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Human biology is tightly linked to proteins, yet most measurements do not precisely determine alternatively spliced sequences or posttranslational modifications. Here, we present the primary structures of ~30,000 unique proteoforms, nearly 10 times more than in previous studies, expressed from 1690 human genes across 21 cell types and plasma from human blood and bone marrow. The results, compiled in the Blood Proteoform Atlas (BPA), indicate that proteoforms better describe protein-level biology and are more specific indicators of differentiation than their corresponding proteins, which are more broadly expressed across cell types. We demonstrate the potential for clinical application, by interrogating the BPA in the context of liver transplantation and identifying cell and proteoform signatures that distinguish normal graft function from acute rejection and other causes of graft dysfunction.

International audience; Recent advances in instrumentation for high-field Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) have enabled access to ∼70 »000 unique molecular formulas in broadband mass spectral characterization of unfractionated/whole asphaltenes. The results accumulated over a decade highlight the need for an asphaltene molecular model that acknowledges the coexistence of (1) monofunctional and polyfunctional species; (2) island and archipelago structural motifs; and (3) heteroatom-depleted/highly aromatic compounds, as well as atypical species with low aromaticity but increased heteroatom content. Collectively, results from FT-ICR MS, preparatory-scale separations (extrography/interfacial material), gel permeation chromatography, precipitation behavior in heptane:toluene, thermal decomposition, and aggregate microstructure by atomic force microscopy (among other techniques), suggest that the strong aggregation of asphaltenes results from the synergy between several intermolecular forces: π-stacking, hydrogen bonding, London forces, and acid/base interactions. This review presents general features of asphaltene molecular composition reported over the past five decades. We focus on mass spectrometry characterization and expose the reasons why early results supported the dominance of single-core motifs. Then, the discussion shifts to recent advances in instrumentation for high-field FT-ICR MS, which have enabled the detection of thousands of species in asphaltene samples, whose molecular composition and fragmentation behavior in ultrahigh vacuum agree with the coexistence of single-core and multicore structural motifs. Furthermore, evidence that highlights the limitations of commercially available/custom-built ion sources and selective ionization effects is presented. Consequently, the limitations require separations (e.g., chromatography, extrography) to gain more-comprehensive molecular-level insights into the composition of these complex organic mixtures. The final sections present evidence for the role of aggregation in selective ionization and suggest that advanced characterization by both thermal desorption/decomposition and liquid chromatography with online FT-ICR MS detection can be employed to mitigate the effects of aggregation and provide unique insights in molecular composition/structure.

High magnetic field Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry provides the highest mass resolving power and mass measurement accuracy for detailed characterization of complex chemical mixtures. Here, we report the coupling of online liquid chromatography of complex mixtures with a 21 tesla FT-ICR mass spectrometer. The high magnetic field enables large ion populations to be analyzed for each spectrum for a high dynamic range, with 3.2 million mass resolving power at m/z 400 (6.2 s transient duration) or 1.6 million (3.1 s transient duration) while maintaining high mass accuracy for molecular formula assignment (root-mean-square assignment error < 0.150 ppm). Thousands of unique elemental compositions are assigned per mass spectrum, which can be grouped by the heteroatom class, double bond equivalents (the number of rings and double bonds to carbon), and carbon number. Figures of merit are discussed, as well as characterization of an Arabian heavy vacuum gas oil in terms of the ring number, compound class, double bond equivalents, and ion type. Consideration of elemental composition and retention order provides additional structural information.

Proton transfer reactions (PTR) have emerged as a powerful tool for the study of intact proteins. When coupled with m/z-selective kinetic excitation, such as parallel ion parking (PIP), one can exert exquisite control over rates of reaction with a high degree of specificity. This allows one to “concentrate”, in the gas-phase, nearly all the signal from an intact protein charge state envelope into a single charge state improving signal-to-noise by 10x or more. While this approach has been previously reported, here we show that implementing these technologies on a 21 T FT-ICR MS provides tremendous advantage for intact protein analysis. Advanced strategies for performing PTR with PIP were developed to complement this unique instrument, including subjecting all analyte ions entering the mass spectrometer to PTR and PIP. This experiment, which we call “PTR-MS(1)-PIP”, generates a pseudo-MS(1) spectrum derived from ions that are exposed to PTR reagent and PIP waveforms, but have not undergone any prior true mass filtering or ion isolation. The result is an extremely rapid and significant improvement in spectral signal-to-noise (S:N) of intact proteins. This permits observation of many more proteoforms and reduces ion injection periods for subsequent tandem mass spectrometry characterization. Additionally, the product ion parking waveform has been optimized to enhance the proton transfer reaction rate without compromise to parking efficiency. We demonstrate that this process, called “rapid park”, can improve reaction rates by 5–10x and explore critical factors discovered to influence this process. Finally, we demonstrate how coupling PTR-MS(1) and rapid park provides a ten-fold reduction in ion injection time, improving the rate of tandem MS sequencing.

The KRAS gene is one of the most frequently mutated oncogenes in human cancer and gives rise to two isoforms, KRAS4A and KRAS4B. KRAS post-translational modifications (PTMs) have the potential to influence downstream signaling. However, the relationship between KRAS PTMs and oncogenic mutations remains unclear, and the extent of isoform-specific modification is unknown. Here, we present the first top-down proteomics study evaluating both KRAS4A and KRAS4B, resulting in 39 completely characterized proteoforms across colorectal cancer cell lines and primary tumor samples. We determined which KRAS PTMs are present, along with their relative abundance, and that proteoforms of KRAS4A versus KRAS4B are differentially modified. Moreover, we identified a subset of KRAS4B proteoforms lacking the C185 residue and associated C-terminal PTMs. By confocal microscopy, we confirmed that this truncated GFP-KRAS4B

Detailed characterization of complex biological surfaces by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) requires instrumentation that is capable of high mass resolving power, mass accuracy, and dynamic range. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) offers the highest mass spectral performance for MALDI MSI experiments, and often reveals molecular features that are unresolved on lower performance instrumentation. Higher magnetic field strength improves all performance characteristics of FT-ICR; mass resolving power improves linearly, while mass accuracy and dynamic range improve quadratically with magnetic field strength. Here, MALDI MSI at 21T is demonstrated for the first time: mass resolving power in excess of 1 600 000 (at m/z 400), root-mean-square mass measurement accuracy below 100 ppb, and dynamic range per pixel over 500:1 were obtained from the direct analysis of biological tissue sections. Molecular features with m/z differences as small as 1.79 mDa were resolved and identified with high mass accuracy. These features allow for the separation and identification of lipids to the underlying structures of tissues. The unique molecular detail, accuracy, sensitivity, and dynamic range combined in a 21T MALDI FT-ICR MSI experiment enable researchers to visualize molecular structures in complex tissues that have remained hidden until now. The instrument described allows for future innovative, such as high-end studies to unravel the complexity of biological, geological, and engineered organic material surfaces with an unsurpassed detail.

