Improved Protein and PTM Characterization with a Practical Electron-Based Fragmentation on Q-TOF Instruments.

Electron-based dissociation (ExD) produces uncluttered mass spectra of intact proteins while preserving labile post-translational modifications. However, technical challenges have limited this option to only a few high-end mass spectrometers. We have developed an efficient ExD cell that can be retrofitted in less than an hour into current LC/Q-TOF instruments. Supporting software has been developed to acquire, process, and annotate peptide and protein ExD fragmentation spectra. In addition to producing complementary fragmentation, ExD spectra enable many isobaric leucine/isoleucine and isoaspartate/aspartate pairs to be distinguished by side-chain fragmentation. The ExD cell preserves phosphorylation and glycosylation modifications. It also fragments longer peptides more efficiently to reveal signaling cross-talk between multiple post-translational modifications on the same protein chain and cleaves disulfide bonds in cystine knotted proteins and intact antibodies. The ability of the ExD cell to combine collisional activation with electron fragmentation enables more complete sequence coverage by disrupting intramolecular electrostatic interactions that can hold fragments of large peptides and proteins together. These enhanced capabilities made possible by the ExD cell expand the size of peptides and proteins that can be analyzed as well as the analytical certainty of characterizing their post-translational modifications.

High-Resolution Ion-Mobility-Enabled Peptide Mapping for High-Throughput Critical Quality Attribute Monitoring.

Characterization and monitoring of post-translational modifications (PTMs) by peptide mapping is a ubiquitous assay in biopharmaceutical characterization. Often, this assay is coupled to reversed-phase liquid chromatographic (LC) separations that require long gradients to identify all components of the protein digest and resolve critical modifications for relative quantitation. Incorporating ion mobility (IM) as an orthogonal separation that relies on peptide structure can supplement the LC separation by providing an additional differentiation filter to resolve isobaric peptides, potentially reducing ambiguity in identification through mobility-aligned fragmentation and helping to reduce the run time of peptide mapping assays. A next-generation high-resolution ion mobility (HRIM) technique, based on structures for lossless ion manipulations (SLIM) technology with a 13 m ion path, provides peak capacities and higher resolving power that rivals traditional chromatographic separations and, owing to its ability to resolve isobaric peptides that coelute in faster chromatographic methods, allows for up to 3× shorter run times than conventional peptide mapping methods. In this study, the NIST monoclonal antibody IgG1κ (NIST RM 8671, NISTmAb) was characterized by LC-HRIM-MS and LC-HRIM-MS with collision-induced dissociation (HRIM-CID-MS) using a 20 min analytical method. This approach delivered a sequence coverage of 96.5%. LC-HRIM-CID-MS experiments provided additional confidence in sequence determination. HRIM-MS resolved critical oxidations, deamidations, and isomerizations that coelute with their native counterparts in the chromatographic dimension. Finally, quantitative measurements of % modification were made using only the m/z-extracted HRIM arrival time distributions, showing good agreement with the reference liquid-phase separation. This study shows, for the first time, the analytical capability of HRIM using SLIM technology for enhancing peptide mapping workflows relevant to biopharmaceutical characterization.

Rapid screening methods for yeast sub-metabolome analysis with a high-resolution ion mobility quadrupole time-of-flight mass spectrometer.

