Charge Detection Mass Spectrometry Research Articles

Coupling Online Size Exclusion Chromatography with Charge Detection-Mass Spectrometry Using Hadamard Transform Multiplexing.

Charge detection mass spectrometry (CD-MS) is a powerful technique for the analysis of large, heterogeneous biomolecules. By directly measuring the charge states of individual ions, CD-MS can measure the masses from spectra where conventional deconvolution approaches fail due to the lack of isotopic resolution or distinguishable charge states. However, CD-MS is inherently slow because hundreds or thousands of spectra need to be collected to produce adequate ion statistics. The slower speed of CD-MS complicates efforts to couple it with online separation techniques, which limit the number of spectra that can be acquired during a chromatographic peak. Here, we present the application of Hadamard transform multiplexing to online size exclusion chromatography (SEC) coupled with Orbitrap CD-MS, with a goal of using SEC for separating complex mixtures prior to CD-MS analysis. We developed a microcontroller to deliver pulsed injections from a large sample loop onto a SEC for online CD-MS analysis. Data showed a series of peaks spaced according to the pseudorandom injection sequence, which were demultiplexed with a Hadamard transform algorithm. The demultiplexed data revealed improved CD-MS signals while preserving retention time information. This multiplexing approach provides a general solution to the inherent incompatibilities of online separations and CD-MS detection that will enable a range of applications.

Multimass Analysis of Adeno-Associated Virus Vectors by Orbitrap-Based Charge Detection Mass Spectrometry.

Adeno-associated virus (AAV) vectors have attracted significant attention as the main platform for gene therapy. To ensure the safety and efficacy of AAV vectors when used as gene therapy drugs, it is essential to assess their critical quality attributes (CQAs). These CQAs include the genome packaging status, the size of the genome encapsidated within the AAV capsid, and the stoichiometry of viral proteins (VPs) that constitute the AAV capsids. Analytical methods have been established for evaluating CQAs, such as analytical ultracentrifugation, capillary gel electrophoresis with laser-induced fluorescence detection, and capillary gel electrophoresis using sodium dodecyl sulfate with UV detection. Here, we present a multimass analysis of AAV vectors using orbitrap-based charge detection mass spectrometry (CDMS), a single-ion mass spectrometry. Orbitrap-based CDMS facilitates the quantitative evaluation of the genome packaging status based on the mass distribution of empty and full particles. Additionally, we established a novel method to analyze the encapsidated genome directly without pretreatment, such as protein digestion or heat treatment, and to estimate the stoichiometric variation of VP for the capsid based on the mass distribution constituted by the single peak corresponding to AAV particles. Orbitrap-based CDMS is a distinctive method that allows multiple mass characterizations of AAV vectors with a small sample volume of 20 μL for 1013 cp/mL in a short time (30 min), and it holds the potential to become a new standard method in the assessment of CQAs for AAV vectors.

High-Throughput Single-Particle Characterization of Aggregation Pathways and the Effects of Inhibitors for Large (Megadalton) Protein Oligomers.

Protein aggregation is involved in many human diseases, but characterizing the sizes and shapes of intermediate oligomers (∼10-100 nm) that are important to the formation of macroscale aggregates like amyloid fibrils is a significant analytical challenge. Here, charge detection mass spectrometry (CDMS) is used to characterize individual conformational states of bovine serum albumin oligomers with up to ∼225 molecules (15 MDa). Elongated, partially folded, and globular conformational families for each oligomer can be readily distinguished based on the extent of charging. The abundances of individual conformers vary with changes in the monomer concentration or by adding aggregation inhibitors, such as SDS, heparin, or MgCl2. These results show the potential of CDMS for investigating intermediate oligomers in protein aggregation processes that are important for understanding aggregate formation and inhibition mechanisms and could accelerate formulation buffer development to prevent the aggregation of biotherapeutics.

