A novel electrophoretic titration method was firstly developed from the concept of moving reaction boundary(MRB)1,2 for protein content and structure analysis.In the developed method,when the voltage was applied,the hydroxide ions in the cathodic vessel moved towards the anode,and neutralized free acidic groups of protein immobilized via highly cross-linked polyacrylamide gel(PAG),generating a MRB between the alkali and the immobilized protein.(i)The boundary moving velocity(VMRB)was as a function of protein content,and an acid–base indicator was used to denote the boundary displacement.As a proof of concept,standard model proteins and biological samples(e.g.,infant milk powder)were chosen for the experiments to study the feasibility of the developed method3,4.The experiments revealed that good linear calibration functions between VMRB and protein content(correlation coefficients R> 0.98).The experiments further demonstrated the following merits of developed method:(1)weak influence of non-protein nitrogen additives(e.g.,melamine)adulterated in protein samples,(2)good agreement with the classic Kjeldahl method(R > 0.99),(3)fast measuring speed in total protein analysis of large samples from the same source,and(4)low limit of detection(0.02–0.15 mg mL-1 for protein content),good precision(R.S.D.of intra-day less than 1.7% and inter-day less than 2.7%),and high recoveries(105–107%).(ii)It was also theoretically revealed that the number of free acidic or alkaline residues of protein was as a function of MRB displacement in the electrophoretic titration system5.As a proof of concept,seven model proteins were chosen for the determination of acidic or alkaline residues of protein via MRB titration.The results showed that the numbers of free acidic and alkaline residues of proteins detected were in good agreement with those obtained from the relevant amino sequences in the NCBI database,demonstrating the feasibility of the developed concept,theory and technique.The general methodology of MRB titration has potential application for inexpensive,facilitative and informative protein structure analysis of free acidic or alkaline residues of protein.