Charge State Assignment Research Articles

Gábor Transform-Based Signal Isolation, Rapid Deconvolution, and Quantitation of Intact Protein Ions with Mass Spectrometry.

High-resolution mass spectrometry (HRMS) is a powerful technique for the characterization and quantitation of complex biological mixtures, with several applications including clinical monitoring and tissue imaging. However, these medical and pharmaceutical applications are pushing the analytical limits of modern HRMS techniques, requiring either further development in instrumentation or data processing methods. Here, we demonstrate new developments in the interactive Fourier-transform analysis for mass spectrometry (iFAMS) software including the first application of Gábor transform (GT) to protein quantitation. Newly added automation tools detect signals from minimal user input and apply thresholds for signal selection, deconvolution, and baseline correction to improve the objectivity and reproducibility of deconvolution. Additional tools were added to improve the deconvolution of highly complex or congested mass spectra and are demonstrated here for the first time. The "Gábor Slicer" enables the user to explore trends in the Gábor spectrogram with instantaneous ion mass estimates accurate to 10 Da. The charge adjuster allows for easy visual confirmation of accurate charge state assignments and quick adjustment if necessary. Deconvolution refinement utilizes a second GT of isotopically resolved data to remove common deconvolution artifacts. To assess the quality of deconvolution from iFAMS, several comparisons are made to deconvolutions using other algorithms such as UniDec and an implementation of MaxEnt in Agilent MassHunter BioConfirm. Lastly, the newly added batch processing and quantitation capabilities of iFAMS are demonstrated and compared to a common extracted ion chromatogram approach.

Approaches for handling high-dimensional cluster expansions of ionic systems

Disordered multicomponent systems attract great interest due to their engineering design flexibility and subsequent rich space of properties. However, detailed characterization of the structure and atomic correlations remains challenging and hinders full navigation of these complex spaces. A lattice cluster expansion is one tool to obtain configurational and energetic resolution. While in theory a cluster expansion can be applied to any system of any dimensionality, the method has primarily been used in binary systems or ternary alloys. Here we apply cluster expansions in high-component ionic systems, setting up the largest cluster expansion ever attempted to our knowledge. In doing so, we address and discuss challenges specific to high-component ionic systems, namely charge state assignments, structural relaxations, and rank-deficient systems. We introduce practical procedures to make the fitting and analysis of complex systems tractable, providing guidance for future computational studies of disordered ionic systems.

Multiply Charged Cation Attachment to Facilitate Mass Measurement in Negative-Mode Native Mass Spectrometry.

Native mass spectrometry (MS) is usually conducted in the positive-ion mode; however, in some cases, it is advantageous to use the negative-ion polarity. Challenges associated with native MS using ensemble measurements (i.e., the measurement of many ions at a time as opposed to the measurement of the charge and the mass-to-charge ratio of individual ions) include narrow charge state distributions with the potential for an overlap in neighboring charge states. These issues can either compromise or preclude confident charge state (and hence mass) determination. Charge state determination in challenging instances can be enabled via the attachment of multiply charged ions of opposite polarity. Multiply charged ion attachment facilitates the resolution of charge states and generates mass-to-charge (m/z) information across a broad m/z range. In this work, we demonstrated the attachment of multiply charged cations to anionic complexes generated under native MS conditions. To illustrate the flexibility available in selecting the mass and charge of the reagents, the 15+ and 20+ charge states of horse skeletal muscle apomyoglobin and the 20+ and 30+ charge states of bovine carbonic anhydrase were demonstrated to attach to model complex anions derived from either β-galactosidase or GroEL. The exclusive attachment of reagent ions is observed with no evidence for proton transfer, which is the key for the unambiguous interpretation of the post-ion/ion reaction product ion spectrum. To illustrate the application to mixtures of complex ions, the 10+ charge state of bovine ubiquitin was attached to mixtures of anions generated from the 30S and 50S particles of the Escherichia coli ribosome. Six and five major components were revealed, respectively. In the case of the 50S anion population, it was shown that the attachment of two 30+ cations of carbonic anhydrase revealed the same information as the attachment of six 10+ cations of ubiquitin. In neither case was the intact 50S particle observed. Rather, particles with different combinations of missing components were observed. This work demonstrated the utility of multiply charged cation attachment to facilitate charge state assignments in native MS ensemble measurements of heterogeneous mixtures.