UniChromCD for Demultiplexing Time-Resolved Charge Detection-Mass Spectrometry Data.

Charge detection mass spectrometry (CD-MS) enables characterization of large, heterogeneous analytes through the analysis of individual ion signals. Because hundreds to thousands of scans must be acquired to produce adequate ion statistics, CD-MS generally requires long analysis times. The slow acquisition speed of CD-MS complicates efforts to couple it with time-dispersive techniques, such as chromatography and ion mobility, because it is not always possible to acquire enough scans from a single sample injection to generate sufficient ion statistics. Multiplexing methods based on Hadamard and Fourier transforms offer an attractive solution to this problem by improving the duty cycle of the separation while preserving retention/drift time information. However, integrating multiplexing with CD-MS data processing is complex. Here, we present UniChromCD, a new module in the open-source UniDec package that incorporates CD-MS time-domain data processing with demultiplexing tools. Following a detailed description of the algorithm, we demonstrate its capabilities using two multiplexed CD-MS workflows: Hadamard-transform size-exclusion chromatography and Fourier-transform ion mobility. Overall, UniChromCD provides a user-friendly interface for analysis and visualization of time-resolved CD-MS data.

Protein Complex Heterogeneity and Topology Revealed by Electron Capture Charge Reduction and Surface Induced Dissociation.

We illustrate the utility of native mass spectrometry (nMS) combined with a fast, tunable gas-phase charge reduction, electron capture charge reduction (ECCR), for the characterization of protein complex topology and glycoprotein heterogeneity. ECCR efficiently reduces the charge states of tetradecameric GroEL, illustrating Orbitrap m/z measurements to greater than 100,000 m/z. For pentameric C-reactive protein and tetradecameric GroEL, our novel device combining ECCR with surface induced dissociation (SID) reduces the charge states and yields more topologically informative fragmentation. This is the first demonstration that ECCR yields more native-like SID fragmentation. ECCR also significantly improved mass and glycan heterogeneity measurements of heavily glycosylated SARS-CoV-2 spike protein trimer and thyroglobulin dimer. Protein glycosylation is important for structural and functional properties and plays essential roles in many biological processes. The immense heterogeneity in glycosylation sites and glycan structure poses significant analytical challenges that hinder a mechanistic understanding of the biological role of glycosylation. Without ECCR, average mass determination of glycoprotein complexes is available only through charge detection mass spectrometry or mass photometry. With narrow m/z selection windows followed by ECCR, multiple glycoform m/z values are apparent, providing quick global glycoform profiling and providing a future path for glycan localization on individual intact glycoforms.

Complementary Nanoparticle Characterization by Resistive-Pulse Sensing, Electron Microscopy, and Charge Detection Mass Spectrometry.

Nanotechnology has provided novel modalities for the delivery of therapeutic and diagnostic agents. In particular, nanoparticles (NPs) can be engineered at a low cost for drug loading and delivery. For example, silica NPs have proven useful as a controlled release platform for anti-inflammatory drugs. Despite the wide-ranging potential applications for NPs, robust characterization across all size ranges remains elusive. Electron microscopy (EM) is the conventional tool for measuring NP diameters. However, imitations in throughput and the inability to provide comprehensive information on physical properties, such as mass and density, without underlying assumptions, hinder a complete analysis. In addition, assessing sample heterogeneity, aggregation, or coalescence in solution by traditional EM analysis is not possible. Resistive-pulse sensing (RPS) provides a high throughput, solution-phase method for characterizing particle heterogeneity based on volume. Complementing these methods, charge detection mass spectrometry (CD-MS), a single particle technique, provides accurate mass information for heterogeneous samples including NPs. By combining EM, RPS and CD-MS, accurate volume, mass, and densities were obtained for silica NPs of various sizes. The results show that the density for 20 nm silica NPs is close to the density of fused silica (2.2 g/cm3). Larger silica NPs were found to have densities that were either smaller or larger, while also falling outside the range of densities usually found for silica colloids and NPs (1.9-2.3 g/cm3). Lower densities are attributed to pores (i.e., porous particles). For one sample, the mass distribution showed two components attributed to two populations of particles in the sample with different densities. The synergistic combination of EM, RPS, and CD-MS measurements outlined here for NP samples, allows much more extensive information to be obtained than from any of the techniques alone.

