Separation and Detection of Intact Noncovalent Protein Complexes from Mixtures by On-Line Capillary Isoelectric Focusing-Mass Spectrometry

摘要: Separation and mass spectrometric analysis of intact noncovalent protein−protein complexes from mixtures is described. Protein were separated using isoelectric focusing in a capillary under native conditions. During the mobilization, molecular masses measured on-line (as they emerged capillary) Fourier transform ion cyclotron resonance (FTICR) spectrometry. An FTICR “in-trap” cleanup procedure was necessary for some to reduce high levels adduction obtain accurate measurements. Optimization conditions different discussed. We have shown that either or their constituent protein subunits can be detected by variation sheath liquid (i.e., NH4OAc vs HOAc) added at electrospray−mass spectrometer interface. Thus, two successive experiments permit fast efficient characterization complex stoichiometry, individual compl...