Study Of New Methods To Assay Protein By Using Peptide

Analytical methods for protein quantification are powerful tools of diagnosis and prognosis in combating diseases such as cancer,Alzheimer's disease,etc.As the major component and active regulator of molecular biosystems,proteins have activities strictly controlled in both the temporal and spacial dimensions.Under pathological conditions,however,the balanced dynamics of protein interactions and activities can be disrupted,leading to identifiable signature of disease development.Screening of these disease markers,as well as marker-based diagnosis,all require highly sensitive analytical tools for protein quantification in the complicated clinical samples.Over the last decade,artificially synthesized targeting ligands,particularly aptamer,have gradually evolved into a powerful tool of molecular recognition,not inferior to the biologically generated antibodies.Although nucleic acid has largely led the way in this development,it can also be noted that short functional peptides frequently act as therapeutic and biomedical agents,targeting many different naturally occurring and artificial protein-ligand interactions.So,as presented in this dissertation,the peptide motifs of protein recognition,metal ion coordination and several other biochemical functions have been assembled and combined to develop multi-functional peptide-based probes and protein sensing interfaces that have been preliminarily optimized to suite the need of clinical detection of disease marker proteins:1.Protein quantification based on peptide-cucurbituril supramolecular interactionThe side chain of aromatic amino acids and some electroactive heterocyclic aromatic compounds can be non-covalently linked together via their simultaneous recognition and "host-guest" inclusion by cucurbit[8]uril,a synthetic macrocyclic molecule;while many protein-peptide interactions is through hydrophobic interactions with aromatic amino acids.Therefore,peptides containing aromatic amino acids are designed as targeting probes and aromatic supramolecular interactions are employed to non-covalently label the probes to generate signal output.In addition,substitution of the electroactive signal reporter by a photo-reactive catalyst can greatly improve sensitivity of the detection,enabling clinical analysis of disease marker proteins in clinical samples of non-small cell lung cancer.2.Protein quantification based on peptide folding and peptide nanostructureUpon binding with ligands,peptides undergo conformational changes and can even fold and assemble into nanostructures.Using the assembly and aggregation of ?-amyloid peptide as the means to amplify signal readout,clinical serum analysis of multiple biomarkers of prostate cancer is realized.Meanwhile,a conformational biosensor is designed for ?-amyloid peptide,the conformational change upon target-binding results in altered kinetics of electron transfer,and frequency synchronization of the electrochemical scans enables the advantageous signal-on working mode of the biosensor.Besides the naturally occurring ?-amyloid peptide,artificially designed peptides are also employed to form a peptide nano-network for the detection of potential marker protein of hepatocellular carcinoma in clinical tissue samples.3.Protein quantification based-on peptide functional motifsPeptides can bind with certain ligands to form functional motifs with targeting or catalytic abilities.Accordingly,a trimeric cupric ion-binding peptide sequence,as the catalytic motif,is combined with a targeting motif to form a bi-functional probe that can recognize target protein as well as to amplify the signal readout.The cupric ion-complexed catalytic motif can generate a large amount of electrochemical reporters via its catalytic activity,enabling highly sensitive detection of cell skeletal proteins in clinical samples.4.Protein quantification with both peptide and small-molecule probe Small-molecule probe can supplement peptide in targeting specific functional sites on the surface of proteins,as well as improving affinity towards certain targets.Employing peptide probes to capture target proteins,while using chemically selective small-molecule probes to recognize modified side-chain groups on the surface of the target protein,the detection of protein nitration is realized.Furthermore,by conjugating the small-molecule probe with large protein molecules,the modified probe can be used together with the original probe to study the relationship between the number of nitrated amino acids and functional state of the target proteins.On the other hand,small molecule modified peptide probe is also constructed to target metastatic marker proteins on the surface of cancer cells.5.Preliminary application in clinical protein bioanaysisThe above designed peptide-based biosensors have been applied in clinical detection of biomarker proteins of cancer and other diseases.To obtain the analytical performance for clinical application,various design strategies and arrangements have been adopted to minimize the interference from the complicated clinical samples,as well as to improve the sensitivity to detect proteins of low abundance.In the detection of clinical tissue samples of liver,lung and breast cancer,fluctuation in the amount of biomarker proteins assayed by the above proposed methods show a parallel with the progress of disease,the detected pattern of biomarker expression is also coherent with the stage,grade and state of metastasis examined by classical pathologic analysis.Furthermore,the detection of multiple protease biomarkers of prostate cancer in clinical serum samples can provide reference for patient sub-grouping and prognosis.