To improve the efficiency, accuracy, reproducibility, throughput and proteome coverage of mass spectrometry-based quantitative approaches, both in vitro and in vivo tagging of particular amino acid residues of cellular proteins have been introduced to assist mass spectrometry for global-scale comparative studies of differentially expressed proteins/modifications between different biologically relevant cell states or cells at different pathological states. The basic features of these methods introduce pair-wise isotope signals of each individual peptide containing a particular type of tagged amino acid (amino acid-coded mass tagging) that originated from different cell states. In this review, the applications of major amino acid-coded mass tagging-based quantitative proteomics approaches, including isotope-coded affinity tag, isobaric tags for relative and absolute quantification (iTRAQ) and stable isotope labeling by amino acids in cell culture are summarized in the context of their respective strengths/weakness in identifying those differentially expressed or post-translational modified proteins regulated by particular cellular stress on a genomic scale in a high-throughput manner. Importantly, these gel-free, in-spectra quantitative mechanisms have been further explored to identify/characterize large-scale protein-protein interactions involving various functional pathways. Taken together, the information about quantitative proteome changes, including multiple regulated proteins and their interconnected relationships, will provide an important insight into the molecular mechanisms, where novel targets for diagnosis and therapeutic intervention will be identified.

A microchip reactor coated with a gold nanoparticle network entrapping trypsin was designed for the efficient on-line proteolysis of low level proteins and complex extracts originating from mouse macrophages. The nanostructured surface coating was assembled via a layer-bylayer electrostatic binding of poly(diallyldimethylammonium chloride) and gold nanoparticles. The assembly process was monitored by UV-visible spectroscopy, atomic force microscopy, and quartz crystal microbalance. The controlled adsorption of trypsin was theoretically studied on the basis of the Langmuir isotherm model, and the fitted ⌫ max and K values were estimated to be 1.2 ؋ 10 ؊7

Inflammation is tightly regulated by nuclear factor-kappa B (NF-kappaB), and if left unchecked excessive NF-kappaB activation for cytokine overproduction can lead to various pathogenic consequences including carcinogenesis. A proinflammatory cytokine, tumor necrosis factor-alpha (TNF-alpha), can be used to explore possible mechanisms whereby unknown functional pathways modulate the NF-kappaB activity for regulating TNF-alpha-induced inflammation. Given the multifunctional nature of 14-3-3 family proteins and the recent finding of their presence in the TNF-alpha/NF-kappaB pathway network, we used a dual-tagging quantitative proteomic method to first profile the TNF-alpha-inducible interacting partners of 14-3-3 epsilon, the least characterized 14-3-3 isomer in the family. For the first time, we found that TNF-alpha stimulation enhances the interactions between 14-3-3 epsilon and some key components in the mitogen-activated protein kinase (MAPK) signal module which is located at the immediate upstream of NF-kappaB, including transforming growth factor-beta activated kinase-1 (TAK1) and its interacting protein, protein phosphatase 2C beta (PPM1B). By using confocal laser scanning, we observed the TNF-alpha-induced colocalizations among 14-3-3 epsilon, TAK1, and protein phosphatase 2C beta (PPM1B), and these interactions were also TNF-alpha-inducible in different cell types. Further, we found that during the full course of the cellular response to TNF-alpha, the interactions between 14-3-3 epsilon and these two proteins were dynamic and were closely correlated with the time course-dependent changes in NF-kappaB activity, suggesting that these 14-3-3 epsilon interactions are the critical points of convergence for TNF-alpha signaling for modulating NF-kappaB activity. We then postulated a mechanistic view describing how 14-3-3 epsilon coordinates its dynamic interactions with TAK1 and PPM1B for differentially modulating TNF-alpha-induced changes in NF-kappaB activity. By using bioinformatics tools, we constructed the network involving most of the 14-3-3 epsilon interacting proteins identified in our proteomic study. We revealed that 14-3-3 epsilon coordinates the cross talks between the MAPK signal module and other molecular pathways/biological processes primarily including protein metabolism and synthesis, DNA repair, and cell cycle regulation where pharmacological targets for therapeutic intervention could be systematically located.

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