Analysis of intact proteins on a chromatographic time scale by electron transfer dissociation tandem mass spectrometry

Direct analysis of intact proteins on a chromatographic time scale is demonstrated on a modified linear ion trap mass spectrometer using sequential ion/ion reactions, electron transfer and proton transfer, to dissociate the sample and to convert the resulting peptide fragments to a mixture of singly and doubly charged species. Proteins are converted to gas-phase, multiply charged, positive ions by electrospray ionization and then allowed to react with fluoranthene radical anions. Electron transfer to the multiply charged protein promotes random fragmentation of amide bonds along the protein backbone. Multiply charged fragment ions are then deprotonated in a second ion/ion reaction with even-electron benzoate anions. m/z values for the resulting singly and doubly charged ions are used to read a sequence of 15–40 amino acids at both the N-terminus and the C-terminus of the protein. This information, along with the measured mass of the intact protein, are employed to identify known proteins and to detect the presence of post-translational modifications. In this study, we analyze intact proteins from the Escherchia coli 70S ribosomal protein complex and identify 46 of the 55 known unique components in a single, 90 min, on-line, chromatography experiment. Truncated versions of the above proteins along with several post-translational modifications are also detected.