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

Abstract

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.

Analyses of Histone Proteoforms Using Front-end Electron Transfer Dissociation-enabled Orbitrap Instruments

“…1C is an average of 20 spectra like that shown in Fig 1A and represents a cumulative acquisition time of 11.2 s (0.56 s/spectrum). The use and benefits of IIPT to charge reduce ETD-produced fragment ions have been described previously (1,23). Briefly, following fragmentation by ETD, fragment ions are allowed to react with a proton transfer reagent.…”

“…The front-end ETD (FETD) source can also be used to ionize ion/ion proton transfer (IIPT) reagents. IIPT reactions are used to disperse fragment ions over the available m/z range in a controlled manner and to simplify ETD fragment ion spectra (1,22,23). Additionally, we have enabled parallel ion parking, first reported by McLuckey and co-workers (24,25), which involves harmonic excitation of selected ions within the ion trap to reduce their reactivity in gas-phase ion/ion reactions.…”

What does the future hold for top down mass spectrometry?

“…In those experiments, proteins were first fractionated by anion-exchange chromatography and then analyzed on an LTQ ion trap with ETD fragmentation following nanoflowLC separation on a C18 column, leading to the identification of 174 proteins from 322 detected different protein forms. Hunt's research group has also demonstrated a high-throughput Top Down analysis of intact small ribosomal proteins from E. coli on an LTQ equipped with ETD fragmentation and PTR charge reduction, resulting in the characterization of 46 of the known 55 70S ribosomal protein complex members, several of which were covalently modified [80]. Although great advancements have been made, there are technological hurdles that must be passed before Top Down MS is turned into a robust proteomic platform.…”

Evolution of instrumentation for the study of gas-phase ion/ion chemistry via mass spectrometry

“…In more recent studies, the same group has used this instrument setup to demonstrate MS n experiments that combine an ETD step followed by a proton-transfer (PT) ion/ion reaction step by adding a second reagent to the CI source to serve as the proton-transfer reactant [37]. Whole protein characterization on a chromatographic scale has been demonstrated using this sequential ETD/PT method [38]. The ion/ion reactions of multiply deprotonated peptide ions with xenon radical cations have also been studied using the same instrument setup [39].…”