Molecular weight determination of adeno-associate virus serotype 8 virus-like particle either carrying or lacking genome via native nES gas-phase electrophoretic molecular mobility analysis and nESI QRTOF mass spectrometry.

Virus‐like particles (VLPs) are proteinaceous shells derived from viruses lacking any viral genomic material. Adeno‐associated virus (AAV) is a non‐enveloped icosahedral virus used as VLP delivery system in gene therapy (GT). Its success as vehicle for GT is due to its selective tropism, high level of transduction, and low immunogenicity. In this study, two preparations of AAV serotype 8 (AAV8) VLPs either carrying or lacking completely genomic cargo (i.e., non‐viral ssDNA) have been investigated by means of a native nano‐electrospray gas‐phase electrophoretic mobility molecular analyzer (GEMMA) (native nES GEMMA) and native nano‐electrospray ionization quadrupole reflectron time‐of‐flight mass spectrometry (MS) (native nESI QRTOF MS). nES GEMMA is based on electrophoretic mobility principles: single‐charge nanoparticles (NPs), that is, AAV8 particle, are separated in a laminar sheath flow of dry, particle‐free air and a tunable orthogonal electric field. Thus, the electrophoretic mobility diameter (EMD) of a bio‐NP (i.e., diameter of globular nano‐objects) is obtained at atmospheric pressure, which can be converted into its MW based on a correlation. First is the native nESI QRTOF. MS's goal is to keep the native biological conformation of an analyte during the passage into the vacuum. Subsequently, highly accurate MW values are obtained from multiple‐charged species after deconvolution. However, once applied to the analysis of megadalton species, native MS is challenging and requires customized instrumental modifications not readily available on standard devices. Hence, the analysis of AAV8 VLPs via native MS in our hands did not produce a defined charge state assignment, that is, charge deconvolution for exact MW determination was not possible. Nonetheless, the method we present is capable to estimate the MW of VLPs by combining the results from native nES GEMMA and native ESI QRTOF MS. In detail, our findings show a MW of 3.7 and 5.0 MDa for AAV8 VLPs either lacking or carrying an engineered genome, respectively.

Mass Analysis of Macro-molecular Analytes via Multiply-Charged Ion Attachment.

A novel gas-phase charge and mass manipulation approach is demonstrated to facilitate the mass measurement of high mass complexes within the context of native mass spectrometry. Electrospray ionization applied to solutions generated under native or near-native conditions has been demonstrated to be capable of preserving biologically relevant complexes into the gas phase as multiply charged ions suitable for mass spectrometric analysis. However, charge state distributions tend to be narrow and extensive salt adduction, heterogeneity, and so on tend to lead to significantly broadened peaks. These issues can compromise mass measurement of high mass bio-complexes, particularly when charge states are not clearly resolved. In this work, we show that the attachment of high mass ions of known mass and charge to populations of ions of interest can lead to well-separated signals that can yield confident charge state and mass assignments from otherwise poorly resolved signals.

Ion trap operational modes for ion/ion reactions yielding high mass-to-charge product ions

To better probe large biomolecular complexes, developments in mass spectrometry (MS) have focused on improving technologies used to generate, transmit, and measure high m/z ions. The additional tandem-MS (MSn) capabilities of ion trap mass spectrometers (ITMS) facilitate experiments that facilitate probing complex biomolecular ions. In particular, charge reduction using gas-phase ion/ion reactions increase separation of charge states generated via electrospray ionization (ESI), which increases confidence in charge state assignments and therefore masses determined from the observed charge states. Current ITMS technologies struggle to generate and measure low charge states of large (>50 kDa) proteins and complexes because of power limitations associated with conventional high-frequency sine wave operation. Other approaches, including frequency scanning techniques and use of digital waveforms, reduce or eliminate some of these limitations. The work presented here studies five different operational modes for a quadrupole ion trap (QIT) mass spectrometer used to generate and measure low charge states of bovine serum albumin (BSA), pyruvate kinase (PK), and GroEL. While digital operation of a QIT presents limitations during the ion/ion reaction period of the experiment, it generally provided the best spectra in terms of resolution and signal at m/z > 50,000.