RATIONALE: The wide chemical diversity and complex matrices inherent to metabolomics still pose a challenge to current analytical approaches for metabolite screening. Although dedicated front-end separation techniques combined with high-resolution mass spectrometry set the benchmark from an analytical point of view, the increasing number of samples and sample complexity demand for a compromise in terms of selectivity, sensitivity and high-throughput analyses. METHODS: Prior to low-field drift tube ion mobility (IM) separation and quadrupole time-of-flight mass spectrometry (QTOFMS) detection, rapid ultrahigh-performance liquid chromatography separation was used for analysis of different concentration levels of dansylated metabolites present in a yeast cell extract. For identity confirmation of metabolites at the MS2 level, an alternating frame approach was chosen and two different strategies were tested: a data-independent all-ions acquisition and a quadrupole broad band isolation (Q-BBI) directed by IM drift separation. RESULTS: For Q-BBI analysis, the broad mass range isolation was successfully optimized in accordance with the distinctive drift time to m/z correlation of the dansyl derivatives. To guarantee comprehensive sampling, a broad mass isolation window of 70 Da was employed. Fragmentation was performed via collision-induced dissociation, applying a collision energy ramp optimized for the dansyl derivatives. Both approaches were studied in terms of linear dynamic range and repeatability employing ethanolic extracts of Pichia pastoris spiked with 1 µM metabolite mixture. Example data obtained for histidine and glycine showed that drift time precision (<0.01 to 0.3% RSD, n = 5) compared very well with the data reported in an earlier IM-TOFMS-based study. CONCLUSIONS: Chimeric mass spectra, inherent to data-independent analysis approaches, are reduced when using a drift time directed Q-BBI approach. Additionally, an improved linear dynamic working range was observed, representing, together with a rapid front-end separation, a powerful approach for metabolite screening.

SPE-IMS-MS: An automated platform for sub-sixty second surveillance of endogenous metabolites and xenobiotics in biofluids.

Characterization of endogenous metabolites and xenobiotics is essential to deconvoluting the genetic and environmental causes of disease. However, surveillance of chemical exposure and disease-related changes in large cohorts requires an analytical platform that offers rapid measurement, high sensitivity, efficient separation, broad dynamic range, and application to an expansive chemical space. Here, we present a novel platform for small molecule analyses that addresses these requirements by combining solid-phase extraction with ion mobility spectrometry and mass spectrometry (SPE-IMS-MS). This platform is capable of performing both targeted and global measurements of endogenous metabolites and xenobiotics in human biofluids with high reproducibility (CV 6 3%), sensitivity (LODs in the pM range in biofluids) and throughput (10-s sample-to-sample duty cycle). We report application of this platform to the analysis of human urine from patients with and without type 1 diabetes, where we observed statistically significant variations in the concentration of disaccharides and previously unreported chemical isomers. This SPE-IMS-MS platform overcomes many of the current challenges of large-scale metabolomic and exposomic analyses and offers a viable option for population and patient cohort screening in an effort to gain insights into disease processes and human environmental chemical exposure.

Broadscale resolving power performance of a high precision uniform field ion mobility-mass spectrometer.

An extensive study of two current ion mobility resolving power theories ("conditional" and "semi-empirical") was undertaken using a recently developed drift tube ion mobility-mass spectrometer. The current study investigates the quantitative agreement between experiment and theory at reduced pressure (4 Torr) for a wide range of initial ion gate widths (100 to 500 µs), and ion mobility values (K0 from 0.50 to 3.0 cm(2) V(-1) s(-1)) representing measurements obtained in helium, nitrogen, and carbon dioxide drift gas. Results suggest that the conditional resolving power theory deviates from experimental results for low mobility ions (e.g., high mass analytes) and for initial ion gate widths beyond 200 µs. A semi-empirical resolving power theory provided close-correlation of predicted resolving powers to experimental results across the full range of mobilities and gate widths investigated. Interpreting the results from the semi-empirical theory, the performance of the current instrumentation was found to be highly linear for a wide range of analytes, with optimal resolving powers being accessible for a narrow range of drift fields between 14 and 17 V cm(-1). While developed using singly-charged ion mobility data, preliminary results suggest that the semi-empirical theory has broader applicability to higher-charge state systems.

Evaluation of drift gas selection in complex sample analyses using a high performance drift tube ion mobility-QTOF mass spectrometer.

