FT-ICR Mass Spectrometer Research Articles

Generation and Characterization of a Distonic Biradical Anion Formed from an Enediynone Prodrug in the Gas Phase

A negatively charged biradical intermediate was successfully generated in the gas phase via cyclization of the deprotonated bicyclo[8.3.0]trideca-12-ene-2,7-diyn-1-one precursor. The inherent negative charge of this biradical allows its characterization via collision-activated dissociation and reactions with a variety of neutral substrates in an FT-ICR mass spectrometer. Although the biradical is unreactive toward reagents that usually react rapidly with positively charged biradicals, such as dimethyl disulfide, it reacts with the halogen-containing substrates carbon tetrachloride, carbon tetrabromide, and bromotrichloromethane via bromine or chlorine atom abstraction, which supports its biradical structure. The results presented in this study indicate that cyclizations commonly used in solution to form biradical intermediates from enediyne compounds may also occur in the gas phase.

Defining the molecular tumor margin regions of clear cell renal carcinoma tissues by MALDI-MS imaging of lipid and glycan species.

421 Background: The frequent tumor recurrence associated with clear cell renal cell carcinoma (ccRCC) suggests that there are underlying molecular processes present in the remaining tissue following nephrectomy that are not identified through conventional histopathological techniques. At the molecular level, transcript, metabolomic, and protein expression patterns have indicated a striking Warburg Effect profile in ccRCC tissues, with major affects on sugar and lipid metabolism. Our group has been applying MALDI mass spectrometry imaging approaches to uniquely profile lipids and glycans associated with disease progression directly in frozen tissue slides. Methods: Frozen ccRCC tissues with tumor, non-tumor adjacent, and tumor margin regions were selected by a pathologist. Lipid profiles from fresh-frozen tissue slides coated in DHB matrix were obtained on a dual source Bruker Solarix 70 FTICR mass spectrometer. Glycans were imaged in a similar fashion in ethanol-washed tissues using on-tissue protein N glycanase F digestion to release surface N-glycans. Detected lipid and glycan ion intensities were converted to a color pixel scale for creating an image of individual peaks, linked directly to the histopathology of the tissue. Results: Five groups of lipid and N-glycan species were identified following MALDI tissue imaging, those present in the immediate margin area of non-tumor tissue adjacent to tumor; only in non-tumor regions; only in tumor regions; primarily in tumor regions but extended beyond the margin; and present throughout the tissue. Specific lipid and glycan species associated with margin and tumor regions are being correlated with disease progression and pathology data. Conclusions: Analysis of the periphery of the tumor tissue and the normal parenchyma or capsule regions at this biomolecule level may better define the metastatic potential of the tumor as compared to analysis of the central tumor region. This approach has the potential to not only improve prognostic assessment and treatment choices, but also to inform on the underlying biology of ccRCC metastasis and new rational targets for therapeutic intervention.

Comparison of the Reactivity of the Three Distonic Isomers of the Pyridine Radical Cation Toward Tetrahydrofuran in Solution and in the Gas Phase

The reactivity of the three distonic isomers of the pyridine radical cation toward tetrahydrofuran is compared in solution and in the gas phase. In solution, the distonic ions were generated by UV photolysis at 300 nm from iodo-precursors in acidic 50:50 tetrahydrofuran/water solutions. In the gas phase, the ions were generated by collisionally activated dissociation (CAD) of protonated iodo-precursors in an FT-ICR mass spectrometer, as described in the literature. The same major reaction, hydrogen atom abstraction, was observed in solution and in the gas phase. Attempts to cleave the iodine atom from the 2-iodopyridinium cation in the gas phase and in solution yielded the 2-pyridyl cation in addition to the desired 2-dehydropyridinium cation. In the gas phase, this ion was ejected prior to the examination of the desired ion's chemical properties. This was not possible in solution. This study suggests that solvation effects are not significant for radical reactions of charged radicals. On the other hand, the even-electron ion studied, the 2-pyridyl cation, shows substantial solvation effects. For example, in solution, the 2-pyridyl cation forms a stable adduct with tetrahydrofuran, whereas in the gas phase, only addition/elimination reactions were observed.