BACKGROUND Hemoglobinopathies and thalassemias are the most common genetically determined disorders. Current screening methods include cation-exchange HPLC and electrophoresis, the results of which can be ambiguous because of limited resolving power. Subsequently, laborious genetic testing is required for confirmation. METHODS We performed a top-down tandem mass spectrometry (MS/MS) approach with a fast data acquisition (3 min), ultrahigh mass accuracy, and extensive residue cleavage by use of positive electrospray ionization 21 Tesla Fourier transform ion cyclotron resonance–tandem mass spectrometry (21 T FT-ICR MS/MS) for hemoglobin (Hb) variant de novo sequencing and β-thalassemia diagnosis. RESULTS We correctly identified all Hb variants in blind analysis of 18 samples, including the first characterization of homozygous Hb Himeji variant. In addition, an Hb heterozygous variant with isotopologue mass spacing as small as 0.0194 Da (Hb AD) was resolved in both precursor ion mass spectrum (MS1) and product ion mass spectrum (MS2). In blind analysis, we also observed that the abundance ratio between intact δ and β subunits (δ/β) or the abundance ratio between intact δ and α subunits (δ/α) could serve to diagnose β-thalassemia trait caused by a mutation in 1 HBB gene. CONCLUSIONS We found that 21 T FT-ICR MS/MS provides a benchmark for top-down MS/MS analysis of blood Hb. The present method has the potential to be translated to lower resolving power mass spectrometers (lower field FT-ICR mass spectrometry and Orbitrap) for Hb variant analysis (by MS1 and MS2) and β-thalassemia diagnosis (MS1).

International audience; The Consortium for Top-Down Proteomics (www.topdownproteomics.org) launched the present study to assess the current state of top-down mass spectrometry (TD MS) and middle-down mass spectrometry (MD MS) for characterizing monoclonal antibody (mAb) primary structures, including their modifications. To meet the needs of the rapidly growing therapeutic antibody market, it is important to develop analytical strategies to characterize the heterogeneity of a therapeutic product's primary structure accurately and reproducibly. The major objective of the present study is to determine whether current TD/MD MS technologies and protocols can add value to the more commonly employed bottom-up (BU) approaches with regard to confirming protein integrity, sequencing variable domains, avoiding artifacts, and revealing modifications and their locations. We also aim to gather information on the common TD/MD MS methods and practices in the field. A panel of three mAbs was selected and centrally provided to 20 laboratories worldwide for the analysis: Sigma mAb standard (SiLuLite), NIST mAb standard, and the therapeutic mAb Herceptin (trastuzumab). Various MS instrument platforms and ion dissociation techniques were employed. The present study confirms that TD/MD MS tools are available in laboratories worldwide and provide complementary information to the BU approach that can be crucial for comprehensive mAb characterization. The current limitations, as well as possible solutions to overcome them, are also outlined. A primary limitation revealed by the results of the present study is that the expert knowledge in both experiment and data analysis is indispensable to practice TD/MD MS.

Fourier transform mass spectrometers routinely provide high mass resolution, mass measurement accuracy, and mass spectral dynamic range. In this work, we utilize 21 T Fourier transform ion cyclotron resonance (FT-ICR) to analyze product ions derived from the application of multiple dissociation techniques and/or multiple precursor ions within a single transient acquisition. This ion loading technique, which we call, "chimeric ion loading", saves valuable acquisition time, decreases sample consumption, and improves top-down protein sequence coverage. In the analysis of MCF7 cell lysate, we show collision-induced dissociation (CID) and electron-transfer dissociation (ETD) on each precursor on a liquid chromatography-mass spectrometry (LC-MS) timescale and improve mean sequence coverage dramatically (CID-only 15% vs chimeric 33%), even during discovery-based acquisition. This approach can also be utilized to multiplex the acquisition of product ion spectra of multiple charge states from a single protein precursor or multiple ETD/proton-transfer reactions (PTR) reaction periods. The analytical utility of chimeric ion loading is demonstrated for top-down proteomics, but it is also likely to be impactful for tandem mass spectrometry applications in other areas.

Asphaltenes are high-boiling and recalcitrant compounds that are generally minor components of crude oil (∼0.1-15.0 wt %) but dominate the composition of heavily weathered spilled petroleum. These solid residues exhibit a high structural complexity, comprised of polycyclic aromatic hydrocarbons (PAHs) that are a mixture of single-core (island) and multicore (archipelago) structural motifs. The mass fraction of each motif is sample-dependent. Thus, knowledge of a potential structural dependence (single- versus multicore) on the production of water-soluble species from asphaltene samples is key to understanding the contribution of photochemically generated dissolved organic matter from oil spills. In this work, asphaltene samples with enriched mass fractions of either island (single-core) or archipelago (multicore) structural motifs are photo-oxidized on artificial seawater by the use of a solar simulator. Molecular characterization of oil- and water-soluble photoproducts, conducted by Fourier transform ion cyclotron resonance mass spectrometry, reveals that island motifs exhibit very limited production of water-soluble species, and their oil-soluble products reflect the molecular composition of the starting material. Conversely, archipelago motifs yield a water-soluble compositional continuum of O

Since the emergence of high resolving power crude oil mass spectrometry two decades ago, hundreds of publications and presentations have detailed petroleum complex mixtures by electrospray ionization (ESI) mass spectrometry (MS). None of these works have reported or detailed ion suppression (also referred to as ionization biasing or matrix effects) which is a well-known feature of ESI. Here, we show the extreme consequences of ionization biasing within a narrow, 1 order of magnitude concentration range for crude oil mixture direct infusion experiments in positive ion (+) ESI. An oil spill contaminant, a crude oil, and an equimolar model compound mixture were electrosprayed at various analyte and acid modifier concentrations for Fourier transform ion cyclotron resonance (FT-ICR) and time-of-flight (TOF) MS analysis. A 3-fold increase in the number of elemental compositions is achieved by optimization of analyte and acid concentration. At high analyte concentration, oxygen heteroatom class (i.e., CcHhOx spe...

