Adding Color to Mass Spectra of Biopolymers: Charge Determination Analysis (CHARDA) Assigns Charge State to Every Ion Peak.

Traditionally, mass spectrometry (MS) output is the ion abundance plotted versus the ionic mass-to-charge ratio m/z. While employing only commercially available equipment, Charge Determination Analysis (CHARDA) adds a third dimension to MS, estimating for individual peaks their charge states z starting from z = 1 and color coding z in m/z spectra. CHARDA combines the analysis of ion signal decay rates in the time-domain data (transients) in Fourier transform (FT) MS with the interrogation of mass defects (fractional mass) of biopolymers. Being applied to individual isotopic peaks in a complex protein tandem (MS/MS) data set, CHARDA aids peptide mass spectra interpretation by facilitating charge-state deconvolution of large ionic species in crowded regions, estimating z even in the absence of an isotopic distribution (e.g., for monoisotopic mass spectra). CHARDA is fast, robust, and consistent with conventional FTMS and FTMS/MS data acquisition procedures. An effective charge-state resolution Rz ≥ 6 is obtained with the potential for further improvements.

Ultralong transients enhance sensitivity and resolution in Orbitrap-based single-ion mass spectrometry.

Orbitrap-based charge detection mass spectrometry utilizes single-molecule sensitivity to enable mass analysis of even highly heterogeneous, high-mass macromolecular assemblies. For contemporary Orbitrap instruments, the accessible ion detection (recording) times are maximally ~1-2 s. Here by modifying a data acquisition method on an Orbitrap ultrahigh mass range mass spectrometer, we trapped and monitored individual (single) ions for up to 25 s, resulting in a corresponding and huge improvement in signal-to-noise ratio (×5 compared with 1 s), mass resolution (×25) and accuracy in charge and mass determination of Orbitrap-based charge detection mass spectrometry.

Ultrahigh-Mass Resolution Mass Spectrometry Imaging with an Orbitrap Externally Coupled to a High-Performance Data Acquisition System.

Matrix-assisted laser desorption ionization (MALDI) mass spectrometry imaging (MSI) is a powerful analytical tool that enables molecular sample analysis while simultaneously providing the spatial context of hundreds or even thousands of analytes. However, because of the lack of a separation step prior to ionization and the immense diversity of biomolecules, such as lipids, including numerous isobaric species, the coupling of ultrahigh mass resolution (UHR) with MSI presents one way in which this complexity can be resolved at the spectrum level. Until now, UHR MSI platforms have been restricted to Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers. Here, we demonstrate the capabilities of an Orbitrap-based UHR MSI platform to reach over 1,000,000 mass resolution in a lipid mass range (600-950 Da). Externally coupling the Orbitrap Q Exactive HF with the high-performance data acquisition system FTMS Booster X2 provided access to the unreduced data in the form of full-profile absorption-mode FT mass spectra. In addition, it allowed us to increase the time-domain transient length from 0.5 to 10 s, providing improvement in the mass resolution, signal-to-noise ratio, and mass accuracy. The resulting UHR performance generates high-quality MALDI MSI images and simplifies the identification of lipids. Collectively, these improvements resulted in a 1.5-fold increase in annotations, demonstrating the advantages of this UHR imaging platform for spatial lipidomics using MALDI-MSI.

Super-Resolution Mass Spectrometry Enables Rapid, Accurate, and Highly Multiplexed Proteomics at the MS2 Level.