Fluorescence-based method for determining the number of ions trapped in a FT-ICR mass spectrometer

A novel technique for determining the number of ions trapped inside a FT-ICR mass spectrometer is proposed. This technique is based on the detection of the spectral power radiated by fluorescing ions trapped inside the mass spectrometer. A pre-calibrated tungsten lamp was used to perform a calibration of the optical system. Using the absolute values of the energy radiated during an experiment, the number of ions trapped inside the cell was estimated. As reference ions, cations of rhodamine 6G and rhodamine B, whose gas-phase properties have been thoroughly studied, were chosen. The rhodamines have very high absorbance and high quantum yield in the gas phase. The method developed was compared to a previously published technique based on induced current measurements. A good agreement between the two techniques was found.

Infrared Matrix-Assisted Laser Desorption Electrospray Ionization (IR-MALDESI) Imaging Source Coupled to a FT-ICR Mass Spectrometer

Mass spectrometry imaging (MSI) allows for the direct monitoring of the abundance and spatial distribution of chemical compounds over the surface of a tissue sample. This technology has opened the field of mass spectrometry to numerous innovative applications over the past 15years. First used with SIMS and MALDI MS that operate under vacuum, interest has grown for mass spectrometry ionization sources that allow for effective imaging but where the analysis can be performed at ambient pressure with minimal or no sample preparation. We introduce here a versatile source for MALDESI imaging analysis coupled to a hybrid LTQ-FT-ICR mass spectrometer. The imaging source offers single shot or multi-shot capability per pixel with full control over the laser repetition rate and mass spectrometer scanning cycle. Scanning rates can be as fast as 1 pixel/second and a spatial resolution of 45μm was achieved with oversampling. Design and integration of a versatile IR-MALDESI imaging source offering multi-shot capability with a commercial FT-ICR mass spectrometer.

First Signal from a Broadband Cryogenic Preamplifier Cooled by Circulating Liquid Nitrogen in a 7 T Fourier Transform Ion Cyclotron Resonance Mass Spectrometer

Despite the outstanding performance of Fourier transform ion cyclotron/mass spectrometry (FTICR/MS), the complexity of the cellular proteome or natural compounds presents considerable challenges. Sensitivity is a key performance parameter of a FTICR mass spectrometer. By improving this parameter, the dynamic range of the instrument can be increased to improve the detection signal of low-abundance compounds or fragment ion peaks. In order to improve sensitivity, a cryogenic detection system was developed by the KBSI (Korean Basic Science Institute) in collaboration with Stahl-Electronics (Mettenheim, Germany). A simple, efficient liquid circulation cooling system was designed and a cryogenic preamplifier implemented inside a FTICR mass spectrometer. This cooling system circulates a cryoliquid from a Dewar to the "liquid circulation unit" through a CF flange to cool a copper block and a cryopreamplifier; the cooling medium is subsequently exhausted into the air. The cryopreamplifier can be operated over a very wide temperature range, from room temperature to low temperature environments (4.2 K). First, ion signals detected by the cryopreamplifier using a circulating liquid nitrogen cooling system were observed and showed a signal-to-noise ratio (S/N) about 130% better than that obtained at room temperature.

Dynamically Harmonized FT-ICR Cell with Specially Shaped Electrodes for Compensation of Inhomogeneity of the Magnetic Field. Computer Simulations of the Electric Field and Ion Motion Dynamics

The recently introduced ion trap for FT-ICR mass spectrometers with dynamic harmonization showed the highest resolving power ever achieved both for ions with moderate masses 500-1000Da (peptides) as well as ions with very high masses of up to 200kDa (proteins). Such results were obtained for superconducting magnets of very high homogeneity of the magnetic field. For magnets with lower homogeneity, the time of transient duration would be smaller. In superconducting magnets used in FT-ICR mass spectrometry the inhomogeneity of the magnetic field in its axial direction prevails over the inhomogeneity in other directions and should be considered as the main factor influencing the synchronic motion of the ion cloud. The inhomogeneity leads to a dependence of the cyclotron frequency from the amplitude of axial oscillation in the potential well of the ion trap. As a consequence, ions in an ion cloud become dephased, which leads to signal attenuation and decrease in the resolving power. Ion cyclotron frequency is also affected by the radial component of the electric field. Hence, by appropriately adjusting the electric field one can compensate the inhomogeneity of the magnetic field and align the cyclotron frequency in the whole range of amplitudes of z-oscillations. A method of magnetic field inhomogeneity compensation in a dynamically harmonized FT-ICR cell is presented, based on adding of extra electrodes into the cell shaped in such a way that the averaged electric field created by these electrodes produces a counter force to the forces caused by the inhomogeneous magnetic field.