We describe complex organic mixture analysis by 21 tesla (T) Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). Ultrahigh mass-resolving power (m/Δm50% > 2 700 000 at m/z 400) and mass accuracy (80 ppb rms) enable resolution and confident identification of tens of thousands of unique elemental compositions. We demonstrate 2.2-fold higher mass-resolving power, 2.6-fold better mass measurement accuracy, and 1.3-fold more assigned molecular formulas compared to our custom-built, state-of-the-art 9.4 T FT-ICR mass spectrometer for petroleum and dissolved organic matter (DOM) analyses. Analysis of a heavy petroleum distillate exemplifies the need for ultrahigh-performance mass spectrometry (49 040 assigned molecular formulas for 21 T versus 29 012 for 9.4 T) and extends the identification of previously unresolved Oo, SsOo, and NOo classes. Mass selective ion accumulation (20 Thompson isolation) of an asphalt volcano sample yields 462 resolved mass spectral peaks at m/z 677 and reveals pre...

High resolution mass spectrometry is a key technology for in-depth protein characterization. High-field Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) enables high-level interrogation of intact proteins in the most detail to date. However, an appropriate complement of fragmentation technologies must be paired with FTMS to provide comprehensive sequence coverage, as well as characterization of sequence variants, and post-translational modifications. Here we describe the integration of front-end electron transfer dissociation (FETD) with a custom-built 21 tesla FT-ICR mass spectrometer, which yields unprecedented sequence coverage for proteins ranging from 2.8 to 29 kDa, without the need for extensive spectral averaging (e.g., ~60% sequence coverage for apo-myoglobin with four averaged acquisitions). The system is equipped with a multipole storage device separate from the ETD reaction device, which allows accumulation of multiple ETD fragment ion fills. Consequently, an optimally large product ion population is accumulated prior to transfer to the ICR cell for mass analysis, which improves mass spectral signal-to-noise ratio, dynamic range, and scan rate. We find a linear relationship between protein molecular weight and minimum number of ETD reaction fills to achieve optimum sequence coverage, thereby enabling more efficient use of instrument data acquisition time. Finally, real-time scaling of the number of ETD reactions fills during method-based acquisition is shown, and the implications for LC-MS/MS top-down analysis are discussed. Graphical Abstract ᅟ.

Stored waveform inverse Fourier transform (SWIFT) is a versatile method to generate complex isolation/ejection waveforms for precursor isolation prior to tandem mass spectrometry experiments. Here, we report ultrahigh resolving power ion isolation by SWIFT on a 21 T Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. Individual histone proteoforms are isolated (0.6 m/z isolation window) with near 100% efficiency using a 52 ms SWIFT isolation, followed by in-cell fragmentation by ultraviolet photodissociation (UVPD). Ion isolation resolving power of 175 000 (m/Δm) is demonstrated by isolation of individual peaks at a spacing of 0.0034 Da at m/z 597 from a complex mixture of Canadian bitumen. An individual m/z ion, which corresponds to a single elemental composition, from a complex mixture is isolated and fragmented by infrared multiphoton dissociation (IRMPD). Theoretical and experimental considerations that limit achievable ion isolation resolving power are discussed.

The current five-year survival rate for systemic AL amyloidosis or multiple myeloma is ∼51%, indicating the urgent need for better diagnosis methods and treatment plans. Here, we describe highly specific and sensitive top-down and middle-down MS/MS methods owning the advantages of fast sample preparation, ultrahigh mass accuracy, and extensive residue cleavages with 21 telsa FT-ICR MS/MS. Unlike genomic testing, which requires bone marrow aspiration and may fail to identify all monoclonal immunoglobulins produced by the body, the present method requires only a blood draw. In addition, circulating monoclonal immunoglobulins spanning the entire population are analyzed and reflect the selection of germline sequence by B cells. The monoclonal immunoglobulin light chain FR2-CDR2-FR3 was sequenced by database-aided de novo MS/MS and 100% matched the gene sequencing result, except for two amino acids with isomeric counterparts, enabling accurate germline sequence classification. The monoclonal immunoglobulin heavy chains were also classified into specific germline sequences based on the present method. This work represents the first application of top/middle-down MS/MS sequencing of endogenous human monoclonal immunoglobulins with polyclonal immunoglobulins background.

The two KRAS isoforms, 4A and 4B, exhibit high sequence identity, differing mainly in their C-terminal “hypervariable regions”, which bear post-translational modifications (PTMs) governing their association with the plasma membrane and downstream signaling effectors. Moreover, the KRAS gene bears three “mutation hotspot” residues (Gly12, Gly13, and Gln61) represented in a high percentage of human cancers. The similarity between KRAS isoforms, in addition to HRAS and NRAS, renders linking KRAS mutations and PTMs significantly challenging by standard proteomic methods based on digestion of proteins into short peptides. However, intact modified protein forms, or proteoforms, of KRAS4A and KRAS4B can be identified and characterized by “top-down” (TD) proteomic methods, elucidating crosstalk between N-terminal mutations and C-terminal PTMs with precise molecular detail. Previously, we employed the TD approach as part of a novel workflow to precisely characterize KRAS4B proteoforms isolated from colorectal cancer cell lines and primary tumor samples by immunoprecipitation (IP) and subsequent analysis on a benchtop Orbitrap mass spectrometer (1). Here, we modified our IP-TD protocol to include KRAS4A, then applied our workflow to the characterization, quantification, and direct comparison of KRAS4A and KRAS4B proteoforms within the context of multiple cancer cell lines. We hypothesized that we would be able to identify distinct proteoform populations for each KRAS isoform, which could then be compared both within and between different contexts. We have thus far identified nine novel proteoforms of KRAS4A, which not only exhibits differential PTM to KRAS4B, but varies widely in both abundance and PTM profile across all cell lines evaluated. We have also applied IP-TD to the analysis of endogenous KRAS4A and KRAS4B proteoforms using a more powerful 21 Tesla FT-ICR mass spectrometer at the National High Magnetic Field Laboratory. The high mass resolving power, mass measurement accuracy, and spectral acquisition rate achieved at high field combined with use of efficient dissociation methods and an external fragment ion accumulator to improve signal-to-noise ratios of MS/MS spectra produced unparalleled results with respect to degree of KRAS proteoform characterization. We were able to identify up to ten new KRAS proteoforms per context, several of which appear to be mutation- or context-specific. These latest advances have broadened our understanding of KRAS biology and provided new insights into the potential roles of KRAS4A and KRAS4B proteoforms in colorectal and other cancers. Reference 1. Ntai I, Fornelli L, DeHart CJ, Hutton JE, Doubleday PF, LeDuc RD, van Nispen AJ, Fellers RT, Whiteley G, Boja ES, Rodriguez H, Kelleher NL. Precise characterization of KRAS4b proteoforms in human colorectal cells and tumors reveals mutation/modification crosstalk. Proc Natl Acad Sci U S A 2018 Apr 17;115(16):4140-5. Citation Format: Caroline J. DeHart, Lissa C. Anderson, Lauren M. Adams, Luca Fornelli, Christopher L. Hendrickson, Neil L. Kelleher. Precise characterization and comparison of KRAS proteoforms by top-down mass spectrometry [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr A26.