In tandem mass spectrometry (MS2)-based multiplexed quantitative proteomics, the complement reporter ion approaches (TMTc and TMTproC) were developed to eliminate the ratio-compression problem of conventional MS2-level approaches. Resolving all high m/z complement reporter ions (∼6.32 mDa-spaced) requires mass resolution and scan speeds above the performance levels of OrbitrapTM instruments. Therefore, complement reporter ion quantification with TMT/TMTpro reagents is currently limited to 5 out of 11 (TMT) or 9 out of 18 (TMTpro) channels (∼1 Da spaced). We first demonstrate that a FusionTM LumosTM Orbitrap can resolve 6.32 mDa-spaced complement reporter ions with standard acquisition modes extended with 3 s transients. We then implemented a super-resolution mass spectrometry approach using the least-squares fitting (LSF) method for processing Orbitrap transients to achieve shotgun proteomics-compatible scan rates. The LSF performance resolves the 6.32 mDa doublets for all TMTproC channels in the standard mass range with transients as short as ∼108 ms (Orbitrap resolution setting of 50,000 at m/z 200). However, we observe a slight decrease in measurement precision compared to 1 Da spacing with the 108 ms transients. With 256 ms transients (resolution of 120,000 at m/z 200), coefficients of variation are essentially indistinguishable from 1 Da samples. We thus demonstrate the feasibility of highly multiplexed, accurate, and precise shotgun proteomics at the MS2 level.

Real-Time Investigation of Primary Ship Engine Emissions by Vacuum Resonance-Enhanced Multiphoton Ionization High-Resolution Orbitrap Mass Spectrometry.

The comprehensive chemical description of air pollution is a prerequisite for understanding atmospheric transformation processes and effects on climate and environmental health. In this study, a prototype vacuum photoionization Orbitrap mass spectrometer was evaluated for field-suitability by an online on-site investigation of emissions from a ship diesel engine. Despite remote measurements in a challenging environment, the mass spectrometric performance could fully be exploited. Due to the high resolution and mass accuracy in combination with resonance-enhanced multiphoton ionization, the aromatic hydrocarbon profile could selectively and sensitively be analyzed. Limitations from commonly deployed time-of-flight platforms could be overcome, allowing to unraveling the oxygen- and sulfur-containing compounds. Scan-by-scan evaluation of the online data revealed no shift in exact m/z, assignment statistics with root mean square error (RMSE) below 0.2 ppm, continuous high-resolution capabilities, and good isotopic profile matches. Emissions from three different feed fuels were investigated, namely, diesel, heavy fuel oil (HFO), and very low sulfur fuel oil (VLSFO). Regulations mainly concern the fuel sulfur content, and thus, exhaust gas treatment or new emerging fuels, such as the cycle-oil-based VLSFO, can legally be applied. Unfortunately, despite lower CHS-class emissions, a substantial amount of PAHs is emitted by the VLSFO with higher aromaticity compared to the HFO. Hence, legislative measures might need to take further chemical criteria into account.

Characterization of the Time-Domain Isotopic Beat Patterns of Monoclonal Antibodies in Fourier Transform Mass Spectrometry.

The time-domain transients in the Fourier transform mass spectrometry (FTMS) analysis of monoclonal antibodies (mAbs) are known to exhibit characteristic isotopic beat patterns. These patterns are defined by the isotopic distributions of all gaseous mAb ions present in the FTMS mass analyzer, originating from single or multiple charge states, and from single or multiple proteoforms. For an isolated charge state of a single proteoform, the mAb isotopic beat pattern resembles narrow splashes of signal amplitude (beats), spaced periodically in the time-domain transient, with broad (often exceeding 1 s) "valleys" between them. Here, we reinforce the importance of isotopic beat patterns for the accurate interpretation and presentation of FTMS data in the analysis of mAbs and other large biopolymers. An updated, mAb-grade version of the transient-mediated FTMS data simulation and visualization tool, FTMS Simulator is introduced and benchmarked. We then apply this tool to evaluate the charge-state dependent characteristics of isotopic beats in mAbs analyses with modern models of Orbitrap and ion cyclotron resonance (ICR) FTMS instruments, including detection of higher-order harmonics. We demonstrate the impact of the isotopic beat patterns on the analytical characteristics of the resulting mass spectra of individual and overlapping mAb proteoforms. The results reported here detail highly nonlinear dependences of resolution and signal-to-noise ratio on the time-domain transient period, absorption or magnitude mode spectra representation, and apodization functions. The provided description and the demonstrated ability to routinely conduct accurate simulations of FTMS data for large biopolymers should aid the end-users of Orbitrap and ICR FTMS instruments in the analysis of mAbs and other biopolymers, including viruses.