Individual Ion Mass Spectrometry Enhances the Sensitivity and Sequence Coverage of Top-Down Mass Spectrometry.

Charge detection mass spectrometry (CDMS) is mainly utilized to determine the mass of intact molecules. Previous applications of CDMS have determined the mass-to-charge ratio and the charge of large polymers, DNA molecules, and native protein complexes, from which corresponding mass values could be assigned. Recent advances have demonstrated that CDMS using an Orbitrap mass analyzer yields the reliable assignment of integer charge states that enables individual ion mass spectrometry (I2MS) and spectral output directly into the mass domain. Here I2MS analysis was extended to isotopically resolved fragment ions from intact proteoforms for the first time. With a radically different bias for ion readout, I2MS identified low-abundance fragment ions containing many hundreds of residues that were undetectable by standard Orbitrap measurements, leading to a doubling in the sequence coverage of triosephosphate isomerase. Thus MS/MS with the detection of individual ions (MS/I2MS) provides a far greater ability to detect high mass fragment ions and exhibits strong complementarity to traditional spectral readout in this, its first application to top-down mass spectrometry.

Information extraction from native mass spectra

Native MS spectra of large protein assemblies are typically characterized by low signal‐to‐noise ratios and broad peak shapes. Accurate peak detection is challenging due to additional issues such as high degrees of peak overlap and highly interleaved charge envelopes. A comprehensive algorithm has been developed to address peak identification, overlapping deconvolution, and charge state assignment in native mass spectra. Our open source software provides a novel and practical tool for those problems and is capable of extracting signals of large protein assemblies from heterogeneous, complex, and noisy native mass spectra.Nonspecifical binding of ligands to large protein complexes is commonly observed in native MS spectra. Here we also present a deconvolution model to discriminate specific bindings of a ligand to a multi‐protein complex target from the nonspecific interactions. Using the deconvolution algorithms with two level modeling steps, specific binding constant(s) can be effectively separated from the nonspecific ones. The nonspecific binding can be modeled by a simple power inverse function. An additional advantage of the method is that the target concentration can be more accurately obtained.Support or Funding InformationThis work was supported by grants from the National Institutes of Health (1S10OD016229, 8P41GM103481, GM49985, GM36659, AI‐21144, NS059690, GM109896, AG021601, AG002132, and AG010770).

Interactions of Intact Unfractionated Heparin with Its Client Proteins Can Be Probed Directly Using Native Electrospray Ionization Mass Spectrometry.

Heparin and related members of the glycosaminoglycan (GAG) family are highly polyanionic linear saccharides that play important roles in a variety of physiological processes ranging from blood coagulation to embryo- and oncogenesis, tissue regeneration, and immune response regulation. These diverse functions are executed via a variety of mechanisms, including protein sequestration, activation, and facilitation of their interactions with cell-surface receptors, but deciphering the specific molecular mechanisms is frequently impossible due to the extremely high degree of GAG heterogeneity. As a result, the vast majority of studies of heparin (or related GAGs) interactions with its client proteins use synthetically produced heparin mimetics with defined structure or short heparin fragments. In this work we use native electrospray ionization mass spectrometry (ESI MS) in combination with limited charge reduction in the gas phase to obtain meaningful information on noncovalent complexes formed by intact unfractionated heparin and antithrombin-III, interaction which is central to preventing blood clotting. Complexes of different stoichiometries are observed ranging from 1:1 to 1:3 (heparin/protein ratio). In addition to binding stoichiometry, the measurements allow the range of heparin chain lengths to be obtained for each complex and the contribution of each complex to the total ionic signal to be calculated. Incorporation of ion mobility measurements in the experimental workflow allows the total analysis time to be shortened very significantly and the charge state assignment for the charge-reduced species to be verified. The possibility to study interactions of intact unfractionated heparin with a client protein carried out directly by native ESI MS without the need to use relatively homogeneous surrogates demonstrated in this work opens up a host of new exciting opportunities and goes a long way toward ameliorating the persistent but outdated view of the intractability of such interactions.

Improved Peak Detection and Deconvolution of Native Electrospray Mass Spectra from Large Protein Complexes.