Charge Detection Mass Spectrometry Enables Molecular Characterization of Nucleic Acid Nanoparticles.

Nucleic acid nanoparticles (NANPs) are increasingly used in preclinical investigations as delivery vectors. Tools that can characterize assembly and assess quality will accelerate their development and clinical translation. Standard techniques used to characterize NANPs, like gel electrophoresis, lack the resolution for precise characterization. Here, we introduce the use of charge detection mass spectrometry (CD-MS) to characterize these materials. Using this technique, we determined the mass of NANPs varying in size, shape, and molecular mass, NANPs varying in production quality due to formulations lacking component oligonucleotides, and NANPs functionalized with protein and nucleic acid-based secondary molecules. Based on these demonstrations, CD-MS is a promising tool to precisely characterize NANPs, enabling more precise assessments of the manufacturing and processing of these materials.

Realization of Higher Resolution Charge Detection Mass Spectrometry.

Charge detection mass spectrometry (CD-MS) allows mass distributions to be measured for heterogeneous samples that cannot be analyzed by conventional MS. With CD-MS, the m/z and charge are measured for individual ions using a detection cylinder embedded in an electrostatic linear ion trap (ELIT). Imprecision in both the m/z and charge measurements contribute to the mass resolution. However, if the charge can be measured with a precision of <0.2 e the charge state can be assigned with a low error rate and the mass resolving power only depends on the m/z resolution. Prior to this work, the best resolving power demonstrated experimentally for CD-MS was 700. Here we demonstrate a resolving power of >14,600, 20-times higher than the previous best. Trajectory simulations were used to optimize the geometry and electrostatic potentials of the ELIT. We found conditions where the energy dependence of the oscillation frequency becomes parabolic, and then operated with a nominal ion energy at the minimum of the parabola. The 14,600 resolving power was obtained with a beam collimator before the ELIT. With the collimator removed, the resolving power of the optimized ELIT is 7300, which is still an order of magnitude higher than the previous best. The resolving power was demonstrated by resolving the isotope distributions for peptides and proteins. High resolution CD-MS measurements were then used to resolve the glycans on a monoclonal antibody and applied to the analysis of hepatitis B virus capsids. The results indicate that procedures for adduct removal need to be improved for the full benefit of the higher resolving power to be realized for higher mass species. However, these results represent a key step toward using CD-MS to analyze very complex protein mixtures where charge states are not well resolved in the m/z spectrum because of congestion from numerous overlapping peaks.

Automated Immunoprecipitation, Sample Preparation, and Individual Ion Mass Spectrometry Platform for Proteoforms.

Charge detection mass spectrometry (CDMS) is a well-established technique that provides direct mass spectral outputs regardless of analyte heterogeneity or molecular weight. Over the past few years, it has been demonstrated that CDMS can be multiplexed on Orbitrap analyzers utilizing an integrated approach termed individual ion mass spectrometry (I2MS). To further increase adaptability, robustness, and throughput of this technique, here, we present a method that utilizes numerous integrated equipment components including a Kingfisher system, SampleStream platform, and Q Exactive mass spectrometer to provide a fully automated workflow for immunoprecipitation, sample preparation, injection, and subsequent I2MS acquisition. This automated workflow has been applied to a cohort of 58 test subjects to determine individualized patient antibody responses to SARS-CoV-2 antigens. Results from a range of serum donors include 37 subject I2MS spectra that contained a positive COVID-19 antibody response and 21 I2MS spectra that contained a negative COVID-19 antibody response. This high-throughput automated I2MS workflow can currently process over 100 samples per week and is general for making immunoprecipitation-MS workflows achieve proteoform resolution.

