| Mass spectrometry has emerged as a powerful tool in the field of proteomics enabling characterization of proteins, their post translational modifications and determining protein-protein interactions. This versatile analytical technology allows both qualitative and quantitative measurements on a large number of proteins that is required to understand the complex molecular biology underlying the various cellular processes such as growth, development and response to stimuli. There are several mass spectrometry-based quantitative proteomic strategies reported in literature, all of which can be broadly classified into two groups: stable isotope-labeling approaches and label-free approaches. While the stable isotope labeling strategies are well established and applied extensively, label-free approaches are relatively new in the field of quantitative proteomics. The aim of this work was to evaluate the use of both labeling and label-free quantitative proteomic approaches with specific application to plant hormone signaling pathways. The use of stable isotope labeling for phosphorylation quantification was evaluated by the development and application of a new Phosphoprotein Acid cleavable Solid-phase Isotope-coded Reagent (PhASIR) to both model phosphopeptides and a phosphoprotein. The difficulties encountered with this approach such as complex labeling chemistries, presence of by-product, poor reproducibility and considerable loss of sample suggested that this approach was not suitable for quantitative phosphoproteomics. On the other hand, label-free quantification strategies are relatively simple, fast and can be easily applied to large-scale proteomic studies. A label-free, intensity-based quantitative strategy using a unique parallel fragmentation and a data-independent acquisition mode, termed LC/MSE was developed to carry out the functional analysis of the Arabidopsis thaliana receptor kinase BRI1 in vivo phosphorylation sites in response to brassinolide treatment. This label-free approach was also applied to carry out a comprehensive analysis of the changes in protein abundance in response to gibberellin treatment in Arabidopsis seedlings. The gel-based, LC/MS E approach termed GeLC/MSE allowed the simultaneous identification and quantification of over 200 gibberellin-responsive cytoplasmic proteins with atleast a greater than two-fold change in abundance. Overall, the LC/MSE approach proved to be a reliable method for label-free quantitative proteomics with the advantages of increased protein/proteome coverage, good analytical reproducibility and accurate quantitative results. |
