Study On Novel Methods For Mass Spectrum Sensing Of Protein Markers

Proteins are a class of important functional executors in the life system,and their activity is always affected by their own state.Proteases exist widely in living organisms and ensure the normal operation of life activities such as material transport,genetic material replication,cell proliferation,differentiation and apoptosis.Therefore,systematic analysis of proteases and their activity is of great significance for monitoring related diseases and developing clinical treatment.The rise of biomass spectrometry with matrix assisted laser desorption ionization means that mass spectrometry has become an important detection method for macromolecule analysis in the field of life analysis.Matrix assisted laser desorption ionization time of flight mass spectrometry(MALDI-TOF MS)has become a powerful tools in the field of life analytical chemistry due to its high sensitivity,high throughout properties,as well as suitable for the analysis of complex samples and strong anti-interference ability.However,in clinical analysis,real biological samples are usually composed of many complex chemicals,and some disease-related biomolecules as markers often need to be directly detected in real samples.Therefore,it is very essential to develop new techniques and methods for highly sensitive and highly throughput quantitative analysis of actual samples.In this paper,a series of new MALDI-MS strategies for quantitative detection of proteases and biomarkers in complex samples were developed by combining mass spectrometry with sensing chip.The specific works are as follows:1.A MALDI-MS sensing chip prepared by non-covalent assembly for quantitation of acid phosphataseAcid phosphatase(ACP)is a ubiquitous kind of natural protease in animals and plants,which plays an important role in many physiological processes.Its abnormal expression in human serum may indicate the occurrence of some diseases.A novel matrix assisted laser desorption ionization mass spectrometry(MALDI-MS)bio-sensing chip was designed for quantitative detection of ACP.The ACP sensing chip is constructed on biotinylated polyethylene(PEG)modified ITO slide,which was further assembled with the biotinylated peptide substrate through non-covalent interaction between biotin and streptavidin.In the presence of ACP,the peptide substrate dephosphorylates under acidic conditions,generating new mass signal.ACP quantitative detection is achieved by the mass signal ratio of the product to the sum of product and substrate peptides.Under the optimal detection condition,the ratio was linearly correlated with ACP concentration at 0.05-1 g/L,and the detection limit(LOD)was 0.04 g/L.The designed ACP sensing chip could be used for the analysis of ACP in clinical complex samples,and has the characteristics of high selectivity,good repeatability and strong anti-interference ability.This work further elucidates the concept of“mass spectrometry biosensing”and the application of MALDI-MS in quantitative analysis,providing a fast and convenient method for clinical quantitative diagnosis of proteases.2.A novel covalent assembly sensing chip for MALDI-MS quantitation of multiple matrix metalloproteinasesAbnormal expression of MMPs is associated with certain diseases.MALDI-MS has the advantages of high sensitivity,high throughout and label free.The appearance of this new"soft-ionization"technology has revolutionized the traditional MS,which is mainly applied to the study of small molecules,and extended to the analysis of biological macromolecules with high polarity,volatility and thermal instability.Due to the limitation of MALDI-MS to interrupt covalent interaction,its detection still has certain deficiency.In order to solve this problem,this work developed a mass spectrometry sensing chip based on"photocleavage molecules",using photocleavage molecule as"switch",so that MALDI-MS can break the covalent connection,thus realizing the quantitative detection of a series of matrix metalloproteinases(MMP-3,MMP-7,MMP-9).3.MALDI-MS based on barcode peptides for quantitative detection of multiplex MMPsMMPs are considered to be an important biomarker for early cancer diagnosis and a target for therapeutic drug development.Therefore,quantitative analysis of the MMPs in cellular processes is crucial for understanding the physiological and pathological state of the cell.In this work,the mass barcoded nanoprobe in response to proteases was used for multiplexing and quantitative detection of MMPs activities in cells.Gold nanoparticles(Au NPs)containing different substrate peptides were attached to the magnetic Fe₃O₄ nanosphere by long PEG chain with photocleavage groups,forming a core-satellite structure.The activity of MMPs was translated into the ratio of the product mass tag to all the mass tags on Au NPs by using the built-in laser interrupting the photocleavage group.This work combines nanotechnology with mass spectrometry and uses the magnetism of Fe₃O₄ nanospheres for rapid purification,opening a new idea for intracellular quantitative detection of MMPs and the development of therapeutic drugs.