Microbial Proteomics: Archaea to Cyanobacteria

This Journal is Korean Society of Photoscience 2012 C 57 Thermococcus onnurineus NA1, sulfur reducing hyperthermophilic archaeon, is capable of H ‐ 2 producing growth and ‐ considered to be hydrogenogenic carboxydotrophy. Utilization of formate as a sole energy source has been well studied in T. onnurineus NA1. However, whether formate could be used as its carbon source remains unanswered question. To obtain a global view on the metabolic characteristics in H2 producing growth, the quantitative proteome analysis of ‐ T. onnurineus NA1 grown on formate, CO and starch was performed by combining 1DSDS PAGE with nano UPLCMS ‐ ‐ E method. Our data propose that CO could be utilized as its sole energy source via electron transport mechanism to produce H2. Additionally, our data propose that CO2 produced from catabolic pathway or CO oxidation by CODH and CO‐ induced hydrogenase activities could subsequently be assimilated into the organic carbon. Overall, proteomic comparison of formate and COgrown cells with starch grown cells revealed that one carbon compound such as formate and CO ‐ ‐ ‐ could be utilized as an efficient substrate to provide cellular carbon and/or energy in T. onnurineus NA1. Unicellular Gloeobacter violaceus PCC 7421 is a Gramnegative photoautotrophic cyanobacterium. ‐ Gloeobacter violaceus PCC 7421 was divided at very early point of the phylogenetic tree and contained very primitive and unusual features like lack of thylakoidal membrane and missing of some photosystem components. In this study we performed proteomic analysis of Gloeobacter violaceus PCC 7421 through the following two approaches. First, we separate the total, soluble and insoluble fractions of Gloeobacter violaceus PCC 7421 and performed the twodimensional ‐ electrophoresis (2DE). Second, insoluble fractions were analyzed by FTICR mass spectrometer. After 2DE separation ‐ ‐ ‐ in gel digested spots were identified by the MALDI/TOF/TOF analysis about 115, 73 and 21 proteins were identified ‐ from total, soluble and insoluble fractions, respectively. Also insoluble fraction was digested using both chemical and tryptic in solution digestion methods to obtain the more peptides with transmembrane regions. Following the FTICR ‐ ‐ analysis 785 proteins were identified and among them 139 proteins with 1 or more (~14) transmembrane domains were analyzed using TMHMM program. Considering the number of theoretical proteins (965) with 1~ TM about 14.4% of that proteins were found. Through these proteomic analyses we could determine the protein preparation methods and provide the 2DE map of ‐ Gloeobacter violaceus PCC 7421. Microbial Proteomics: Archaea to Cyanobacteria

Analysis of carbon and nitrogen co-metabolism in yeast by ultrahigh-resolution mass spectrometry applying 13C- and 15N-labeled substrates simultaneously

Alternative metabolic pathways inside a cell can be deduced using stable isotopically labeled substrates. One prerequisite is accurate measurement of the labeling pattern of targeted metabolites. Experiments are generally limited to the use of single-element isotopes, mainly (13)C. Here, we demonstrate the application of direct infusion nanospray, ultrahigh-resolution Fourier transform ion cyclotron resonance-mass spectrometry (FTICR-MS) for metabolic studies using differently labeled elemental isotopes simultaneously--i.e., (13)C and (15)N--in amino acids of a total protein hydrolysate. The optimized strategy for the analysis of metabolism by a hybrid linear ion trap-FTICR-MS comprises the collection of multiple adjacent selected ion monitoring scans. By limiting both the width of the mass range and the number of ions entering the ICR cell with automated gain control, sensitive measurements of isotopologue distribution were possible without compromising mass accuracy and isotope intensity mapping. The required mass-resolving power of more than 60,000 is only achievable on a routine basis by FTICR and Orbitrap mass spectrometers. Evaluation of the method was carried out by comparison of the experimental data to the natural isotope abundances of selected amino acids and by comparison to GC/MS results obtained from a labeling experiment with (13)C-labeled glucose. The developed method was used to shed light on the complexity of the yeast Saccharomyces cerevisiae carbon-nitrogen co-metabolism by administering both (13)C-labeled glucose and (15)N-labeled alanine. The results indicate that not only glutamate but also alanine acts as an amino donor during alanine and valine synthesis. Metabolic studies using FTICR-MS can exploit new possibilities by the use of multiple-labeled elemental isotopes.

