Biospecific Element-Tagging And ICPMS-based Bioanalysis

Recently,element-tagging based inductively coupled plasma mass spectrometry(ICPMS)has been becoming more and more popular in scientific community for bio-quantitative bioanalysis.As we know the advantage(for example,labeling-free)when the natural existing elements(e.g.S,P,Se,Fe,Mn,Co,Ni,and Zn)in biomolecules are utilized for their quantification using ICPMS,but these element-labels suffer from several intrinsic disadvantages such as low ionization efficiency in ICP ion-source and thus high detection limit,polyatomic ion interferences and possible instability of metal ions that exist in the biomolecules by coordination binding.In order to overcome these disadvantages,exogenous element-tagging strategies have been developed and predominated in the field of element-tagging based ICPMS quantitative bio-analysis.Among which Lanthanide-tagging based ICPMS quantitative bio-analysis is of the merits of no biological background,high sensitivity,wide dynamic linear range and almost no matrix effect,especially ensure the accuracy and credibility of determination via the isotope dilution techniques.From the moment,element-tagging based ICPMS quantitative bioanalysis has been moving from the analysis of model nucleic acids and proteins in-vitro to that of biomolecules in living systems.This in vivo quantitative bioanalysis will provide more clues and suggestions to the biological problems.However,the key point of the element-tagging based ICPMS quantitative bioanalysis is how to tag/label the biomolecules with the elemental reporters,in which the labeling/tagging should be qualified with biospecificity,fast kinetics,and exact binding stoichiometry as well as good biocompatibility.In such circumstances,the biospecific element-tagging strategies development has been an important but challenging research area.Considering that selectivity and sensitivity are the two pillars of analytical chemistry,we developed biospecific europium-tagging strategies that utilize the features of activity-based inhibitions and metabolic pathways of biomolecules together with the bioorthogonal click chemistry for the highly sensitive and specific quantification of targeted biomolecules and their activity evaluation as well as counting their host cells and bacteria using ICPMS.The established methodology was then applied to investigate the related biological functions and behaviors of the targeted biomolecules under external stimuli.The details were presented in this PhD thesis that includes six chapters.Chapter 1.I brifly summarized the state of the art and discussed the remaining problems and challenges in the field of the element-tagging based ICPMS quantitative bio-analysis.The bioorthogonal click chemistry and their applications in chemical biology were also introduced.Based upon,research proposal of my PhD study was then presented.Chapter 2.P450 3A4(CYP3A4)is one of the most important isoforms in the human cytochrome P450 superfamily.It was used as an example in order to demonstrate an activity-based labeling and then click chemistry(CC)mediated element-tagging strategy for simultaneously specific quantification and activity measurement of an enzyme using species-unspecific isotope dilution inductively coupled plasma mass spectrometry(SUID-ICPMS).A dual functional hexynylated 17a-ethynylestradiol activity-based probe was synthesized for specifically labeling CYP3A4 and then CC-mediated Eu-tagging with an azido-DOTA-Eu complex for CYP3A4 quantification and activity measurement in human liver microsome and serum samples using 153Eu SUID ICPMS.The LOD(3a)of CYP3A4 reached 20.3 finol when monitoring 151/153Eu ICPMS signals,in addition to the merits of specificity and simultaneous activity measurement achieved.Chapter 3.A novel activity-based and Cu-free click chemistry(CC)mediated methodology for glutathione Stransferase omega 1(GSTO1)quantification using species unspecific isotope dilution inductively coupled plasma mass spectrometry(SUID-ICPMS)was developed,in which dibenzylcyclooctynemodified 2-chloroacetamide(DBCO-ChAcA)was designed and synthesized as a navigator towards GSTO1 for subsequent N3-DOTA-Eu-tagging via Cu-free CC.Using 153Eu-SUID ICPMS coupled with size exclusion chromatography(SEC),the LOD(3?)of GSTO1 reached 6.9 fmol with an RSD of 2.4%at the 0.1 ?M level(n = 5)considering the recovery of GSTO1 on the SEC was 96.5 ± 2.4%.The GSTO1 contents in the cells of human hepatocellular carcinoma C7721 and breast carcinoma MCF-7 as well as normal hepatic C7701 without or with cis-platin administration were quantified to be from 1.2 ?g/10,000 cells(n = 3,RSD = 4.5%)corresponding to 1.2 ×10-2 ng per cell to 4.76 ?g/10,000 cells(n = 3,RSD = 2.9%)corresponding to 4.76 × 10-2 ng per cell.For a comparative study,DBCO-ChAcA-fluor 488-based fluorescence microscopy could not alone visualize GSTO1 in the cells but could together with those from the small SH contain molecules such as GSH and that from extra N3-fluor 488 in the cells.Chapter 4.Bacterial resistance has been a global crisis.D-Alanine-crosslinked peptidoglycan(PG)layers underpinning the cytoplasmic membrane with membrane-spanning proteins set the first barrier for antibiotic resistance.I developed a Eu-tagging strategy for quantifying the D-Alanine in vivo in the PG-based bacterial cell wall.Alkyne-bearing D-Alanine(ADA)metabolism-incorporation enables us to perform a ADA-specific Eu-tagging with azide-DOTA-Eu and to count the D-Alanine using 153Eu-isotope dilution ICPMS.Escherichia coli and Staphylococcus aureus were exemplified to investigate density of D-Alanine(dDA)dynamics during the different growth phases and under the administration of antibiotics.This quantitative dDA provides a molecular insight for understanding the role of D-Alanine in building the PG layers and the mechanisms of antibiotic resistance,which,in turn,elevate discovery and design of novel antibiotics and strategically combinatorial use of the established antibiotics.Additionally,dDA allowed us to count the bacterial number,providing a simple and promising bacterium counting method.Chapter 5.Although we believe that the cell surface sialic acids(Sias)are playing an important role in cell-cell interactions and related tumor metastasis processes,acquisition of their quantitative information has yet been a challenge to date.I developed a new analytical platform for Sias-specific imaging and absolute quantification.N-Azidoacetyl-Mannosamine tetraacylated was used as a metabolic sugar substrate to bioassemble azido-Sias on the surface of cells via the metabolic pathway of Sias de novo synthesis.These azido-Sias allow us to perform a duplex Sias-specific analysis with various fluorescent and elemental reporters such as DIBO-Alexa Fluor 647,DBCO-DOTA-Eu and DBCO-PEG4-BODIPY,which can be easily labeled and/or tagged through an effective copper-free bioorthogonal click reaction.Compared to the previous reported strategies,we absolutely quantified the cell surface Sias with the LODs(3a)down to 8.9 fmol and 0.24 pmol using 153Eu-and 10B-species unspecific isotope dilution ICPMS,in addition to their red-and green-CLSM profiling,achieving a specific targeting and two-dimension analysis.Such a platform enables us to evaluate Sias regulation under the administration of paclitaxel,finding that 1 ?M paclitaxel induced a significant Sias decrease of 67%on the surface of hepatic tumor cell SMMC-7721,while had no obvious adverse effect to that of para-carcinomatous liver cell L02.Besides Sias,we believe that this metabolism-based click-mediated platform will provide opportunities to study other monosaccharides and their corresponding biological roles when more corresponding chemically modified sugar substrates and specific bioorthogonal reactions are developed.Chapter 6.I summarized the achievments during my PhD study,and pointed out the issues that need to be solved in the future.Trends of the element-tagging based ICPMS quantitative bioanalysis were also prospected.