Structural Analysis of Monoclonal Antibodies with Top-down and Middle-down Electron Transfer Dissociation Mass Spectrometry: The First Decade.

Monoclonal antibodies (mAbs) are protein biotherapeutics with a proven efficacy toward fighting life-threatening diseases. Their exceptional healing potential drives the annual increase in the number of novel mAbs and other antibody-like molecules entering clinical trials and the number of approved mAb-based drugs. Mass spectrometry (MS) offers high selectivity and specificity for the potentially unambiguous identification and comprehensive structural characterization of proteins, including at the proteoform level. It is thus not surprising that MS-based approaches are playing a central role in the biopharma laboratories, complementing and advancing traditional biotherapeutics characterization workflows. A combination of MS approaches is required to comprehensively characterize mAbs' structures: the commonly employed bottom-up MS approaches are efficiently complemented with mass measurements at the intact and subunit (middle-up) levels, together with product ion analysis following gas-phase fragmentation of precursor ions performed at the intact (top-down) and subunit (middle-down) levels. Here we overview our group's contribution to increasing the efficiency of these approaches and the development of the novel strategies over the past decade. Our particular focus has been on the top-down and middle-down MS methods that utilize electron transfer dissociation (ETD) for gas-phase protein ion fragmentation. Several approaches pioneered by our group, particularly an ETD-based middle-down approach, constitute a part of commercial software solutions for the mAb's characterization workflows.

Fourier transform ion cyclotron resonance mass spectrometry at the true cyclotron frequency.

Ion cyclotron resonance (ICR) cells provide stability and coherence of ion oscillations in crossed electric and magnetic fields over extended periods of time. Using the Fourier transform enables precise measurements of ion oscillation frequencies. These precisely measured frequencies are converted into highly accurate mass-to-charge ratios of the analyte ions by calibration procedures. In terms of resolution and mass accuracy, Fourier transform ICR mass spectrometry (FT-ICR MS) offers the highest performance of any MS technology. This is reflected in its wide range of applications. However, in the most challenging MS application, for example, imaging, enhancements in the mass accuracy of fluctuating ion fluxes are required to continue advancing the field. One approach is to shift the ion signal power into the peak corresponding to the true cyclotron frequency instead of the reduced cyclotron frequency peak. The benefits of measuring the true cyclotron frequency include increased tolerance to electric fields within the ICR cell, which enhances frequency measurement precision. As a result, many attempts to implement this mode of FT-ICR MS operation have occurred. Examples of true cyclotron frequency measurements include detection of magnetron inter-harmonics of the reduced cyclotron frequency (i.e., the sidebands), trapping field-free (i.e., screened) ICR cells, and hyperbolic ICR cells with quadrupolar ion detection. More recently, ICR cells with spatially distributed ion clouds have demonstrated attractive performance characteristics for true cyclotron frequency ion detection. Here, we review the corresponding developments in FT-ICR MS over the past 40 years.

The Human Proteoform Project: Defining the human proteome.

Proteins are the primary effectors of function in biology, and thus, complete knowledge of their structure and properties is fundamental to deciphering function in basic and translational research. The chemical diversity of proteins is expressed in their many proteoforms, which result from combinations of genetic polymorphisms, RNA splice variants, and posttranslational modifications. This knowledge is foundational for the biological complexes and networks that control biology yet remains largely unknown. We propose here an ambitious initiative to define the human proteome, that is, to generate a definitive reference set of the proteoforms produced from the genome. Several examples of the power and importance of proteoform-level knowledge in disease-based research are presented along with a call for improved technologies in a two-pronged strategy to the Human Proteoform Project.