Charge Detection Mass Spectrometry Reveals Favored Structures in the Assembly of Virus-Like Particles: Polymorphism in Norovirus GI.1.

The main capsid protein (CP) of norovirus, the leading cause of gastroenteritis, is expected to self-assemble into virus-like particles with the same structure as the wild-type virus, a capsid with 180 CPs in a T = 3 icosahedron. Using charge detection mass spectrometry (CD-MS), we find that the norovirus GI.1 variant is structurally promiscuous, forming a wide variety of well-defined structures, some that are icosahedral capsids and others that are not. The structures that are present evolve with time and vary with solution conditions. The presence of icosahedral T = 3 and T = 4 capsids (240 CPs) under some conditions was confirmed by cryo-electron microscopy (cryo-EM). The cryo-EM studies also confirmed the presence of an unexpected prolate geometry based on an elongated T = 4 capsid with 300 CPs. In addition, CD-MS measurements indicate the presence of well-defined peaks with masses corresponding to 420, 480, 600, and 700 CPs. The peak corresponding to 420 CPs is probably due to an icosahedral T = 7 capsid, but this could not be confirmed by cryo-EM. It is possible that the T = 7 particles are too fragile to survive vitrification. There are no mass peaks associated with the T = 9 and T = 12 icosahedra with 540 and 720 CPs. The larger structures with 480, 600, and 700 CPs are not icosahedral; however, their measured charges suggest that they are hollow shells. The use of CD-MS to monitor virus-like particles assembly may have important applications in vaccine development and quality control.

Charge Detection Mass Spectrometry Reveals Conformational Heterogeneity in Megadalton-Sized Monoclonal Antibody Aggregates.

Aggregation of protein-based therapeutics can occur during development, production, or storage and can lead to loss of efficacy and potential toxicity. Native mass spectrometry of a covalently linked pentameric monoclonal antibody complex with a mass of ∼800 kDa reveals several distinct conformations, smaller complexes, and abundant higher-order aggregates of the pentameric species. Charge detection mass spectrometry (CDMS) reveals individual oligomers up to the pentamer mAb trimer (15 individual mAb molecules; ∼2.4 MDa) whereas intermediate aggregates composed of 6-9 mAb molecules and aggregates larger than the pentameric dimer (1.6 MDa) were not detected/resolved by standard mass spectrometry, size exclusion chromatography (SEC), capillary electrophoresis (CE-SDS), or by mass photometry. Conventional quadrupole time-of-flight mass spectrometry (QTOF MS), mass photometry, SEC, and CE-SDS did not resolve partially or more fully unfolded conformations of each oligomer that were readily identified using CDMS by their significantly higher extents of charging. Trends in the charge-state distributions of individual oligomers provides detailed insight into how the structures of compact and elongated mAb aggregates change as a function of aggregate size. These results demonstrate the advantages of CDMS for obtaining accurate masses and information about the conformations of large antibody aggregates despite extensive overlapping m/z values. These results open up the ability to investigate structural changes that occur in small, soluble oligomers during the earliest stages of aggregation for antibodies or other proteins.

Thermal Remodeling of Human HDL Particles Reveals Diverse Subspecies.

High-density lipoproteins (HDL) are micelle-like particles consisting of a core of triglycerides and cholesteryl esters surrounded by a shell of phospholipid, cholesterol, and apolipoproteins. HDL is considered "good" cholesterol, and its concentration in plasma is used clinically in assessing cardiovascular health. However, these particles vary in structure, composition, and therefore function, and thus can be resolved into subpopulations, some of which have specific cardioprotective properties. Mass measurements of HDL by charge detection mass spectrometry (CD-MS) previously revealed seven distinct subpopulations which could be delineated by mass and charge [Lutomski, C. A. et al. Anal. Chem. 2018]. Here, we investigate the thermal stabilities of these subpopulations; upon heating, the particles within each subpopulation undergo structural rearrangements with distinct transition temperatures. In addition, we find evidence for many new families of structures within each subpopulation; at least 15 subspecies of HDL are resolved. These subspecies vary in size, charge, and thermal stability. While this suggests that these new subspecies have unique molecular compositions, we cannot rule out the possibility that we have found evidence for new structural forms within the known subpopulations. The ability to resolve new subspecies of HDL particles may be important in understanding and delineating the role of unique particles in cardiovascular health and disease.