SummaryPost-translational modifications (PTMs) are found on most proteins, particularly on “hub” proteins like the tumor suppressor p53, which has over 100 possible PTM sites. Substantial crosstalk between PTM sites underlies the ability of such proteins to integrate diverse signals and coordinate downstream responses. However, disentangling the combinatorial explosion in global PTM patterns across an entire protein (“modforms”) has been challenging, as conventional peptide-based mass spectrometry strategies (so-called “bottom-up” MS) destroy such global correlations. Alternatively, direct analysis of intact and modified proteins using “top-down” MS retains global information. Here, we applied both strategies to recombinant p53 phosphorylatedin vitrowith Chk1 kinase, which exhibited 41 modified sites by bottom-up MS, but no more than 8 modified sites per molecule detected by top-down MS. This observation that many low-abundance modifications comprise relatively few modforms above a 1% threshold indicates that endogenous p53 PTM complexity may be more definable than previously thought.

A three-dimensional code based on the particle-in-cell algorithm modified to account for the inhomogeneity of the magnetic field was applied to determine the effect of Z1, Z2, Z3, Z4, X, Y, ZX, ZY, XZ2 YZ2, XY and X2–Y2 components of an orthogonal magnetic field expansion on ion motion during detection in an FT-ICR cell. Simulations were performed for magnetic field strengths of 4.7, 7, 14.5 and 21 Tesla, including experimentally determined magnetic field spatial distributions for existing 4.7 T and 14.5 T magnets. The effect of magnetic field inhomogeneity on ion cloud stabilization (“ion condensation”) at high numbers of ions was investigated by direct simulations of individual ion trajectories. Z1, Z2, Z3 and Z4 components have the largest effect (especially Z1) on ion cloud stability. Higher magnetic field strength and lower m/z demand higher relative magnetic field homogeneity to maintain cloud coherence for a fixed time period. The dependence of mass resolving power upper limit on Z1 inhomogeneity is evaluated for different magnetic fields and m/z. The results serve to set the homogeneity requirements for various orthogonal magnetic field components (shims) for future FT-ICR magnet design.

We thank Dr. Laszlo Prokai, Dr. Jae-Seok Kim, and Dr. Hyun Sik Kim for their insightful editorial commentaries on our recent publication “ Diagnosis of hemoglobinopathy and β-thalassemia by 21 Tesla Fourier transform ion cyclotron resonance mass spectrometry and tandem mass spectrometry of hemoglobin from blood ” (1-3). As described by the editorial authors, 21 Tesla Fourier transform ion cyclotron resonance mass spectrometry (21 T FT-ICR MS) and top-down MS/MS set a new benchmark for precision diagnosis of hemoglobinopathies and thalassemia with fast sample preparation (dilute and infuse) and data acquisition (3 min).

We present laser desorption atmospheric pressure photochemical ionization mass spectrometry (LD/APPCI MS) for rapid throughput analysis of complex organic mixtures, without the need for matrix, electric discharge, secondary electrospray, or solvents/vaporizers. Analytes dried on a microscope slide are vaporized in transmission geometry by a laser beam aligned with the atmospheric pressure inlet of the mass spectrometer. The laser beam initiates a cascade of reactions in the region between the glass slide and MS inlet, leading to generation of reagent ions for chemical ionization of vaporized analyte. Positive analyte ions are generated predominantly by proton transfer, charge exchange, and hydride abstraction, whereas negative ions are generated by electron capture or proton transfer reactions, enabling simultaneous analysis of saturated, unsaturated, and heteroatom-containing hydrocarbons. The absence of matrix interference renders LD/APPCI MS particularly useful for analysis of small molecules (2000 Da) such as those present in petroleum crude oil and petroleum deposits. [M + H](+) and M(+•) dominate the positive-ion mass spectra for olefins and polyaromatic hydrocarbons, whereas saturated hydrocarbons are observed mainly as [M - H](+) and/or M(+•). Heteroatom-containing hydrocarbons are observed predominantly as [M + H](+). [M - H](-) and M(-•) are the dominant negative ions observed for analytes of lower gas-phase basicity or higher electron affinity than O2. The source was coupled with a 9.4 T Fourier transform ion cyclotron resonance mass spectrometer (FTICR MS) to resolve and identify thousands of peaks from Athabasca bitumen heavy vacuum gas oil distillates (400-425 and 500-538 °C), enabling simultaneous characterization of their polar and nonpolar composition. We also applied LD/APPCI FTICR MS for rapid analysis of sodium and calcium naphthenate deposits with little to no sample pretreatment to provide mass spectral fingerprints that enable reliable compositional characterization.

Fullerenes that encapsulate clusters of atoms represent a fundamental interest in chemistry, materials, and carbon science due to their unique properties and nanoscale structures. Numerous cages that feature a combination of carbon, metal, and heteroatom-based clusters have been discovered since, for example, Sc3NC@C80 was first reported. Nanocarbon reactions that underlie formation of such compounds, however, are not well understood. Here, we experimentally investigate intramolecular reactions of metallofullerenes in the gas phase by means of laser-based techniques, analyzed by high magnetic field Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry. A variety of structurally defined clusterfullerene cages (e.g., Sc3N@C68, Sc3N@C78, etc.) are probed under energetic conditions to decipher reactions that may lead to encapsulation of C atoms, as well as clusterfullerenes that include metal-loss products. An aim of the present work is to identify mechanistic processes and principles that guide formation of metallofullerenes that entrap carbon, metal, and heteroatom-based clusters.