Drug-to-Antibody Ratio Estimation via Proteoform Peak Integration in the Analysis of Antibody-Oligonucleotide Conjugates with Orbitrap Fourier Transform Mass Spectrometry.

The therapeutic efficacy and pharmacokinetics of antibody-drug conjugates (ADCs) in general, and antibody-oligonucleotide conjugates (AOCs) in particular, depend on the drug-to-antibody ratio (DAR) distribution and average value. The DAR is considered a critical quality attribute, and information pertaining to it needs to be gathered during ADC/AOC development, production, and storage. However, because of the high structural complexity of ADC/AOC samples, particularly in the initial drug-development stages, the application of the current state-of-the-art mass spectrometric approaches can be limited for DAR analysis. Here, we demonstrate a novel approach for the analysis of complex ADC/AOC samples, following native size-exclusion chromatography Orbitrap Fourier transform mass spectrometry (FTMS). The approach is based on the integration of the proteoform-level mass spectral peaks in order to provide an estimate of the DAR distribution and its average value with less than 10% error. The peak integration is performed via a truncation of the Orbitrap's unreduced time-domain ion signals (transients) before mass spectra generation via FT processing. Transient recording and processing are undertaken using an external data acquisition system, FTMS Booster X2, coupled to a Q Exactive HF Orbitrap FTMS instrument. This approach has been applied to the analysis of whole and subunit-level trastuzumab conjugates with oligonucleotides. The obtained results indicate that ADC/AOC sample purification or simplification procedures, for example, deglycosylation, could be omitted or minimized prior to the DAR analysis, streamlining the drug-development process.

Vacuum Laser Photoionization inside the C-trap of an Orbitrap Mass Spectrometer: Resonance-Enhanced Multiphoton Ionization High-Resolution Mass Spectrometry.

State-of-the-art mass spectrometry with ultraviolet (UV) photoionization is mostly limited to time-of-flight (ToF) mass spectrometers with 1000-10 000 m/Δm mass resolution. However, higher resolution and higher spectral dynamic range mass spectrometry may be indispensable in complex mixture characterization. Here, we present the concept, implementation, and initial evaluation of a compact ultrahigh-resolution mass spectrometer with gas-phase laser ionization. The concept is based on direct laser photoionization in the ion accumulation and ejection trap (C-trap) of an Orbitrap mass spectrometer. Resonance-enhanced multiphoton ionization (REMPI) using 266 nm UV pulses from a frequency-quadrupled Nd:YAG laser was applied for selective and efficient ionization of monocyclic and polycyclic aromatic hydrocarbons. The system is equipped with a gas inlet for volatile compounds and a heated gas chromatography coupling. The former can be employed for rapid system m/z-calibration and performance evaluation, whereas the latter enables analysis of semivolatile and higher-molecular-weight compounds. The capability to evaluate complex mixtures is demonstrated for selected petrochemical materials. In these experiments, several hundred to over a thousand compounds could be attributed with a root-mean-square mass error generally below 1 ppm and a mass resolution of over 140 000 at 200 m/z. Isobaric interferences could be resolved, and narrow mass splits, such as 3.4 mDa (SH4/C3), are determined. Single laser shots provided limits of detection in the 20-ppb range for p-xylene and 1,2,4-trimethylbenzene, similar to compact vacuum REMPI-ToF systems.

Improved Uranium Isotope Ratio Analysis in Liquid Sampling-Atmospheric Pressure Glow Discharge/Orbitrap FTMS Coupling through the Use of an External Data Acquisition System.

