The Enrichment And Identification Of Metallothionein By Functionalized Nano-Magnetic Particles And MALDI-TOF /MS

Metallothionein?MT?is a low molecular weight protein with the ability of binding metal ions and high inducibility.Therefore,MT is often regarded as an important biomarker for assessment of heavy metal pollution in water environment.The application of MT is very extensive.It has good prospects in medicine,health food,anti radiation and cosmetics.Content of metallothionein in organism is low in nature,The traditional metallothionein enrichment is induced by heavy metal stress,and then MT is extracted from the digestive gland,hepatopancreas and other parts.The traditional extraction of protein is homogenate the tissue with Tris-HCl buffer or sucrose buffer and then centrifuge,after that,the separation and purification of protein is achieved by high performance liquid chromatography or gel filtration and ion exchange chromatography.This method require a long time,complex operation,much loss and high protein content.At present,the superior technology for detecting low abundance proteins is MALDI-TOF/MS analysis.But it has high requirements on the sample.Salt and other small molecules can increase noise,and high abundance proteins often suppress the signal peaks of low abundance proteins,this can lead to a bad detection result.Therefore,the development of low abundance protein enrichment is a research hotspot at present.The development of magnetic composite nanoparticles provides a new idea for the enrichment of low abundance proteins.Herein,we prepared a core-shell nanoparticle:gold-coated iron oxide nanoparticles?Fe₃O₄@Au NP?.It possessed the advantages of fast response to magnetic fields and optical properties attributing to Fe₃O₄ and Au nanoparticles separately.Fe₃O₄@Au nanoparticles can be used to enrich MT simply through Au–S interaction,and the purified proteins were determined by Matrix assisted laser desorption ionization time of flight mass spectrometry?MALDI-TOF/MS?analyses.First,the synthesis and characterization of Fe₃O₄@Au core shell were researched.The Fe₃O₄ nanoparticles were synthesized by chemical coprecipitation,and then Fe₃O₄nanoparticles was silanated.Amido groups modified on the surface of Fe₃O₄nanoparticles.We synthesized gold nanoparticles by sodium citrate reduction method.Fe₃O₄@Au core shell nanoparticles were synthesized through strong adsorption between Au and amino groups.Through the characterization,we found that the synthesized Fe₃O₄@Au core shell nanoparticles had better magnetic responsiveness.However,the Fe₃O₄@Au core shell nanoparticles solution is unstable due to incomplete coating.Therefore,we synthesized and characterized Fe₃O₄@Au core shell nanoparticles by second methods.First,we synthesized Fe₃O₄ nanoparticles,Citric acid ion modified on the surface of Fe₃O₄ nanoparticles.A gold shell was directly modified on the surface of Fe₃O₄ nanomaterials by reduction of citric acid on HAuCl₄·3H₂O.Through the characterization,the synthesized Fe₃O₄@Au core shell nanoparticles not only has good magnetic response and uniform particle size,but also has good dispersion and are not easy to reunite.Therefore,we chose the second synthesized Fe₃O₄@Au core shell nanoparticles for subsequent MT enrichment and mass spectrometry detection.In this study,the mixture of BSA and MT was incubated and washed with Fe₃O₄@Au core shell nanoparticles.The content of MT in mixture was identified by mass spectrometry.It was found that when the BSA samples were not incubated with Fe₃O₄@Au core shell nanoparticles,the strongest peak of BSA appeared in m/z 66415.8.The rabbit liver MT samples were fully mixed with BSA according to the proportion of1:1.The results of mass spectrometric identification of the mixture showed that the strongest peak of BSA was visible within the range of m/z 6000⁷0000,and there was no peak of MT in the m/z 6000⁷000.The peak signal of the MT was completely suppressed by the signal peak of the BSA.Then we added the Fe₃O₄@Au core shell nanoparticles into BSA+MT mixed solution and incubated 5 min.We applied an magnetic field to separate the target protein.After removing the supernatant,the Fe₃O₄@Au-MT were directly detected by mass spectrometry.We found that a clear mass spectrum of MT appeared in m/z 6000⁷000,and there was no obvious mass spectrum in m/z 15000⁷000 range.The results showed that after the enrichment of core shell particles,the BSA of the mixture was removed after washing,and the location of BSA mass spectrum disappeared.The 1 mg/mL MT solution was diluted to 100 ng/mL,0.1 ng/mL,1 pg/mL,10fg/mL series mixed solution with matrix solution?1 mg/mL BSA?,According to this method,Fe₃O₄@Au core shell nanoparticles were added to mixed solution and incubated 5 min,and then the purified proteins were determined by MALDI-TOF/MS analyses.When the concentration of MT was 100 ng/mL,the peak of mass spectrum was stronger,but with the decrease of MT concentration,the intensity of signal peak decreases.After repeated measurements,a weak mass spectrum can be seen when the concentration of MT is 10 fg/mL.When the concentration of MT is below 10 fg/mL,the peak of mass spectrum is not detected.The results in this work showed that the Fe₃O₄@Au nanoparticles could directly enrich MT from the complex solutions and the sensitivity has been greatly improved compared with the single mass spectrometry technology.The detection limit can be up to 10 fg/mL through the identification of MALDI-TOF/MS.