Fourier transform mass spectrometry (FTMS) of the isolated isotopic distribution for a highly charged biomolecule produces time-domain signal containing large amplitude signal "beats" separated by extended periods of much lower signal magnitude. Signal-to-noise ratio for data sampled between beats is low because of destructive interference of the signals induced by members of the isotopic distribution. Selective blanking of the data between beats has been used to increase spectral signal-to-noise ratio. However, blanking also eliminates signal components and, thus, can potentially distort the resulting FT spectrum. Here, we simulate the time-domain signal from a truncated isotopic distribution for a single charge state of an antibody. Comparison of the FT spectra produced with or without blanking and with or without added noise clearly show that blanking does not improve mass accuracy and introduces spurious peaks at both ends of the isotopic distribution (thereby making it more difficult to identify posttranslational modifications and/or adducts). Although the artifacts are reduced by use of multiple Gaussian (rather than square wave) windowing, blanking appears to offer no advantages for identifying true peaks or for mass measurement.

Top-down electron capture dissociation (ECD) Fourier transform ion cyclotron resonance (FTICR) mass spectrometry was performed for structural analysis of an intact monoclonal antibody (IgG1kappa (κ) isotype, ~148 kDa). Simultaneous ECD for all charge states (42+ to 58+) generates more extensive cleavages than ECD for an isolated single charge state. The cleavages are mainly localized in the variable domains of both heavy and light chains, the respective regions between the variable and constant domains in both chains, the region between heavy-chain constant domains CH2 and CH3, and the disulfide bond (S-S)-linked heavy-chain constant domain CH3. The light chain yields mainly N-terminal fragment ions due to the protection of the interchain disulfide bond between light and heavy chain, and limited cleavage sites are observed in the variable domains for each chain, where the S-S spans the polypeptide backbone. Only a few cleavages in the S-S-linked light-chain constant domain, hinge region, and heavy-chain constant domains CH1 and CH2 are observed, leaving glycosylation uncharacterized. Top-down ECD with a custom-built 9.4 T FTICR mass spectrometer provides more extensive sequence coverage for structural characterization of IgG1κ than does top-down collision-induced dissociation (CID) and electron transfer dissociation (ETD) with hybrid quadrupole time-of-flight instruments and comparable sequence coverage for top-down ETD with orbitrap mass analyzers.

Successful high-throughput characterization of intact proteins from complex biological samples by mass spectrometry requires instrumentation capable of high mass resolving power, mass accuracy, sensitivity, and spectral acquisition rate. These limitations often necessitate the performance of hundreds of LC–MS/MS experiments to obtain reasonable coverage of the targeted proteome, which is still typically limited to molecular weights below 30 kDa. The National High Magnetic Field Laboratory (NHMFL) recently installed a 21 T FT-ICR mass spectrometer, which is part of the NHMFL FT-ICR User Facility and available to all qualified users. Here we demonstrate top-down LC-21 T FT-ICR MS/MS of intact proteins derived from human colorectal cancer cell lysate. We identified a combined total of 684 unique protein entries observed as 3238 unique proteoforms at a 1% false discovery rate, based on rapid, data-dependent acquisition of collision-induced and electron-transfer dissociation tandem mass spectra from just 40 LC–MS/MS experiments. Our identifications included 372 proteoforms with molecular weights over 30 kDa detected at isotopic resolution, which substantially extends the accessible mass range for high-throughput top-down LC–MS/MS.

With the rapid growth of therapeutic monoclonal antibodies (mAbs), stringent quality control is needed to ensure clinical safety and efficacy. Monoclonal antibody primary sequence and post-translational modifications (PTM) are conventionally analyzed with labor-intensive, bottom-up tandem mass spectrometry (MS/MS), which is limited by incomplete peptide sequence coverage and introduction of artifacts during the lengthy analysis procedure. Here, we describe top-down and middle-down approaches with the advantages of fast sample preparation with minimal artifacts, ultrahigh mass accuracy, and extensive residue cleavages by use of 21 tesla FT-ICR MS/MS. The ultrahigh mass accuracy yields an RMS error of 0.2-0.4 ppm for antibody light chain, heavy chain, heavy chain Fc/2, and Fd subunits. The corresponding sequence coverages are 81%, 38%, 72%, and 65% with MS/MS RMS error ~4 ppm. Extension to a monoclonal antibody in human serum as a monoclonal gammopathy model yielded 53% sequence coverage from two nano-LC MS/MS runs. A blind analysis of five therapeutic monoclonal antibodies at clinically relevant concentrations in human serum resulted in correct identification of all five antibodies. Nano-LC 21 T FT-ICR MS/MS provides nonpareil mass resolution, mass accuracy, and sequence coverage for mAbs, and sets a benchmark for MS/MS analysis of multiple mAbs in serum. This is the first time that extensive cleavages for both variable and constant regions have been achieved for mAbs in a human serum background. Graphical Abstract ᅟ.

Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) typically utilizes an m/z-independent excitation magnitude to excite all ions to the same cyclotron radius, so that the detected signal magnitude is directly proportional to the relative ion abundance. However, deleterious space charge interaction between ion clouds is maximized for clouds of equal radius. To minimize ion cloud interactions, we induce an m/z-dependent ion radius distribution (30%-45% of the maximum cell radius) that results in a 3-fold increase in mass spectral dynamic range for complex mixtures, consistent with increased ion cloud lifetime for less-abundant ion clouds. Further, broadband frequency-sweep (chirp) excitation that contains the second and/or third harmonic frequency of an excited ion cloud swept from low-to-high frequency produces systematic variations in accurate mass measurement not observed when the sweep direction is reversed. The ion cyclotron radius distribution induces an m/z-dependent frequency shift that can be corrected to provide a root-mean-square (rms) mass measurement error of