Native electrospray-ionization mass spectrometry (native MS) measures biomolecules under conditions that preserve most aspects of protein tertiary and quaternary structure, enabling direct characterization of large intact protein assemblies. However, native spectra derived from these assemblies are often partially obscured by low signal-to-noise as well as broad peak shapes because of residual solvation and adduction after the electrospray process. The wide peak widths together with the fact that sequential charge state series from highly charged ions are closely spaced means that native spectra containing multiple species often suffer from high degrees of peak overlap or else contain highly interleaved charge envelopes. This situation presents a challenge for peak detection, correct charge state and charge envelope assignment, and ultimately extraction of the relevant underlying mass values of the noncovalent assemblages being investigated. In this report, we describe a comprehensive algorithm developed for addressing peak detection, peak overlap, and charge state assignment in native mass spectra, called PeakSeeker. Overlapped peaks are detected by examination of the second derivative of the raw mass spectrum. Charge state distributions of the molecular species are determined by fitting linear combinations of charge envelopes to the overall experimental mass spectrum. This software is capable of deconvoluting heterogeneous, complex, and noisy native mass spectra of large protein assemblies as demonstrated by analysis of (1) synthetic mononucleosomes containing severely overlapping peaks, (2) an RNA polymerase II/α-amanitin complex with many closely interleaved ion signals, and (3) human TriC complex containing high levels of background noise. Graphical Abstract ᅟ.

Analysis of Native-Like Proteins and Protein Complexes Using Cation to Anion Proton Transfer Reactions (CAPTR).

Mass spectra of native-like protein complexes often exhibit narrow charge-state distributions, broad peaks, and contributions from multiple, coexisting species. These factors can make it challenging to interpret those spectra, particularly for mixtures with significant heterogeneity. Here we demonstrate the use of ion/ion proton transfer reactions to reduce the charge states of m/z-selected, native-like ions of proteins and protein complexes, a technique that we refer to as cation to anion proton transfer reactions (CAPTR). We then demonstrate that CAPTR can increase the accuracy of charge state assignments and the resolution of interfering species in native mass spectrometry. The CAPTR product ion spectra for pyruvate kinase exhibit ~30 peaks and enable unambiguous determination of the charge state of each peak, whereas the corresponding precursor spectra exhibit ~6 peaks and the assigned charge states have an uncertainty of ±3%. 15+ bovine serum albumin and 21+ yeast enolase dimer both appear near m/z 4450 and are completely unresolved in a mixture. After a single CAPTR event, the resulting product ions are baseline resolved. The separation of the product ions increases dramatically after each subsequent CAPTR event; 12 events resulted in a 3000-fold improvement in separation relative to the precursor ions. Finally, we introduce a framework for interpreting and predicting the figures of merit for CAPTR experiments. More generally, these results suggest that CAPTR strongly complements other mass spectrometry tools for analyzing proteins and protein complexes, particularly those in mixtures. Graphical Abstract ᅟ.

Photolytic determination of charge state for large proteins and fragments in an ion trap mass spectrometer.

One of the major shortcomings of linear ion trap mass spectrometers is poor resolution. Failure to resolve isotopic peaks makes charge state determination for large proteins very difficult, hindering the ability to perform top-down proteomics. Peptides, proteins and corresponding fragments modified with para-iodobenzoate were trapped and irradiated with 266 nm light from an Nd:YAG laser. Loss of iodine due to photodissociation was then used to assign charge states by measuring the corresponding m/z shifts. Initial experiments on small peptides illustrate the feasibility of the method. Further studies performed on larger proteins in higher charge states yielded similar results, revealing that fragment ions over a significant mass range either remain in or are quickly cooled to the laser overlap region of the ion trap. Rapid charge state assignment for both whole molecules and collision-induced dissociation (CID) fragments can be obtained by photoactivation of chromophores with labile carbon-iodine bonds.