Identification of A-series oligomeric procyanidins from pericarp of Litchi chinensis by FT-ICR-MS and LC-MS

Procyanidin oligomers from litchi pericarp (Litchi chinensis) were isolated and identified by online analysis. Accurate molecular weight (MW) distribution and composition of the procyanidin extracts were recorded on an Apex-Qe–FT–ICR mass spectrometer. The MW range of litchi procyanidins was from 289.07151 to 1439.30529 m/z and composed of A- and B-series oligomeric procyanidins. The variety of the main procyanidins (DP < 5) was identified by HPLC and LC–ESI-MS analysis. There are more than 41.7% of A-series procyanidins in the oligomeric procyanidins, however, B-series procyanidins takes only 24.1% of the litchi pericarp extracts. In this study, we determined the effects of (−)-epicatechin, A-type procyanidin dimer (A2) and trimer, epicatechin-(4β→8, 2β→O→7)-epicatechin-(4β→8)-epicatechin from litchi pericarp on free radical scavenging. The results showed that A-type procyanidin dimer and trimer both had a strong scavenging ability on DPPH and OH, indicating that litchi is a new plant source of A-type procyanidins and natural antioxidant.

Ion–molecule reactions for the differentiation of primary, secondary and tertiary hydroxyl functionalities in protonated analytes in a tandem mass spectrometer

A mass spectrometric method utilizing gas-phase ion-molecule reactions of 1-butanethiol and di-tert-butyl peroxide has been developed for the differentiation of primary, secondary and tertiary hydroxyl functionalities in protonated analytes in a FT-ICR mass spectrometer.

Spectroscopic evidence for an oxazolone structure in anionic b-type peptide fragments.

Infrared spectra of anionic b-type fragments generated by collision induced dissociation (CID) from deprotonated peptides are reported. Spectra of the b2 fragments of deprotonated AlaAlaAla and AlaTyrAla have been recorded over the 800–1800 cm–1 spectral range by multiple-photon dissociation (MPD) spectroscopy using an FTICR mass spectrometer in combination with the free electron laser FELIX. Structural characterization of the b-type fragments is accomplished by comparison with density functional theory calculated spectra at the B3LYP/6-31++G(d,p) level for different isomeric structures. Although diketopiperazine structures represent the energetically lowest isomers, the IR spectra suggest an oxazolone structure for the b2 fragments of both peptides. Deprotonation is shown to occur on the oxazolone α-carbon, which leads to a conjugated structure in which the negative charge is practically delocalized over the entire oxazolone ring, providing enhanced gas-phase stability.Electronic supplementary materialThe online version of this article (doi:10.1007/s13361-011-0297-3) contains supplementary material, which is available to authorized users.

An atmospheric pressure ionization source based on desorption electrospray ionization technology (DESI) for ion cyclotron resonance mass spectrometry

An atmospheric pressure ionization source based on desorption electrospray ionization technology for a bench-top hybrid FTICR mass spectrometer is described. The ion source was characterized using low-molecular-weight-weight pharmaceutical samples. The dependences of signal intensities on various experimental parameters (solvent composition, surface temperature, spray voltage, etc.) were studied. Based on the results obtained, plausible mechanisms of desorption electrospray ionization for the analytes under the study are discussed.

Novel Cysteine Tags for the Sequencing of Non-Tryptic Disulfide Peptides of Anurans: ESI-MS Study of Fragmentation Efficiency

Mass spectrometry faces considerable difficulties in de novo sequencing of long non-tryptic peptides with S-S bonds. Long disulfide-containing peptides brevinins 1E and 2Ec from frog Rana ridibunda were reduced and alkylated with nine novel and three known derivatizing agents. Eight of the novel reagents are maleimide derivatives. Modified samples were subjected to MS/MS studies on FT-ICR and Orbitrap mass spectrometers using CAD/HCD or ECD/ETD techniques. Procedures, fragmentation patterns, and sequence coverage for two peptides modified with 12 tags are described. ECD/ETD and CAD fragmentation revealed complementary sequence information. Higher-energy collisionally activated dissociation (HCD) sufficiently enhanced y-ions formation for brevinin 1E, but not for brevinin 2Ec. Some novel tags [N-benzylmaleimide, N-(2,6-dimethylphenyl)maleimide] along with known N-phenylmaleimide and iodoacetic acid showed high total sequence coverage taking into account combined ETD and HCD fragmentation. Moreover, modification of long (34 residues) brevinin 2Ec with N-benzylmaleimide or N-(2,6-dimethylphenyl)maleimide yielded high sequence coverage and full C-terminal sequence determination with ECD alone.