Isotope ratio (IR) analysis of natural abundance uranium presents a formidable challenge for mass spectrometry (MS): the required spectral dynamic range needs to enable the quantitatively accurate measurement of the 234UO2 species present at ∼0.0053% isotopic abundance. We address this by empowering a benchtop Orbitrap Fourier transform mass spectrometer (FTMS) coupled with the liquid sampling-atmospheric pressure glow discharge (LS-APGD) ion source and an external high-performance data acquisition system, FTMS Booster X2. The LS-APGD microplasma has demonstrated impressive capabilities regarding elemental and IR analysis when coupled with Orbitrap FTMS. Despite successes, there are limitations regarding the dynamic range and mass resolution that stem from space charge effects and data acquisition and processing restrictions. To overcome these limitations, the FTMS Booster was externally interfaced to an LS-APGD Q Exactive Focus Orbitrap FTMS to obtain time-domain signals (transients) and to process unreduced data. The unreduced time-domain data acquisition with user-controlled processing permit the evaluation of the effects of in-hardware transient phasing, increased transient lengths, advanced transient coadding, varying the length of a transient to be processed with a user-defined time increment, and the use of absorption-mode FT (aFT) processing methods on IR analysis. The added capabilities extend the spectral dynamic range of the instrument to at least 4-5 orders of magnitude and provide a resolution improvement from ∼70k to 900k m/Δm at 200 m/z. The empowered LS-APGD Orbitrap platform allows for the simultaneous measurement of 234UO2 and the prominent 235UO2 and 238UO2 isotopic species at their natural abundances, ultimately yielding improvements in performance when compared to previous uranium IR results on this same Q Exactive Focus instrument.

Fast Afucosylation Profiling of Glycoengineered Antibody Subunits by Middle-Up Mass Spectrometry.

Middle-up LC-MS antibody characterization workflows using reduction or IdeS digestion for a focused assessment of N-glycan profiling of three representative glycoengineered monoclonal antibodies (mAbs), namely, obinutuzumab (GlycomAb technology, Glycart/Roche), benralizumab (Potelligent Technology, BioWa, Kyowa Kirin) and mAb B (kifunensine) and compared to mAb A, produced in a common CHO cell line. In addition, EndoS or EndoS2 enzyme are used for quantitative determination of Fc-glycan core afucosylation and high mannose for these antibodies, as requested by health authorities for Fc-competent therapeutics mAbs critical quality attributes (CQAs).

Untargeted analysis of pure snail slime and snail slime-induced Au nanoparticles metabolome with MALDI FT-ICR MS.

Chronic wounds result from the failure of the normal wound healing process. Any delay during the tissue repair process could be defined as chronic wound healing, potentially having a highly detrimental impact on human health. To face this problem, in the last years, the use of different technologies alternative to therapeutic agents is gaining more attention. The Helix aspersa snail slime-based products are increasingly being used for skin injury, thanks to their ability to make tissue repair processes faster. To date, a comprehensive overview of pure snail slime metabolome is not available. Besides, Au nanoparticles (AuNPs) technology is spreading rapidly in the medical environment, and the search for AuNPs "green" synthetic routes that involve natural products as precursor agents is demanded, alongside with a deep comprehension of the kind of species that actively take part in synthesis and product stabilization. The aim of this work is to characterize the metabolic profile of a pure snail slime sample, by an untargeted high-resolution mass spectrometry-based analysis. In addition, insights on AuNPs synthesis and stabilization by the main components of pure snail slime used to induce the synthesis were obtained. The untargeted analysis provided a large list of important classes of metabolites, that is, fatty acid derivatives, amino acids and peptides, carbohydrates and polyphenolic compounds that could be appreciated in both samples of slime, with and without AuNPs. Moreover, a direct comparison of the obtained results suggests that mostly nitrogen and sulfur-bearing metabolites take part in the synthesis and stabilization of AuNPs.

Imaging of Triglycerides in Tissues Using Nanospray Desorption Electrospray Ionization (Nano-DESI) Mass Spectrometry.