A Remodeled Protein Arginine Methyltransferase 1 (PRMT1) Generates Symmetric Dimethylarginine

Protein arginine methylation is emerging as a significant post-translational modification involved in various cell processes and human diseases. As the major arginine methylation enzyme, protein arginine methyltransferase 1 (PRMT1) strictly generates monomethylarginine and asymmetric dimethylarginine (ADMA), but not symmetric dimethylarginine (SDMA). The two types of dimethylarginines can lead to distinct biological outputs, as highlighted in the PRMT-dependent epigenetic control of transcription. However, it remains unclear how PRMT1 product specificity is regulated. We discovered that a single amino acid mutation (Met-48 to Phe) in the PRMT1 active site enables PRMT1 to generate both ADMA and SDMA. Due to the limited amount of SDMA formed, we carried out quantum mechanical calculations to determine the free energies of activation of ADMA and SDMA synthesis. Our results indicate that the higher energy barrier of SDMA formation (ΔΔG(‡) = 3.2 kcal/mol as compared with ADMA) may explain the small amount of SDMA generated by M48F-PRMT1. Our study reveals unique energetic challenges for SDMA-forming methyltransferases and highlights the exquisite control of product formation by active site residues in the PRMTs.

Identification and quantification of poly(amidoamine) PAMAM dendrimers of generations 0 to 3 by liquid chromatography/hybrid quadrupole time‐of‐flight mass spectrometry in aqueous medium

Poly(amidoamine) PAMAM dendrimers are highly water soluble and are used as flexible scaffolding or nanocontainers to conjugate, complex or encapsulate therapeutic drugs to overcome intrinsically weak characteristics such as solubilization in aqueous medium. To provide a reliable method for the quantitation of PAMAM dendrimers in aqueous medium, we report here a validation study which was developed in a complex wastewater matrix to evaluate the matrix effect in the electrospray ionization (ESI) source. PAMAM dendrimers (generations G0 to G3) were identified and quantitated in aqueous medium using liquid chromatography interfaced to a hybrid quadrupole/time-of-flight mass analyzer. This approach used the high resolving power of isotopic clusters and mass accuracy of the instrument, with especial attention to the tandem mass spectrometric (MS/MS) capabilities. The formation of multiply charged ions of PAMAM dendrimers in the ESI source and their later fragmentation allowed fragmentation paths to be determined and structural assignments to be made. The analytical strategy allowed dendrimer identification with a high degree of confidence obtained by accurate mass and high resolution with mass errors below 5 ppm and 10 ppm in MS and MS/MS modes. The parameters of validation in spiked matrix were: limits of quantification in the range of 0.12 to 1.25 μM depending on the generation, linearity (R >0.996), repeatability (R.S.D. <6.7%) and reproducibility (R.S.D. <10.8%). Accurate mass measurement, elemental composition, and charge state assignment through the resolution of isotopic clusters of product and precursor ions, confers enhanced confidence on PAMAM dendrimer characterization. This selectivity provided high discriminating capacity of PAMAM dendrimers against matrix interferences. Because of the reliable and reproducible quantitation by LC/ESI-QTOF-MS, analysis of PAMAM dendrimers in an aqueous matrix is feasible.

Use of an Artificial Immune System Derived Method for the Charge State Assignment of Small-Molecule Mass Spectra

Knowing the charge state of an ion in a mass spectrum is crucial to being able to assign a formula to it. For many small-molecule peaks in complex mass spectra, the intensities of the isotopic peaks are too low to allow the charge state to be calculated from isotopic spacings, which is the basis of the conventional method of determining the charge state of an ion. A novel artificial intelligence derived method for identifying the charge state of ions, in the absence of any isotopic information or a series of charge states, has been developed using an artificial immune system approach. This technique has been tested against synthetic and real data sets and has proven successful in identifying the majority of multiply charged ions, thereby significantly improving the peak assignment rate and confidence.

Interpreting the Charge State Assignment in Electrospray Mass Spectra of Bioparticles

In electrospray ionization mass spectra of heterogeneous protein complexes and other bioparticles, accurate mass determination is often hampered by the inaccuracy in determination of the charge states for individual signals. Here, we describe an algorithm that automatically minimizes the standard deviation in a series of related ion peaks with varying numbers of charges. The algorithm assumes that the mass is invariant and allows the determination of the correct charge state in a peak series. The analysis results in a periodic pattern, which can be interpreted as a harmonic oscillator, when the minimum standard deviation of a charge state series is found. We observed that a mass resolution of much less than 1000 in the acquired mass spectra is sufficient to achieve a correct charge state assignment. Moreover, the boundaries of mixed species can be identified by examining the loss of periodicity in the pattern of the analysis. We tested our algorithm successfully on novel spectra and on spectra reported in the literature with sample masses up to several million Dalton, e.g., viral particles, polyethylene glycol polymers, and polystyrene nanoparticles.