Electrically Compensated Fourier Transform Ion Cyclotron Resonance Cell for Complex Mixture Mass Analysis

Complex natural organic mixtures such as petroleum require ultrahigh mass spectral resolution to separate and identify thousands of elemental compositions. Here, we incorporate a custom-built, voltage-compensated ICR cell for Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS), based on a prior design by Tolmachev to produce optimal mass resolution. The compensated ICR cell installed in a custom-built 9.4 T FTICR mass spectrometer consists of seven cylindrical segments with axial proportions designed to generate a dc trapping potential that approaches an ideal three-dimensional axial quadrupolar potential. However, the empirically optimized compensation voltages do not correspond to the most quadrupolar trapping field. The compensation electrodes minimize variation in the reduced cyclotron frequency by balancing imperfections in the magnetic and electric field. The optimized voltages applied to compensation electrodes preserve ion cloud coherence for longer transient duration by approximately a factor of 2, enabling separation and identification of isobaric species (compounds with the same nominal mass but different exact mass) common in petroleum, such as C(3) vs SH(4) (separated by 3.4 mDa) and SH(3)(13)C vs (12)C(4) (separated by 1.1 mDa). The improved performance of the ICR cell provides more symmetric peak shape and better mass measurement accuracy. A positive ion atmospheric pressure photoionization (APPI) petroleum spectrum yields more than 26,000 assigned peaks, Fourier-limited resolving power of 800,000 at m/z 500 (6.6 s transient duration), and 124 part per billion root mean square (rms) error. The tunability of the compensation electrodes is critical for optimal performance.

Encapsulation of Metal Cations by the PhePhe Ligand: A Cation−π Ion Cage

Structures and binding thermochemistry are investigated for protonated PhePhe and for complexes of PhePhe with the alkaline-earth ions Ba(2+) and Ca(2+), the alkali-metal ions Li(+), Na(+), K(+), and Cs(+), and the transition-metal ion Ag(+). The two neighboring aromatic side chains open the possibility of a novel encapsulation motif of the metal ion in a double cation-π configuration, which is found to be realized for the alkaline-earth complexes and, in a variant form, for the Ag(+) complex. Experimentally, complexes are formed by electrospray ionization, trapped in an FT-ICR mass spectrometer, and characterized by infrared multiple photon dissociation (IRMPD) spectroscopy using the free electron laser FELIX. Interpretation is assisted by thermochemical and IR spectral calculations using density functional theory (DFT). The IRMPD spectrum of protonated PhePhe is reproduced with good fidelity by the calculated spectrum of the most stable conformation, although the additional presence of the secondmost stable conformation is not excluded. All metal-ion complexes have charge-solvated binding modes, with zwitterion (salt bridge) forms being much less stable. The amide oxygen always coordinates to the metal ion, as well as at least one phenyl ring (cation-π interaction). At least one additional chelation site is always occupied, which may be either the amino nitrogen or the carboxy carbonyl oxygen. The alkaline-earth complexes prefer a highly compact caged structure with both phenyl rings providing cation-π stabilization in a "sandwich" configuration (OORR chelation). The alkali-metal complexes prefer open-cage structures with only one cation-π interaction, except perhaps Cs(+). The Ag(+) complex shows a unique preference for the closed-cage amino-bound NORR structure. Ligand-driven perturbations of normal-mode frequencies are generally found to correlate linearly with metal-ion binding energy.

