Separation And Characterization Of Proteins By The System Of Asymmetrical Flow Field-Flow Fractionation-Mass Spectrometry

The development of proteomics is promoting the progress of all life sciences, which research results are widely used in medical and industrial fields. The study on protein complexes is important in proteomics because protein conformation, self-dissociation and dissociation are strongly related to the biological activity. The separation of proteins using other methods, such as liquid chromotagraphy (LC), is seriously limited, owing to the possible interaction between the proteins and the stationary phase and/or to the possible presence of organic modifiers in the mobile phase, which can cause degradation. So, there is an urgent need to develop a protein separation techniaue. which can both acquire high purification and recovery, and maintain the protein activity and stability; especially it is compatible with mass spectrometry.Firstlv. this paper introduces the relevant theories of asymmetrical flow field-flow fractionation; the theory formula of resolution was derived according to the theory of fluid mechanics and mass transfer separation. Then, the effects of experimental parameters, including injection volume, cross flow rate, detector flow rate, focusing time, membrane material, buffer concentration and channel spacer, on the asymmetrical flow field-flow fractionation were studied by separating the mixed proteins (myoglobin and bovine serum albumin); moreover, the experimental results and theoretical analysis results were compared. The mass recovery and activity recovery of nrotein in the asymmetrical flow field-flow fractionation were tested, which results were compared with liquid chromotagraphy (LC). It showed that when separated proteins by AF4, the loss of protein molecules was very little, and at the same time, the structure of protein molecules could be well protected.A new combination system of the asymmetrical flow field-flow fractionation-electrospray ionization mass spectrometry (AF4-ESI-MS) was designed on the basis of the significant advantages of AF4 and ESI-MS techniques in separation and characterization of proteins than other technologies. Firstly, the solution of ammonium acetate salt was selected as the mobile phase, and myoglobin and bovine serum albumin mixed proteins was separated and characterized successfully under the optimal separation conditions. Then, the combination system was used to separate and characterize the mixed proteins sample cytochrome C and myoglobin with smaller particle size differences, and the rapid separation and characterization were carried out in 30 min. The relative deviation of the relative molecular weight between characterized and retrieved was 0.023%for myoglobin, 0.009% for bovine serum albumin and 0.008% for cytochrome C, and all the relative standard deviation of the relative molecular weight was less than 0.01%, which showed the combination system has high accuracy and reliability. In addition, the asymmetrical flow field-flow fractionation was off-line coupled tomatrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) for the characterization of intact protein, which results was consistent with that of AF4-ESI-MS.