On-Line Hollow-Fiber Flow Field-Flow Fractionation-Electrospray lonization/ Time-of-Flight Mass Spectrometry of Intact Proteins

摘要

Capabilities of mass spectrometry for the analysis of intact proteins can be increased through separation methods.Flow field-flow fractionation(FIFFF)is characterized by the particularly "soft"separation mechanism,which is ideally suited to maintain the native structure of intact proteins.This work describes the original on-line coupling between hollow-fiber FIFFF(HF F1FFF),the microcolumn variant of FIFFF,and electrospray ionization/time-of-flight mass spectrometry(ESI/TOFMS)for the analysis and characterization of intact proteins.The results show that the native(or pseudonative)structure of horse heart myoglobin and horseradish peroxidase is maintained.Sample desalting is also observed for horse heart myoglobin.Correlation between the molar mass values independently measured by HF FIFFF retention and ESI/ TOFMS allows us to confirm the protein aggregation features of bovine serum albumin and to indicate possible changes in the quaternary structure of human hemoglobin.