Nonpolar triglycerides (TGs) are rarely detected in mass spectrometry imaging (MSI) experiments unless they are abundant in the sample. Herein, we use nanospray desorption electrospray ionization (nano-DESI) to explore the role of the solvent composition and ionic dopants on the detection of TGs in a murine gastrocnemius muscle tissue used as a model system. We evaluated three solvent mixtures for their ability to extract nonpolar TG species: MeOH:H2O 9:1 (v/v), MeOH:DCM 6:4 (v/v) and MeOH:AcN:tol 5:3.5:1.5 (v/v/v). We observe that TGs are mainly detected as [M+K]+ adducts and their extraction efficiency is improved using less polar solvents: MeOH:DCM and MeOH:AcN:tol. We also explore whether the ionization efficiency of TGs may be improved by doping the MeOH:AcN:tol solvent with ammonium formate (AF) and other ionic additives. However, the formation of [M+NH4]+ adducts of TGs is less efficient than the formation of [M+K]+ adducts in the range of AF concentrations from 0.1 to 10 mM. Chemical derivatization using 100 µM of Girard T reagent predominately generates reaction products of phosphatidylcholine rather than TG species. Moreover, the presence of the Girard T reagent suppresses ion signals of all the species in the spectrum including TGs. Nano-DESI MSI experiments performed using MeOH:AcN:tol solvent enable imaging of TGs without any detectable adverse effect on signals of other lipids and metabolites. Specifically, 10 out of 14 TG species were detected exclusively using MeOH:AcN:tol and the sensitivity towards other TGs was improved by at least an order of magnitude. Although polyunsaturated TGs may be detected using both solvents, saturated and monounsaturated TGs are only detected using MeOH:AcN:tol. Our results provide a direct path for the improved detection of TGs in tissue imaging experiments using liquid-based ambient ionization techniques.

Narrow Aperture Detection Electrodes ICR Cell with Quadrupolar Ion Detection for FT-ICR MS at the Cyclotron Frequency.

Ion signal detection at the true (unperturbed) cyclotron frequency instead of the conventional reduced cyclotron frequency has remained a formidable challenge since the inception of Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). Recently, routine FT-ICR MS at the true cyclotron frequency has become a reality with the implementation of ICR cells with narrow aperture detection electrodes (NADEL). Here, we describe the development and implementation of the next generation of these cells, namely, a 2xNADEL ICR cell, which comprises four flat detect and four ∼45° cylindrical excite electrodes, enabling independent ion excitation and quadrupolar ion detection. The performance of the 2xNADEL ICR cell was evaluated on two commercial FT-ICR MS platforms, 10 T LTQ FT from Thermo Scientific and 9.4 T SolariX XR from Bruker Daltonics. The cells provided accurate mass measurements in the analyses of singly and multiply charged peptides (root-mean-square, RMS, mass error Δm/m of 90 ppb), proteins (Δm/m = 200 ppb), and petroleum fractions (Δm/m < 200 ppb). Due to the reduced influence of measured frequency on the space charge and external (trapping) electric fields, the 2xNADEL ICR cells exhibited stable performance in a wide range of trapping potentials (1-20 V). Similarly, in a 13 h rat brain MALDI imaging experiment, the RMS mass error did not exceed 600 ppb even for low signal-to-noise ratio analyte peaks. Notably, the same set of calibration constants was applicable to Fourier spectra in all pixels, reducing the need for recalibration at the individual pixel level. Overall, these results support further experimental development and fundamentals investigation of this promising technology.

Interlaboratory Study for Characterizing Monoclonal Antibodies by Top-Down and Middle-Down Mass Spectrometry.

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.

Transient-Mediated Simulations of FTMS Isotopic Distributions and Mass Spectra to Guide Experiment Design and Data Analysis.