Protein complex heterogeneity and topology revealed by electron capture charge reduction and surface induced dissociation.

We illustrate the utility of native mass spectrometry (nMS) combined with a fast, tunable gas-phase charge reduction, electron capture charge reduction (ECCR), for the characterization of protein complex topology and glycoprotein heterogeneity. ECCR efficiently reduces the charge states of tetradecameric GroEL, illustrating Orbitrap m/z measurements to greater than 100,000 m/z. For pentameric C-reactive protein and tetradecameric GroEL, our novel device combining ECCR with surface induced dissociation (SID) reduces the charge states and yields more topologically informative fragmentation. This is the first demonstration that ECCR yields more native-like SID fragmentation. ECCR also significantly improved mass and glycan heterogeneity measurements of heavily glycosylated SARS-CoV-2 spike protein trimer and thyroglobulin dimer. Protein glycosylation is important for structural and functional properties and plays essential roles in many biological processes. The immense heterogeneity in glycosylation sites and glycan structure poses significant analytical challenges that hinder a mechanistic understanding of the biological role of glycosylation. Without ECCR, average mass determination of glycoprotein complexes is available only through charge detection mass spectrometry or mass photometry. With narrow m/z selection windows followed by ECCR, multiple glycoform m/z values are apparent, providing quick global glycoform profiling and providing a future path for glycan localization on individual intact glycoforms.

Compaction, Relaxation, and Linearization of Megadalton-Sized DNA Plasmids: DNA Structures Probed by CD-MS.

High purity plasmid DNA is a raw material for recombinant protein production as well as an active ingredient in DNA vaccines. There are four primary plasmid structures that can be observed in a typical plasmid formulation: supercoiled, relaxed (circular), linearized, and condensed. Determining what structures are present in a sample is important, as the structure can affect activity; the supercoiled structure has the highest activity, and >90% supercoiled is desired for industry standards. Recently, charge detection mass spectrometry (CD-MS) was used to distinguish two of the structures, supercoiled and condensed, by measuring the charge deposited on the ions by positive mode electrospray. Here, CD-MS is used to probe the structures of DNA plasmids during compaction with polycations, and through enzymatic treatment to relax and linearize plasmids. We find that all four structural types for plasmid DNA have unique charging profiles that can be distinguished using CD-MS. The extent of mechanical shearing of the DNA plasmids during electrospray is strongly influenced by the structural type.

Submicrometer Particle Impact Dynamics and Chemistry.

Experimental studies of the collision phenomena of submicrometer particles is a developing field. This review examines the range of phenomena that can be observed with new experimental approaches. The primary focus is on single-particle impact studies enabled by charge detection mass spectrometry (CDMS) implemented using the Aerosol Impact Spectrometer (AIS) at the University of California, San Diego. The AIS combines electrospray ionization, aerodynamic lens techniques, CDMS, and an electrostatic linear accelerator to study the dynamics of particle impact over a wide range of incident velocities. The AIS has been used for single-particle impact experiments on positively charged particles of diverse composition, including polystyrene latex spheres, tin particles, and ice grains, over a wide range of impact velocities. Detection schemes based on induced charge measurements and time-of-flight mass spectrometry have enabled measurements of the impact inelasticity through the determination of the coefficient of restitution, measurements of the angular distributions of scattered submicrometer particles, and the chemical composition and dissociation of solute molecules in hypervelocity ice grain impacts.

