIC is a more appropriate labeling reagent for quantitative proteomics applications.

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