Predator data station: A fast data acquisition system for advanced FT-ICR MS experiments

Here, we present the Predator data station, a control system for FT-ICR mass spectrometers that champions speed and experimental flexibility while simultaneously providing stability, ease of use, and the ability to integrate more advanced hardware as it becomes available. The Predator is the first FT-ICR MS data station comprised solely of fast PCI, PXI, and yet faster PXI Express-based commercial data acquisition hardware. Increased data transfer speed is required because recorded transient data count increases linearly at higher magnetic field (higher measured frequency) with extended transient duration for FT-ICR MS instruments. The application of new cell designs with additional compensation voltages, experimental techniques to increase resolution, and experimental techniques that minimize/reject variations in ion abundance exemplify the scope of recent Predator data station implementations. When the above techniques are applied simultaneously, the results give rise to sub-30 ppb rms mass error for 5250 assigned peaks in a petroleum FT-ICR mass spectrum. The Predator data station is designed for facile implementation with any FT-ICR MS instrument. The Predator hardware provides 17 analog voltage outputs and 18 digital TTL outputs synchronized to a single timing source. SWIFT, chirp, and single frequency excitation waveforms are generated by a 100 MSample/s arbitrary waveform generator with a minimum 32 MB of onboard memory and the potential of terabytes of virtual memory via first in-first out (FIFO) buffering. Transient detection is facilitated by a 2-channel, 100 MSample/s digitizer with a minimum of 32 MB of onboard memory per channel. FIFO buffering implementation allows TB transient collection as well. Commercial hardware, royalty-free software solutions, and commercially produced custom printed circuit boards (PCB) for the cell controller ensure open availability. The present data complement numerous extant publications: the Predator data station has been the sole data station for the National High Magnetic Field Laboratory (NHMFL) 9.4 T FT-ICR MS instrument since July 2004, and several additional Predator data stations are in operation elsewhere.

Fragmentation of xanthene dyes by laser activation and collision‐induced dissociation on a high‐resolution Fourier transform ion cyclotron resonance mass spectrometer

Surprising fragmentation reactions in different xanthene dyes have been investigated by means of photodissociation and collision-induced dissociation in a 9.4 T FT-ICR mass spectrometer. It is shown that extensive rearrangement reactions lead to the formation of unexpected fragments which are identified for the first time by the use of high resolving mass power. The observed reactions are an example of the fragmentation of a quinoidal even-electron cation.

Electrochemistry-electrospray ionization FT ICR mass spectrometry (EC ESI MS) of guanine–tyrosine and guanine–glutathione crosslinks formed on-line

Crosslinking of guanine with tyrosine and glutathione is studied by on-line electrochemistry electrospray ionization mass spectrometry (EC ESI MS) in the positive ion mode with a high resolution FT ICR mass spectrometer without the use of an external oxidizer in the sample. Dimeric adducts of guanine with tyrosine ( m/ z 331) and ( m/ z 333) and a dimer of guanine with glutathione ( m/ z 459) are detected in the mass spectra of guanine–tyrosine and guanine–glutathione mixtures in addition to dimers of guanine ( m/ z 303) and tyrosine ( m/ z 361) and ( m/ z 363) and glutathione dimers ( m/ z 613) and ( m/ z 615). Guanine tetramers ( m/ z 627) and tetramers of guanine with 1 or 2 tyrosines, but not with three tyrosines, were also identified in the mass spectra of guanine–tyrosine mixtures. Formation of radicals and other oxidation products during positive ion mode ESI can drive the formation of covalent dimer adducts of guanine with tyrosine and glutathione. When low EC cell voltage is applied in on-line EC ESI MS, changes in ion intensities reflect changes in the oxidation conditions and are more apparent for 2e, 2H + than for the 1e, 1H + oxidation processes. Efficient oxidation during ESI is indicated for analytes with low redox potentials.

Native Electrospray and Electron-Capture Dissociation in FTICR Mass Spectrometry Provide Top-Down Sequencing of a Protein Component in an Intact Protein Assembly

The intact yeast alcohol dehydrogenase (ADH) tetramer of 147 kDa was introduced into a FTICR mass spectrometer by native electrospray. Electron capture dissociation of the entire 23+ to 27+ charge state distribution produced the expected charge-reduced ions and, more unexpectedly, 39 c-type peptide fragments that identified N-terminus acetylation and the first 55 amino acids. The results are in accord with the crystal structure of yeast ADH, which shows that the C-terminus is buried at the assembly interface, whereas the N-terminus is exposed, allowing ECD to occur. This remarkable observation shows promise that a top-down approach for intact protein assemblies will be effective for characterizing their components, inferring their interfaces, and obtaining both proteomics and structural biology information in one experiment.