Azo-coupling Strategy For Derivatization Of 3-Nitrotyrosine-Containing Protein

Protein nitration which is most caused by the reactive nitrogen species(RNS)was closely related to over 50 human diseases including neurodegenerative diseases,aging,cancer and cardiovascular diseases.Due to its irreversible modification and stability,3-nitrotyrosine(3-NT)has been used as a stable marker of RNS,oxidative/nitrative stress and protein nitration stress in many biological,pathological and medical researches.The detection of 3-NT and 3-NT-containing protein mostly depends on the instrumental analysis and 3-NT antibody-based biochemical analysis.These methods have extremely low detection limitation and high sensitivity.However,the complicated operation,time consuming,fussy sample pretreatment process and the treated sample cannot be used for other test are remain big problems in 3-NT detection.Protein chemical modification has become a good choice to label,detect and enrich 3-NT-containing protein due to its simple operation,high chemical selectivity and broad compatibility.Several specific labelling methods have been developed these years all start with reducing 3-NT to 3-Aminotyrosine(3-AT)and do the following specific reaction with the 2-Amino-4-alkylphenol.Inspired by the high yield of azo dyes synthesis,we developed a fast,high yield diazo coupling strategy for specifically labelling 3-NT-containing protein in proteome.The strategy was investigated in specificity,lower labelling limitation and compatibility.After being tested in small molecular level,3-NT-containing peptide and protein,we have a preliminary application of this azo-coupling strategy in labelling the 3-NT-containing protein in nitro-HepG2 cell(nitrited by peroxynitrite).The main contents are summarized as follows:Chapter 1: A brief introduction of the background and aim of this sunject.It contained a brief introduction of the influence and detection of reactive oxygen/nitrogen species.Then we explained the mechanism of tyrosine nitration,the physicochemical properties of 3-NT and the effects of tyrosine nitration on protein structure and function by listing related researches.Finally,some recent developed 3-NT labelling approaches were present detailedly.Chapter 2: Based on the high performance azo-coupling reaction under mild condition,we developed the protein modification strategy for specific labelling 3-NT-containing protein.After labelling the 3-NT-containing peptide and protein,our strategy labelled 3-NT-containing preotin in nitro-HepG2 cell successfully.With the simple operation and relatively low cost,the picomole level of PTN is able to be derivatized within 2 h and compatible with different kinds of proteins from biological samples.As a cleavable linkage,the azo bond minimized the influence caused by sample derivatization to the following antibody-related analysis.Furthermore,with the introduction of the bioorthogonal azide group,samples can be used in multiple studies with only being derivatized once.The detailed experimental precudure,original data and the discussion of results were shown in this chapter.Chapter 3: A summary of the azo-coupling strategy for 3-NT-selective labelling of proteins in proteome and perspective view about potential reaction that can be used for 3-NT-containing protein labelling.