Characterization of Mass, Diameter, Density, and Surface Properties of Colloidal Nanoparticles Enabled by Charge Detection Mass Spectrometry.

A variety of scattering-based, microscopy-based, and mobility-based methods are frequently used to probe the size distributions of colloidal nanoparticles with transmission electron microscopy (TEM) often considered to be the "gold standard". Charge detection mass spectrometry (CDMS) is an alternative method for nanoparticle characterization that can rapidly measure the mass and charge of individual nanoparticle ions with high accuracy. Two low polydispersity, ∼100 nm diameter nanoparticle size standards with different compositions (polymethyl methacrylate/polystyrene copolymer and 100% polystyrene) were characterized using both TEM and CDMS to explore the merits and complementary aspects of both methods. Mass and diameter distributions are rapidly obtained from CDMS measurements of thousands of individual ions of known spherical shape, requiring less time than TEM sample preparation and image analysis. TEM image-to-image variations resulted in a ∼1-2 nm range in the determined mean diameters whereas the CDMS mass precision of ∼1% in these experiments leads to a diameter uncertainty of just 0.3 nm. For the 100% polystyrene nanoparticles with known density, the CDMS and TEM particle diameter distributions were in excellent agreement. For the copolymer nanoparticles with unknown density, the diameter from TEM measurements combined with the mass from CDMS measurements enabled an accurate measurement of nanoparticle density. Differing extents of charging for the two nanoparticle standards measured by CDMS show that charging is sensitive to nanoparticle surface properties. A mixture of the two samples was separated based on their different extents of charging despite having overlapping mass distributions centered at 341.5 and 331.0 MDa.

Higher-order structure and proteoforms of co-occurring C4b-binding protein assemblies in human serum

The complement is a conserved cascade that plays a central role in the innate immune system. To maintain a delicate equilibrium preventing excessive complement activation, complement inhibitors are essential. One of the major fluid-phase complement inhibitors is C4b-binding protein (C4BP). Human C4BP is a macromolecular glycoprotein composed of two distinct subunits, C4BPα and C4BPβ. These associate with vitamin K-dependent protein S (ProS) forming an ensemble of co-occurring higher-order structures. Here, we characterize these C4BP assemblies. We resolve and quantify isoforms of purified human serum C4BP using distinct single-particle detection techniques: charge detection mass spectrometry, and mass photometry accompanied by high-speed atomic force microscopy. Combining cross-linking mass spectrometry, glycoproteomics, and structural modeling, we report comprehensive glycoproteoform profiles and full-length structural models of the endogenous C4BP assemblies, expanding knowledge of this key complement inhibitor’s structure and composition. Finally, we reveal that an increased C4BPα to C4BPβ ratio coincides with elevated C-reactive protein levels in patient plasma samples. This observation highlights C4BP isoform variation and affirms a distinct role of co-occurring C4BP assemblies upon acute phase inflammation.

Not All Arms of IgM Are Equal: Following Hinge-Directed Cleavage by Online Native SEC-Orbitrap-Based CDMS.

Immunoglobulins M (IgM) are key natural antibodies produced initially in humoral immune response. Due to their large molecular weights and extensive glycosylation loads, IgMs represent a challenging target for conventional mass analysis. Charge detection mass spectrometry (CDMS) may provide a unique approach to tackle heterogeneous IgM assemblies, although this technique can be quite laborious and technically challenging. Here, we describe the use of online size exclusion chromatography (SEC) to automate buffer exchange and sample introduction, and demonstrate its adaptability with Orbitrap-based CDMS. We discuss optimal experimental parameters for online SEC-CDMS experiments, including ion activation, choice of column, and resolution. Using this approach, CDMS histograms containing hundreds of individual ion signals can be obtained in as little as 5 min from single injections of <1 μg of sample. To demonstrate the unique utility of online SEC-CDMS, we performed real-time kinetic monitoring of pentameric IgM digestion by the protease IgMBRAZOR, which cleaves specifically in the hinge region of IgM. Several digestion intermediates corresponding to processive losses of F(ab')2 subunits could be mass-resolved and identified by SEC-CDMS. Interestingly, we find that for the J-chain linked IgM pentamer, cleavage of one of the F(ab')2 subunits is much slower than the other four F(ab')2 subunits, which we attribute to the symmetry-breaking interactions of the J-chain within the pentameric IgM structure. The online SEC-CDMS methodologies described here open new avenues into the higher throughput automated analysis of heterogeneous, high-mass protein assemblies by CDMS.