We present atmospheric pressure laser-induced acoustic desorption chemical ionization (AP/LIAD-CI) with O(2) carrier/reagent gas as a powerful new approach for the analysis of saturated hydrocarbon mixtures. Nonthermal sample vaporization with subsequent chemical ionization generates abundant ion signals for straight-chain, branched, and cycloalkanes with minimal or no fragmentation. [M - H](+) is the dominant species for straight-chain and branched alkanes. For cycloalkanes, M(+•) species dominate the mass spectrum at lower capillary temperature (100 °C) and [M - H](+) at higher temperature (200 °C). The mass spectrum for a straight-chain alkane mixture (C(21)-C(40)) shows comparable ionization efficiency for all components. AP/LIAD-CI produces molecular weight distributions similar to those for gel permeation chromatography for polyethylene polymers, Polywax 500 and Polywax 655. Coupling of the technique to Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) for the analysis of complex hydrocarbon mixtures provides unparalleled mass resolution and accuracy to facilitate unambiguous elemental composition assignments, e.g., 1754 peaks (rms error = 175 ppb) corresponding to a paraffin series (C(12)-C(49), double-bond equivalents, DBE = 0) and higher DBE series corresponding to cycloparaffins containing one to eight rings. Isoabundance-contoured plots of DBE versus carbon number highlight steranes (DBE = 4) of carbon number C(27)-C(30) and hopanes of C(29)-C(35) (DBE = 5), with sterane-to-hopane ratio in good agreement with field ionization (FI) mass spectrometry analysis, but performed at atmospheric pressure. The overall speciation of nonpolar, aliphatic hydrocarbon base oil species offers a promising diagnostic probe to characterize crude oil and its products.

An absorption-mode Fourier transform ion cyclotron resonance (FT-ICR) mass spectrum exhibits two types of baseline distortion: a slow periodic oscillation even where no signal peaks are present, and additional distortion near signal peaks and proportional to signal magnitude. These distortions interfere with automated peak-picking, unless the baseline systematic variation is much less than baseline rms random noise. We previously showed that the slow oscillation is removed by low-pass filtering. Here, we present a fast, robust, and automated algorithm that flattens the absorption-mode spectral baseline, even in the vicinity of signal peaks. The method begins by defining baseline data values, followed by linear interpolation to generate baseline data values between the defined values, then boxcar smoothing to generate a final baseline spectrum, and final subtraction of that baseline from the original spectrum to yield a baseline-flattened absorption-mode spectrum. We apply the algorithm to a crude oil spectrum (with 8000 peaks) and to a ribonuclease A protein spectrum (with multiply-charged ion isotopic distributions). We identify many more peaks (crude oil) without loss of mass accuracy, and obtain more accurate isotopic distributions (RNase A).

Current high-throughput top-down proteomic platforms provide routine identification of proteins less than 25 kDa with 4-D separations. This short communication reports the application of technological developments over the past few years that improve protein identification and characterization for masses greater than 25 kDa. Advances in separation science have allowed increased numbers of proteins to be identified, especially by nanoliquid chromatography (nLC) prior to mass spectrometry (MS) analysis. Further, a goal of high-throughput top-down proteomics is to extend the mass range for routine nLC MS analysis up to 80 kDa because gene sequence analysis predicts that ~70% of the human proteome is transcribed to be less than 80 kDa. Normally, large proteins greater than 50 kDa are identified and characterized by top-down proteomics through fraction collection and direct infusion at relatively low throughput. Further, other MS-based techniques provide top-down protein characterization, however at low resolution for intact mass measurement. Here, we present analysis of standard (up to 78 kDa) and whole cell lysate proteins by Fourier transform ion cyclotron resonance mass spectrometry (nLC electrospray ionization (ESI) FTICR MS). The separation platform reduced the complexity of the protein matrix so that, at 14.5 T, proteins from whole cell lysate up to 72 kDa are baseline mass resolved on a nano-LC chromatographic time scale. Further, the results document routine identification of proteins at improved throughput based on accurate mass measurement (less than 10 ppm mass error) of precursor and fragment ions for proteins up to 50 kDa.

At sufficiently high mass accuracy, it is possible to distinguish phosphorylated from unmodified peptides by mass measurement alone. We examine the feasibility of that idea, tested against a library of all possible in silico tryptic digest peptides from the human proteome database. The overlaps between in silico tryptic digest phosphopeptides generated from known phosphorylated proteins (1–12 sites) and all possible unmodified human peptides are considered for assumed mass error ranges of ±10, ±50, ±100, ±1000, and ±10,000 ppb. We find that for mass error ±50 ppb, 95% of all phosphorylated human tryptic peptides can be distinguished from nonmodified peptides by accurate mass alone throughout the entire nominal mass range. We discuss the prospect of on-line LC MS/MS to identify phosphopeptide precursor ions in MS1 for selected dissociation in MS2 to identify the peptide and site(s) of phosphorylation.

Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) provides the highest mass resolving power and mass measurement accuracy for unambiguous identification of biomolecules. Previously, the highest-mass protein for which FTICR unit mass resolution had been obtained was 115 kDa at 7 T. Here, we present baseline resolution for an intact 147.7 kDa monoclonal antibody (mAb), by prior dissociation of noncovalent adducts, optimization of detected total ion number, and optimization of ICR cell parameters to minimize space charge shifts, peak coalescence, and destructive ion cloud Coulombic interactions. The resultant long ICR transient lifetime (as high as 20 s) results in magnitude-mode mass resolving power of ∼420 000 at m/z 2 593 for the 57+ charge state (the highest mass for which baseline unit mass resolution has been achieved), auguring for future characterization of even larger intact proteins and protein complexes by FTICR MS. We also demonstrate up to 80% higher resolving power by phase...

Valence parity provides a way to distinguish between N-terminal and C-terminal electron capture dissociation/electron transfer dissociation (ECD/ETD) product ions based on their number of hydrogen plus nitrogen atoms determined by accurate mass measurement and forms a basis for de novo peptide sequencing. The effect of mass accuracy (0.1-1 ppm error) on c'/z(•) overlap and unique elemental composition overlap is evaluated for a database of c'/z(•) product ions each based on all possible amino acid combinations and four subset databases containing the same c' ions but with z(•) ions determined by in silico digestion with trypsin, Glu-C, Lys-C, or chymotrypsin. High mass accuracy reduces both c'/z(•) overlap and unique elemental composition overlap. Of the four proteases, trypsin offers slightly better discrimination between N- and C-terminal ECD/ETD peptides. Interestingly, unique elemental composition overlap curves for c'/c' and z(•)/z(•) peptide ions exhibit discontinuities at certain nominal masses for 0.1-1.0 ppm mass error. Also, as noted in the companion article (Polfer et al. Anal. Chem.2011, DOI: 10.1021/ac201624t), the number of ECD/ETD product ion amino acid compositions as a function of nominal mass increases exponentially with mass but with a superimposed modulation due to higher prevalence of certain elemental compositions.

Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry relies upon linearity between the ion cyclotron excitation and the observed response. However, nonlinearities result from non-ideal applied electric and magnetic fields and Coulombic interactions. Here, we report nonlinear response at low excitation electric field magnitude due to Coulombic shielding. The measured ICR signal magnitude exhibits an excitation voltage threshold that increases monotonically with the number of shielding ions (i.e., unexcited ions). If shielding ions are not present, ICR signal magnitude versus excitation voltage is linear (e.g., for quadrupole-isolated ions of nearly a single m/z). Finally, we show that shielding results in a reduced cyclotron radius at low excitation voltage, resulting in an increased rate of transient decay; thereby exacerbating response nonlinearity and excitation threshold for long data acquisition period.

It has been known for 35 years that phase correction of FTICR data can in principle produce an absorption-mode spectrum with mass resolving power as much as a factor of 2 higher than conventional magnitude-mode display, an improvement otherwise requiring a (much more expensive) increase in magnetic field strength. However, temporally dispersed excitation followed by time-delayed detection results in steep quadratic variation of signal phase with frequency. Here, we present a robust, rapid, automated method to enable accurate broadband phase correction for all peaks in the mass spectrum. Low-pass digital filtering effectively eliminates the accompanying baseline roll. Experimental FTICR absorption-mode mass spectra exhibit at least 40% higher resolving power (and thus an increased number of resolved peaks) as well as higher mass accuracy relative to magnitude mode spectra, for more complete and more reliable elemental composition assignments for mixtures as complex as petroleum.

We have observed new mechanistic insight into the formation of metallic nitride clusterfullerenes from doped graphite by use of laser-based plasma synthesis techniques, analyzed by ultrahigh resolution 9.4 tesla Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry. These online chemical sampling techniques provide a route to investigate underexplored nanocarbon reactions that take place under the harsh conditions of metallofullerene synthesis, and are expected to be applicable to macroscopic production methods. Here, we report efforts to form various clusterfullerenes by laser vaporization and extend these techniques as new avenues for investigation of exohedral functionalization (e.g., fluorination of Sc3N@C80) and heteroatom-doping of endohedral fullerenes. This work was supported by NSF Division of Materials Research through DMR-11-57490 and the State of Florida.

Dissociation kinetics of the K(+) loss reaction of three potassiated tertiary amino acids (Scheme 1) were studied by infrared multiple photon dissociation (IRMPD) in a Fourier transform ion cyclotron resonance (FT ICR)-MS instrument. The aim of the study was to probe if a kinetic study by IRMPD can yield useful information on the ion structure of the precursor ion species. The measured activation energy values determined by IRMPD are related to the potassium affinity, DeltaH(K(+)), of N-methyl proline determined by threshold collision-induced dissociation experiments. By appropriate scaling with this reference value, the experimentally determined activation energy values for the K(+) loss are transformed into respective potassium affinities, DeltaH(K(+))(IRMPD). These values match the calculated potassium affinity values for salt bridge (SB) structures, DeltaH(K(+))(SB), substantially better than those for canonical structures with a single formal charge site (charge solvation (CS)), thereby allowing structure identification. This conclusion is consistent with other spectroscopic data, which yielded unambiguous evidence of these tertiary amino acids adopting SB structures in the gas phase. This study demonstrates that IRMPD can be applied to determine individual ion structures in the gas phase, given that adequate reference values are available for proper scaling.

Successful electron capture dissociation (ECD) Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) applications to peptide and protein structural analysis have been enabled by constant progress in implementation of improved electron injection techniques. The rate of ECD product ion formation has been increased to match the liquid chromatography and capillary electrophoresis timescales, and ECD has been combined with infrared multiphoton dissociation in a single experimental configuration to provide simultaneous irradiation, fast switching between the two techniques, and good spatial overlap between ion, photon, and electron beams. Here we begin by describing advantages and disadvantages of the various existing electron injection techniques for ECD in FT-ICR MS. We next compare multiple-pass and single-pass ECD to provide better understanding of ECD efficiency at low and high negative cathode potentials. We introduce compressed hollow electron beam injection to optimize the overlap of ion, photon, and electron beams in the ICR ion trap. Finally, to overcome significant outgassing during operation of a powerful thermal cathode, we introduce nonthermal electron emitter-based electron injection. We describe the first results obtained with cold cathode ECD, and demonstrate a general way to obtain low-energy electrons in FT-ICR MS by use of multiple-pass ECD.

We describe the design and current performance of a 14.5 T hybrid linear quadrupole ion trap Fourier transform ion cyclotron resonance mass spectrometer. Ion masses are routinely determined at 4-fold better mass accuracy and 2-fold higher resolving power than similar 7 T systems at the same scan rate. The combination of high magnetic field and strict control of the number of trapped ions results in external calibration broadband mass accuracy typically less than 300 ppb rms, and a resolving power of 200,000 (m/Delta m50% at m/z 400) is achieved at greater than 1 mass spectrum per second. Novel ion storage optics and methodology increase the maximum number of ions that can be delivered to the FTICR cell, thereby improving dynamic range for tandem mass spectrometry and complex mixture applications.

We present a method to distinguish N-terminal from C-terminal product ions in electron capture dissociation (ECD) MS/MS due to the change in relative abundances of even-electron (prime) and odd-electron (radical) product ions produced in consecutive ECD and activated ion-ECD mass spectra. The method is based on the rate and direction of hydrogen atom transfer between N-terminal and C-terminal ECD products and its dependence on ion internal energy. We demonstrate that increasing ion internal energy by vibrational activation prior to ECD results in decreased ratio of radical/prime N-terminal product ions (c*/c' ratio), but increased ratio of radical/prime C-terminal product ions (z*/z' ratio) in many cases. The combination of AI-ECD and ECD promises to increase the confidence of mass spectrometry-based peptide sequencing and protein identification.

