Solid-phase alkylation reagent for in-situ capture and digestion of proteome

• 作 者: Huiming Yuan Lihua Zhang Yukui Zhang
• 机 构: KeyLaboratoryofSeparationScienceforAnalyticalchemistry,DalianInstituteofChemicalPhysiscs,ChineseAcad
• 出 处: 2016年分析化学前沿国际研讨会及中美分析化学研讨会
• 发表日期: 2016年6期
• 关键词: Proteome Solid-phase alkylation in-situ capture and digestion.

　　For bottom-up based proteome analysis,sample preparation including protein extraction,purication and digestion is indispensable prior to LC-MS analysis.However,traditional in-solution protocols such as FASP,suffer from not only time-consuming,but also low protein/peptide recovery and irreproducibility.Herein,a novel iodo-functionalized core-shell silica material was prepared by a surface-initiated atom transfer radical polymerization(ATRP),by which proteins dissolved in 4%SDS(w/v)could be convalently bound via thiol alkylation,and then washed with 50%methanol and 50 mM NH4HCO3(pH =8.0),finally digested by an ultraviolet assisted enzymatic hydrolysis.The whole procedure could be completed within 3.5 hours.To evaluate the performance of such a protocol,105 HeLa cells were used as a model sample.By our method,the HeLa cells were firstly dissolved in 4%SDS and 50 mM trichloroethyl phosphate,and then placed into a 95℃ water bath for 30 min,then the proteins were alkylated with iodo-functionalized core-shell silica material for 1.5 h.After wahsed with 50%methanol and 50 mM NH4HCO3(pH =8.0),the proteins were digested by trypsin via an ultraviolet assisted enzymatic hydrolysis for 30 min.Compared to FASP protocol,not only the number of identified proteins could be increased by 1.5 times,but also the sample preparation time was shortened by 1/12.Furthermore,our developed approach was also successfully applied for the proteomic analysis of formalin fixed and paraffi-embeded tissue slice,demonstrating the potential for the treatment of clinical trace sample.