The Proteomic Profiling Of Newly Synthesized Proteins Of HEK293 Cells

In both normal and pathological states, cells respond quikly to environmental stimulis by synthesizing new proteins. The selective identification of newly synthesized proteins has been hindered by the fact that all proteins, new and old, share the same pool of amino acids and thus are chemically indistinguishable.The proteome is a dynamic entity, tightly regulated by protein synthesis and degradation to maintain homeostasis in cells, tissues, and organisms.Cells respond to fluctuations in their environment by changing the ensemble of proteins they express. Alterations in protein synthesis, degradation and post-translational modifications enable cells to adapt to changing environmental conditions.Hence, a major endeavor in biology is the comparison of two or more protein complements in biological systems, for example, the cancerous versus non-cancerous state. Mass spectrometry (MS)-based proteomics has become an important and versatile technique to characterize the expression and functional modification of proteins, complementing the genomics efforts. Many methods of biochemical and analytical tools for protein separation, fractionation and modification have been developed and combined to enable proteomic analysis. Resolving techniques such as one and two-dimensional gel electrophoresis and multidimensional liquid chromatography, work in conjunction with MS to decipher the protein of a cell or a whole organism.Post-translational modifications such as phosphorylation or ubiquitination readily provide a suitable handle for enrichment of the "phosphoproteome" or for proteins for degradation, reducing sample complexity by selectively enriching for newly synthesized proteins is troublesome, as all proteins—old and new—share the same pool of 20 amino acids. Nonetheless, the specific enrichment and identification of recently synthesized proteins would complement the range of differential proteomic profiling methods and deepen our insights into the spatial and temporal dynamics of proteomes. HPG labelling technique was developed to specifically identify the subpopulation of newly synthesized proteins. The core of the HPG labelling technique capitalizes on the manifold potential of small bioorthogonal chemoselective groups.These groups deliver unique chemical functionality to their target molecules, which subsequently can be tagged with exogenously delivered probes for detection or isolation in a highly selective, manner. In the first step of HPG labelling technique, newly synthesized proteins are labeled using the alkyne-bearing artificial amino acid HPG, endowing them with novel alkyne functionality, which distinguishes them from the pool of pre-existing proteins. Moreover, despite general perception, alkynes are nontoxic and stable. Using the copper-catalyzed alkyne-azide ligation, the reactive alkyne group of HPG is covalently coupled to a fluorescent tag or analkyne-bearing biotin-Flag tag in the second step. This tag enables subsequent detection, affinity purification and MS identification of HPG-labeled proteins. The detection of the flurescence enable us to pick out the newly synthesized proteins.The enrichment for newly synthesized proteins decreases the complexity of the sample, fostering the identification of proteins expressed at low levels.HPG is an effective surrogate for methionine, an essential amino acid, and does not require any further manipulations to be accepted as a substrate by the methionyl-tRNA synthetase. Labeling with HPG is very similar to the traditional metabolic labeling with radioactive amino acids (35S-labeled methionine or cysteine) and has been tested in a variety of cell lines. The presence and incorporation of HPG is non-toxic and does not affect global rates of protein synthesis or degradation. The copper-catalyzed alkyne-azide ligation, also known as "click chemistry," can be performed on denatured proteins in the presence of detergents, such as SDS, promoting the likewise identification of diverse classes of proteins, that is, membranous and soluble, acidic and basic as well as highand low-molecular-weight proteins.The identification of newly synthesized proteins with HPG labelling technique is limited to proteins that possess at least one methionine residue, excluding the 1.02% of all entries in a human protein database, which do not contain a single methionine. Given that 5.08% of the human proteome possess only a single, N-terminal methionine and that this residue may be subject to removal by post-translational processing, at least 94% of themammalian proteomes are candidates for identification by HPG labelling technique.The core of the HPG labelling technique—the chemoselective tagging of HPG-incorporated proteins is not restricted to the mere identification of newly synthesized proteins in a 2D proteomics approach. It also offers the possibility to work in combination with other proteomic approaches, such as isotope-coded affinity tags, isobaric tags for relative and absolute quantification or phospho-and MuDPIT(multi-dimention protein identification technology), to directly compare different proteomes in a single MS experiment or to facilitate the identification of even more and more specific subpopulations of the proteome, respectively. Furthermore, subcellular fractionation and immunopurification of protein complexes can be followed by HPG labelling technique to assess the temporal and spatial dynamics of certain subcellular compartments, organelles and protein-protein interaction networks.2DE is the only method so far to separate thousands of proteins in the same gel at the same time. It provides us a convenient mean and a high resolution method to get the protein purified. The 2D-Gel consists of a 1st electricity focusing and the 2st electrophoresis process. The 1st dimension separate the protein according the PI and the second dimension according the MW of the protein. The 2D-Gel coupled silver stain can resolve 1000-3 OOOspots in a single gel in common. The detection limit can reach the nano-gram protein. But the 2D-Gel also has some disadvantaged:It is hard to detect the low abundance protein and the hydrophobic and membrane proteins. Also, the process is laborious and it is hard to make them highthrouput.Using the HPG labelling technology, we can easily pull the newly synthesized proteins from the whole protein poll by tagging the protein with the HPG. After 2D gel separation, we can pick the newly synthesized fluorescent spot for MS identification. In this way; we can easily find the temporal and spatial newly synthesized proteins.Based on the background knowledge above, our project select the HEK 293 cell, starting with the starving treatment, then labeled the cell with HPG, followed with the click-chemistry tagging on the fluorescent tag. Then the protein was extract from the cell and the 2D GEL separation was performed. After staining the gel the correspondent spots with the fluorescent was cut and sent for MS-identification. Then we carried out the bioinformatics search about the function about the protein. Based on the researches above, we have some conclusion:1. By optimizing the time of the pulse and the concentration of the probe, we find a sound condition to label the newly synthesized protein.2. By comparing the protein labeled with HPG only and HPG with protein synthesis inhibitor, we confirm the labeled fluorescence proteins are newly synthesized proteins.3. We get the good labeling efficacy by optimizing the condition of the click-chemistry.4. We set up the 2D work flow to separate the newly synthesized protein and get the profiling of the HEK293cell lines.5.40 differential protein spots were analyzed by mass spectrometry.26 proteins were identified after deleting keratin and identical proteins.6. We analyze the protein function by searching the protein database.