A recently developed uniform-field high resolution ion mobility (IM) quadrupole time of flight (Q-TOF) mass spectrometer is used for evaluating the utility of alternate drift gases for complex sample analyses. This study provides collision cross section comparison for 275 total pesticides including structural isomers in nitrogen, helium, carbon dioxide, nitrous oxide and sulfur hexafluoride drift gases. Furthermore, a set of small molecules and Agilent tune mix compounds were used to study the trends in experimentally derived collision cross section values in argon and the alternate drift gases. Two isomeric trisaccharides, melezitose and raffinose, were used to evaluate the effect of the drift gasses for mobility separation. The hybrid ion mobility Q-TOF mass analyzer used in this study consists of a low pressure uniform field drift tube apparatus coupled to a high resolution Q-TOF mass spectrometer. Conventionally, low pressure ion mobility instruments are operated using helium drift gas to obtain optimal structural information and collision cross-section (CCS) values that compare to theoretical CCS values. The instrument employed in this study uses nitrogen as the standard drift gas but also allows the utility of alternate drift gases for improved structural analysis and selectivity under certain conditions. The use of alternate drift gases with a wide range of polarizabilities allows the evaluation of mobility separation power in terms of induced dipole interactions between the drift gas and the analyte ions.

Conformational ordering of biomolecules in the gas phase: nitrogen collision cross sections measured on a prototype high resolution drift tube ion mobility-mass spectrometer.

Ion mobility-mass spectrometry measurements which describe the gas-phase scaling of molecular size and mass are of both fundamental and pragmatic utility. Fundamentally, such measurements expand our understanding of intrinsic intramolecular folding forces in the absence of solvent. Practically, reproducible transport properties, such as gas-phase collision cross-section (CCS), are analytically useful metrics for identification and characterization purposes. Here, we report 594 CCS values obtained in nitrogen drift gas on an electrostatic drift tube ion mobility-mass spectrometry (IM-MS) instrument. The instrument platform is a newly developed prototype incorporating a uniform-field drift tube bracketed by electrodynamic ion funnels and coupled to a high resolution quadrupole time-of-flight mass spectrometer. The CCS values reported here are of high experimental precision (±0.5% or better) and represent four chemically distinct classes of molecules (quaternary ammonium salts, lipids, peptides, and carbohydrates), which enables structural comparisons to be made between molecules of different chemical compositions for the rapid "omni-omic" characterization of complex biological samples. Comparisons made between helium and nitrogen-derived CCS measurements demonstrate that nitrogen CCS values are systematically larger than helium values; however, general separation trends between chemical classes are retained regardless of the drift gas. These results underscore that, for the highest CCS accuracy, care must be exercised when utilizing helium-derived CCS values to calibrate measurements obtained in nitrogen, as is the common practice in the field.

Front-end electron transfer dissociation: a new ionization source.

Electron transfer dissociation (ETD), a technique that provides efficient fragmentation while depositing little energy into vibrational modes, has been widely integrated into proteomics workflows. Current implementations of this technique, as well as other ion-ion reactions like proton transfer, involve sophisticated hardware, lack robustness, and place severe design limitations on the instruments to which they are attached. Described herein is a novel, electrical discharge-based reagent ion source that is located in the first differentially pumped region of the mass spectrometer. The reagent source was found to produce intense reagent ion signals over extended periods of time while having no measurable impact on precursor ion signal. Further, the source is simple to construct and enables implementation of ETD on any instrument without modification to footprint. Finally, in the context of hybrid mass spectrometers, relocation of the reagent ion source to the front of the mass spectrometer enables new approaches to gas phase interrogation of intact proteins.

Basic vapor exposure for tuning the charge state distribution of proteins in negative electrospray ionization: elucidation of mechanisms by fluorescence spectroscopy.