Prediction of Drift Time in Ion Mobility-Mass Spectrometry Based on Peptide Molecular Weight

A computational model is introduced for predicting peptide drift time in ion mobility-mass spectrometry (IMMS). Each peptide was represented using a numeric descriptor: molecular weight. A simple linear regression predictor was constructed for peptides drift time prediction. Three datasets with different charge state assignments were used for the model training and testing. The dataset one contains 212 singly charged peptides, dataset two has 306 doubly charged peptides, and dataset three contains 77 triply charged peptides. Our proposed method achieved a prediction accuracy of 86.3%, 72.6%, and 59.7% for the dataset one, two and three, respectively. Peptide drift time prediction in IMMS will improve the confidence of peptide identifications by limiting the peptide search space during MS/MS database searching and therefore, reducing false discovery rate (FDR) of protein identification.

Electrospray Ionization Mass Spectrometry of Highly Heterogeneous Protein Systems: Protein Ion Charge State Assignment via Incomplete Charge Reduction

Correct mass and charge assignment for large highly heterogeneous macromolecular ions (e.g., large glycoproteins with significant carbohydrate content) presents a great challenge in native electrospray ionization mass spectrometry (ESI MS). A new approach to this problem combines complexity reduction (mass-selection of a narrow distribution of ionic species from a heterogeneous mixture) and gas-phase ion chemistry (electron-transfer reactions) to induce partial reduction of the ionic charge. The resulting spectra are devoid of complexity and are easy to interpret, leading to correct mass assignment. The new method is tested using several glycoproteins and their complexes, for which standard deconvolution approaches do not work.

Tandem Mass Spectrometry of Thiolate-Protected Au Nanoparticles NaxAu25(SC2H4Ph)18−y(S(C2H4O)5CH3)y

We report the first collision-induced dissociation tandem mass spectrometry (CID MS/MS) of a thiolate-protected Au nanoparticle that has a crystallographically determined structure. CID spectra assert that dissociation pathways for the mixed monolayer Na(x)Au(25)(SC(2)H(4)Ph)(18-y)(S(C(2)H(4)O)(5)CH(3))(y) centrally involve the semi-ring Au(2)L(3) coordination (L = some combination of the two thiolate ligands) that constitutes the nanoparticle's protecting structure. The data additionally confirm charge state assignments in the mass spectra. Prominent among the fragments is [Na(2)AuL(2)](1+), one precursor of which is identified as another nanoparticle fragment in the higher m/z region. Another detected fragment, [Na(2)Au(2)L(3)](1+), represents a mass loss equivalent to an entire semi-ring, whereas others suggest involvement (fragmentation/rearrangement) of multiple semi-rings, e.g., [NaAu(3)L(3)](1+) and [NaAu(4)L(4)](1+). The detailed dissociation/rearrangement mechanisms of these species are not established, but they are observed in other mass spectrometry experiments, including those under non-CID conditions, namely, electrospray ionization mass spectrometry (ESI-MS) with both time-of-flight (TOF) and FT-ICR analyzers. The latter, previously unreported results show that even soft ionization sources can result in Au nanoparticle fragmentation, including that yielding Au(4)L(4) in ESI-TOF of a much larger thiolate-protected Au(144) nanoparticle under non-CID conditions.

Prediction of peptide drift time in ion mobility-mass spectrometry

Background Understanding the proteome, the structure and function of each protein, and the interactions among proteins will give clues to search useful targets and biomarkers for pharmaceutical design. Peptide drift time prediction in IMMS will improve the confidence of peptide identification by limiting the peptide search space during MS/MS database searching and therefore reducing false discovery rate (FDR) of protein identification. A peptide drift time prediction method was proposed here using an artificial neural networks (ANN) regression model. We test our proposed model on three peptide datasets with different charge state assignment (see Table 1). The results can be found in Figure 1, where a higher prediction performance was achieved, over 0.9 for CI and C2, as well as 0.75 for C3.