Fourier transform mass spectrometry (FTMS) applications require accurate analysis of extremely complex mixtures of species in wide mass and charge state ranges. To optimize the related FTMS data analysis accuracy, parameters for data acquisition and the allied data processing should be selected rationally, and their influence on the data analysis outcome is to be understood. To facilitate this selection process and to guide the experiment design and data processing workflows, we implemented the underlying algorithms in a software tool with a graphical user interface, FTMS Isotopic Simulator. This tool computes FTMS data via time-domain data (transient) simulations for user-defined molecular species of interest and FTMS instruments, including diverse Orbitrap FTMS models, followed by user-specified FT processing steps. Herein, we describe implementation and benchmarking of this tool for analysis of a wide range of compounds as well as compare simulated and experimentally generated FTMS data. In particular, we discuss the use of this simulation tool for narrowband, broadband, and low- and high-resolution analysis of small molecules, peptides, and proteins, up to the level of their isotopic fine structures. By demonstrating the allied FT processing artifacts, we raise awareness of a proper selection of FT processing parameters for modern applications of FTMS, including intact mass analysis of proteoforms and top-down proteomics. Overall, the described transient-mediated approach to simulate FTMS data has proven useful for supporting contemporary FTMS applications. We also find its utility in fundamental FTMS studies and creating didactic materials for FTMS teaching.

Trace-Level Persistent Organic Pollutant Analysis with Gas-Chromatography Orbitrap Mass Spectrometry-Enhanced Performance by Complementary Acquisition and Processing of Time-Domain Data.

The range of commercial techniques for high-resolution gas-chromatography-mass spectrometry (GC-MS) has been recently extended with the introduction of GC Orbitrap Fourier transform mass spectrometry (FTMS). We report on progress with quantitation performance in the analysis of persistent organic pollutants (POP), by averaging of time-domain signals (transients), from a number of GC-FTMS experiment replicates. Compared to a standard GC-FTMS measurement (a single GC-FTMS experiment replicate, mass spectra representation in reduced profile mode), for the 10 GC-FTMS technical replicates of ultratrace POP analysis, sensitivity improvement of up to 1 order of magnitude is demonstrated. The accumulation method was implemented with an external high-performance data acquisition system and dedicated data processing software to acquire the time-domain data for each GC-FTMS replicate and to average the acquired GC-FTMS data sets. Concomitantly, the increased flexibility in ion signal detection allowed the attainment of ultrahigh-mass resolution (UHR), approaching R = 700 000 at m/z = 200.

Monitoring glycation levels of a bispecific monoclonal antibody at subunit level by ultrahigh-resolution MALDI FT-ICR mass spectrometry.

Bispecific monoclonal antibodies (BsAbs) are engineered proteins with multiple functionalities and properties. The "bi-specificity" of these complex biopharmaceuticals is a key characteristic for the development of novel and more effective therapeutic strategies. The high structural complexity of BsAbs poses a challenge to the analytical methods needed for their characterization. Modifications of the BsAb structure, resulting from enzymatic and non-enzymatic processes, further complicate the analysis. An important example of the latter type of modification is glycation, which can occur in the manufacturing process, during storage in the formulation or in vivo after application of the drug. Glycation affects the structure, function, and stability of monoclonal antibodies, and consequently, a detailed analysis of glycation levels is required. Mass spectrometry (MS) plays a key role in the structural characterization of monoclonal antibodies and top-down, middle-up and middle-down MS approaches are increasingly used for the analysis of modifications. Here, we apply a novel middle-up strategy, based on IdeS digestion and matrix-assisted laser desorption ionization (MALDI) Fourier transform ion cyclotron resonance (FT-ICR) MS, to analyze all six different BsAb subunits in a single high-resolution mass spectrum, namely two light chains, two half fragment crystallizable regions and two Fd' regions, thus avoiding upfront chromatography. This method was used to monitor glycation changes during a 168 h forced-glycation experiment. In addition, hot spot glycation sites were localized using top-down and middle-down MALDI-in-source decay FT-ICR MS, which provided complementary information compared to standard bottom-up MS.