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Development Of New Methods To Study The Detection Of Protein Modification And Quantification

Posted on:2013-01-04Degree:DoctorType:Dissertation
Country:ChinaCandidate:Y Y XuFull Text:PDF
GTID:1110330371486843Subject:Biochemistry and Molecular Biology
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Protein, as one of the main components of living organisms, plays a key role in various activities of life. It not only directly reflects life phenomena, but also executes a variety of cell functions. Post-translational modification of protein includes the proteolytic cleavage and chemical modification at specific sites of newly synthesized proteins. Studies have shown that the status of protein post-translational modification, as well as the concentration of protein biomarkers, is directly related to human metabolism, disease diagnosis, prognosis and efficient evaluation of response to drug intervention. Many kinds of diseases, including cancer, diabetes, metabolic and neurological syndrome all correlate with the abnormal modification and expression of the proteins. Therefore, development of simple, rapid, sensitive and accurate method for protein modification and quantification has become the focus of protein research, which has great significance in life science and clinical diagnosis. In this thesis, the author will present the following findings. 1. Simulation and assay of protein biotinylation with electrochemical technique.Protein biotinylation plays an important role in metabolism and transcription regulation, so study of protein biotinylation has received more and more interests. In this work, the bifunctional Escherichia coli biotin-inducible repressor protein A (BirA) and its substrate for protein biotinylation, a unique peptide with a specific sequence, are introduced as a model to electrochemically simulate the committed step in fatty acid biosynthesis. With the help of gold nanoparticles and peroxidase-labeled streptavdin involved in the electrochemical system, protein biotinylation is achieved on the surface of the working electrode, and the process of protein biotinylation can be electrochemically assayed by the obtained electrochemical response. Therefore, a new method to assay protein biotinylation is proposed and this work may provide a new perspective for understanding protein biotinylation in vitro.2. Gold nanoparticles based colorimetric assay of protein poly(ADP-ribosyl)ation.Protein poly(ADP-ribosyl)ation (PARylation) is a unique post-translational modification process catalyzed by poly(ADP-ribose) polymerases (PARP) involved in various cellular processes. In this chapter, auto-PARylation which is vital in poly(ADP-ribose) metabolism is studied with gold nanoparticles (Au-NPs). When the catalytic substrate of protein PARylation, nicotinamide adenine dinucleotide (NAD), is not cleaved into nicotinamide and ADP-ribose unit, the negative-charged NAD molecules are adsorbed on the surface of Au-NPs, keeping Au-NPs stable, so the color of the test solution is red. Nevertheless, after PARylation takes place, Au-NPs will aggregate due to unprotection of NAD, thus the color of the test solution changes to be blue. Furthermore, the spectrophotometric time-dependent curve of Au-NPs may character the process of auto-PARylation more sensitively and specifically. Due to the unnecessasity of coupled enzymes or modified catalytic substrates, this work may be followed by investigating the PARylation of other proteins in the future.3. Sensitive detection of CD147and its expression on cancer cells with electrochemical technique.Cluster of differentiation147(CD147), also known as extracellular matrix metalloproteinase inducer (EMMPRIN), plays an essential role in tumor progression and metastasis, the expression of which on cell surface is a critical clinical testing index for cancer therapy. In this work, an electrochemical method to assay CD147/EMMPRIN expression on tumor cell surface is proposed. While the oxidation of3,3',5,5'-tetramethylbenzidine (TMB) catalyzed by horseradish peroxidase (HRP) can be employed for electrochemical measurement, the signal enhancement amplified by Au-NPs can be also utilized in this study. Therefore, under optimized conditions, the fabricated biosensor responds linearly to the CD147/EMMPRIN concentration from125to1000pg/mL with a detection limit as low as52pg/mL. Moreover, the CD147/EMMPRIN expressed on a single breast cancer cell can be calculated as2.57×104molecules/cell. So, the proposed strategy in this study with considerable potential for monitoring the dynamic protein expression on cancer cells may contribute to the effective diagnosis and treatment for cancers in the future.4. Protein detection based on small molecule-linked DNA with electrochemical technique.In this chapter, based on small molecule-linked DNA and the nicking endonuclease-assisted amplification (NEA) strategy, a novel electrochemical method for protein detection is proposed in this work. Specifically, the small molecule-linked DNA (probe1) can be protected from exonuclease-catalyzed digestion upon binding to the protein target of the small molecule, so the DNA strand may hybridize with another DNA strand (probe2) that is previously immobilized onto an electrode surface. Consequently, the NEA process is triggered, resulting in continuous removal of the DNA strands from the electrode surface, and the blocking effect against the electrochemical species [Fe(CN)6]3-/4-becomes increasingly lower; thus, increased electrochemical waves can be achieved. Because the whole process is activated by the target protein, an electrochemical method for protein quantification is developed. Taking folate receptor (FR) as an example in this work, we can determine the protein in a linear range from0.3to15ng/mL with a detection limit of0.19ng/mL. Furthermore, because the method can be used for the assay of FR in serum samples and for the detection of other proteins such as streptavidin by simply changing the small molecule moiety of the DNA probes, this novel method is expected to have great potential applications in the future.5. Sensitive detection for protease activity with electrochemical technique.In this study, we have designed and synthesized a positively charged peptide, which has a cysteine at one end, an arginine at the other end. The peptide can self-assemble onto a gold electrode through the special force of Au-S bond and the modified electrode shows positive charge, which has a strong electrostatic repulsion of the positively charged signal molecules. When the modified electrode incubates with protease, arginine leaves from the electrode surface leading to the weakened repulsion, thus the electrochemical signal increases. Therefore, taking trypsin as an example, under optimized conditions, the fabricated biosensor responds to trypsin concentration from0.5to15μg/mL. Furthermore, based on the same principle, via the change of cleavage sequence of the peptide, the system is applicable for the detection of other protease activities.
Keywords/Search Tags:Protein modification, protein quantification, protease, biotin ligase, PARP, extracellular matrix metalloproteinase inducer (EMMPRIN, CD147), tumorbiomarker, protein biomarker, folate receptor, small molecule linked DNA ligand, Au-NPs
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