Point mutation in a virus-like capsid drives symmetry reduction to form tetrahedral cages

Protein capsids are a widespread form of compartmentalization in nature. Icosahedral symmetry is ubiquitous in capsids derived from spherical viruses, as this geometry maximizes the internal volume that can be enclosed within. Despite the strong preference for icosahedral symmetry, we show that simple point mutations in a virus-like capsid can drive the assembly of unique symmetry-reduced structures. Starting with the encapsulin from Myxococcus xanthus, a 180-mer bacterial capsid that adopts the well-studied viral HK97 fold, we use mass photometry and native charge detection mass spectrometry to identify a triple histidine point mutant that forms smaller dimorphic assemblies. Using cryoelectron microscopy, we determine the structures of a precedented 60-mer icosahedral assembly and an unexpected 36-mer tetrahedron that features significant geometric rearrangements around a new interaction surface between capsid protomers. We subsequently find that the tetrahedral assembly can be generated by triple-point mutation to various amino acids and that even a single histidine point mutation is sufficient to form tetrahedra. These findings represent a unique example of tetrahedral geometry when surveying all characterized encapsulins, HK97-like capsids, or indeed any virus-derived capsids reported in the Protein Data Bank, revealing the surprising plasticity of capsid self-assembly that can be accessed through minimal changes in the protein sequence.

Charge Detection Mass Spectrometry for Megadalton Polymer Characterization and Measurement of Electrospray-Generated Charged Droplet Dynamics with a New "Direct Visualization of the Rayleigh Limit" Approach to Aid in m/z Calibration.

A charge detector has been constructed and mounted inside the vacuum housing of a commercial mass spectrometer (Micromass-Waters Quattro I, Waters Corp., Manchester, UK). The in-house built single-pass charge detector is composed of a designed, complete electronics system that includes a low-noise charge amplifier. Communication to the data acquisition system was enabled, and analog and digital filters were devised, followed by their tuning and programming. Data treatment scripts for data analysis and plotting were automated, and the assembled system was calibrated and tested. The instrument has an acquisition speed of ∼200 detection events/s, and it permits detection down to ∼510 charges (= three times RMS noise) for a single measured particle. The charge detector was employed to determine the oligomer distribution of a megadalton polymer, polyethylene glycol (PEG). The PEG size distribution exhibits a maximum at ∼ m/z 5910 with the oligomeric population mass distribution peaking near 4.45 MDa. In studies of methanol droplet dynamics, "charge vs time-of-flight" plots enabled clear visualization of the zone near the Rayleigh limit to droplet charging. The highest population of methanol droplets near the Rayleigh limit carried 5000-7000 charges. This corresponds to droplet weights of 10-20 GDa, with the high-end tail extending above 70 GDa. This visualization of the most highly charged droplets (that bear numbers of charges near those defined by the Rayleigh equation) was exploited as a calibration aid for our charge detector, which lacks a means of precisely defining ion energy. A maximum m/z error of -12.3% was calculated for the method, i.e., less than the potential error in assigning the true level of charging of the most highly charged droplets relative to the Rayleigh limit. With these limitations in mind, the introduced method will provide a new means for aiding the calibration of m/z values in charge detectors.