Molecular characterization of sulfur-containing species in petroleum is important because sulfur-containing compounds are detrimental to the environment and the refining processes. In a recent report, the sulfur-containing compounds in a vacuum bottom residue (VBR) were methylated to enhance their detectability by electrospray ionization (ESI) mass analysis. The most abundant sulfur compounds exhibited relatively low double bond equivalents (4 < DBE < 12). Alternatively, atmospheric pressure photoionization (APPI) mass analysis can provide molecular characterization without chemical derivatization. Here, we compare the sulfur speciation of a petroleum vacuum bottom residue by ESI and APPI with a 9.4 T Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer. Even after methylation, ions produced by APPI extend to much higher DBE than by ESI. Moreover, analysis of the saturates and aromatics fractions of underivatized VBR by APPI shows comparable ionization efficiency across a broad DBE range. ...

Applying an inverted voltage to the "sidekick" electrodes during ion cyclotron resonance detection improves both Fourier transform ion cyclotron resonance (FT-ICR) mass spectral signal-to-noise ratio (at fixed resolving power) and resolving power (at fixed signal-to-noise ratio). The time-domain signal duration increases by up to a factor of 2. The method has been applied to 7-T FT-ICR MS of electrosprayed positive ions from substance P and human growth hormone protein ( approximately 22 000 Da, m/Deltam50% 200 000), without the need for pulsed cooling gas inside the ICR trap. The modification can be easily adapted to any FT-ICR instrument equipped with sidekick electrodes. The present effects are shown to be comparable to electron field modification by injection of an electron beam during ICR detection, reported by Kaiser and Bruce (Kaiser, N. K.; Bruce, J. E. Anal. Chem. 2005, 77, 5973-5981.). Although the exact mechanism is not fully understood, computer simulations show that a flattening of the radial potential gradient along the magnetic field direction in the ICR trap may contribute to the effects. This study not only provides a way to enhance the quality of FT-ICR mass spectra but also offers insight into understanding of ion motions inside an ICR ion trap.

The effect of peptide dication charge location on electron capture dissociation (ECD) fragmentation pattern is investigated. ECD fragmentation patterns are compared for peptides with amide and free acid C-terminal groups. ECD of free acid compared with C-terminally amidated peptides with basic residues near the N-terminus demonstrates increased formation of a-type ions. Similarly, ECD of free acid compared with C-terminally amidated peptides with basic residues near the C-terminus exhibits increased formation of y-type ions. Alteration of the peptide sequence to inhibit the formation of charged side chains (i.e., amino acid substitution and acetylation) provides further evidence for charge location effect on ECD. We propose that formation of zwitterionic peptide structures increases the likelihood of amide nitrogen protonation (versus basic side chains), which is responsible for the increase in a- and y-type ion formation.

Electron capture dissociation (ECD) efficiency in a 9.4 T Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer varies periodically with the time interval between ion and electron injection. The observed modulation frequency correlates to within 1% with ion magnetron frequency, most probably due to misalignment between the ion beam and the electron beam. The optimum ECD conditions are obtained by correctly phasing electron injection with the ion magnetron motion. Displacement of the trapped ion cloud by variation of the ICR trap radial electric field decreases ECD efficiency modulation amplitude. Experiments directly suggest that only ions interacting with electrons at the moment of electron injection participate in ECD reactions.

The National High Magnetic Field Laboratory (NHMFL) has identified as one of its next high-field persistent-magnet targets a 21 T superconducting magnet system to be used in a Fourier Transform Ion Cyclotron Resonance (FT-ICR) Mass Spectrometer. The required fundamental parameters of a 21 T central field and 110 mm warm bore diameter are similar to those already successfully demonstrated in the NHMFL's Ultra-Wide Bore 900 MHz Nuclear Magnetic Resonance magnet system. However, there are several ancillary requirements of the FT-ICR spectrometer that require technical attention such as fringe field restrictions requiring shielding, length of uniformity zone, magnitude of inhomogeneity, horizontal orientation of the magnet axis and a limitation on the length of the cryostat. This paper describes the overall system requirements, features and status of the superconducting magnet conceptual design proposed for meeting these requirements

Untreated and hydrotreated nitrogen-concentrated distillation fractions (483−504 °C) from Syncrude oil have been analyzed by electron ionization, field desorption ionization, and electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry. The routinely attainable mass resolving power ≥300 000 at m/z 300 and sub-ppm mass accuracy allowed for resolution and elemental compositional assignment of various compound classes. Multiple closely spaced mass doublets (with mass differences

We analyzed three vacuum gas oil distillation fractions, 295−319 °C, 319−456 °C, and 456−543 °C, with a home-built external electron ionization (EI) 7 T Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer. EI at 10 eV allows soft ionization of aromatic compounds in the vacuum gas oil range. Unambiguous elemental composition assignment provides insight into compositional variations at the molecular level; for example, ultrahigh resolving power (m/Δm50% ≈ 300 000 at m/z 300) and mass accuracy (

Amide hydrogen/deuterium exchange is a powerful biophysical technique for probing changes in protein dynamics induced by ligand interaction. The inherent low throughput of the technology has limited its impact on drug screening and lead optimization. Automation increases the throughput of H/D exchange to make it compatible with drug discovery efforts. Here we describe the first fully automated H/D exchange system that provides highly reproducible H/D exchange kinetics from 130 ms to 24 h. Throughput is maximized by parallel sample processing, and the system can run H/D exchange assays in triplicate without user intervention. We demonstrate the utility of this system to differentiate structural perturbations in the ligand-binding domain (LBD) of the nuclear receptor PPARgamma induced upon binding a full agonist and a partial agonist. PPARgamma is the target of glitazones, drugs used for treatment of insulin resistance associated with type II diabetes. Recently it has been shown that partial agonists of PPARgamma have insulin sensitization properties while lacking several adverse effects associated with full agonist drugs. To further examine the mechanism of partial agonist activation of PPARgamma, we extended our studies to the analysis of ligand interactions with the heterodimeric complex of PPARgamma/RXRalpha LBDs. To facilitate analysis of H/D exchange of large protein complexes, we performed the experiment with a 14.5-T Fourier transform ion cyclotron resonance mass spectrometer capable of measuring mass with accuracy in the ppb range.