Manipulation for simplifying or increasing the observed charge state distributions of proteins can be highly desirable in mass spectrometry experiments. In the present work, we implemented a vapor introduction technique to an Agilent Jet Stream ESI (Agilent Technologies, Santa Clara, CA, USA) source. An apparatus was designed to allow for the enrichment of the nitrogen sheath gas with basic vapors. An optical setup, using laser-induced fluorescence and a pH-chromic dye, permits the pH profiling of the droplets as they evaporate in the electrospray plume. Mechanisms of pH droplet modification and its effect on the protein charging phenomenon are elucidated. An important finding is that the enrichment with basic vapors of the nitrogen sheath gas, which surrounds the nebulizer spray, leads to an increase in the spray current. This is attributed to an increase in the electrical conductivity of water-amine enriched solvent at the tip exit. Here, the increased current results in a generation of additional electrolytically produced OH(-) ions and a corresponding increase in the pH at the tip exit. Along the electrospray plume, the pH of the droplets increases due to both droplet evaporation and exposure to basic vapors from the seeded sheath gas. The pH evolution in the ESI plume obtained using pure and basic seeded sheath gas was correlated with the evolution of the charge state distribution observed in mass spectra of proteins, in the negative ion mode. Taking advantage of the Agilent Jet Stream source geometry, similar protein charge state distributions and ion intensities obtained with basic initial solutions, can be obtained using native solution conditions by seeding the heated sheath gas with basic vapors.

Profiling an electrospray plume by laser-induced fluorescence and Fraunhofer diffraction combined to mass spectrometry: influence of size and composition of droplets on charge-state distributions of electrosprayed proteins.

We investigated how physico-chemical properties of charged droplets are affected by the electrospray process, using simultaneous in situ measurements by laser-induced fluorescence (LIF), Fraunhofer diffraction and mass spectrometry. For this purpose, we implemented a laser-induced-fluorescence profiling setup in conjunction with a fast, high-resolution particle sizing scheme on a modified Agilent Jet Stream electrospray source coupled to a single quadrupole mass analyser. The optical setup permits us to profile the solvent fractionation and the size of the droplets as they evaporate in an electrospray plume by measuring both the angular scattering pattern and emission spectra of a solvatochromic fluorescent dye. Mass spectra are recorded simultaneously. These mass spectrometry and optical spectroscopy investigations allow us to study the relation between the observed charge-state distributions of protein anions and physico-chemical properties of evaporating droplets in the spray plume. By mixing water with methanol, a refolding of cytochrome C is observed as the water percentage increases in the plume due to the preponderant evaporation of volatile methanol.

Top-down protein fragmentation by infrared multiphoton dissociation in a dual pressure linear ion trap.

Infrared multiphoton dissociation (IRMPD) was implemented in a novel dual pressure linear ion trap for rapid top-down proteomics. The high pressure cell provided improved trapping and isolation efficiencies while the isotopic profiles of 10+ charged ions could be resolved by mass analysis in the low pressure cell that enabled effective top down protein identification. Striking differences between IRMPD in the low pressure cell and CID in the high pressure cell were observed for proteins ranging from 8.6 to 29 kDa. Because of secondary dissociation, IRMPD yielded product ions in significantly lower charge states as compared to CID, thus facilitating more accurate mass identification and streamlining product ion assignment. This outcome was especially useful for database searching of larger proteins (approximately 29 kDa) as IRMPD substantially improved protein identification and scoring confidence. Also, IRMPD showed an increased selectivity toward backbone cleavages N-terminal to proline and C-terminal to acidic residues (especially for the lowest charge states), which could be useful for a priori spectral predictions and enhanced database searching for protein identification.

Infrared multiphoton dissociation of peptide cations in a dual pressure linear ion trap mass spectrometer.

A dual pressure linear ion trap mass spectrometer was modified to permit infrared multiphoton dissociation (IRMPD) in each of the two cells-the first a high pressure cell operated at nominally 5 x 10(-3) Torr and the second a low pressure cell operated at nominally 3 x 10(-4) Torr. When IRMPD was performed in the high pressure cell, most peptide ions did not undergo significant photodissociation; however, in the low pressure cell peptide cations were efficiently dissociated with less than 25 ms of IR irradiation regardless of charge state. IRMPD of peptide cations allowed the detection of low m/z product ions including the y(1) fragments and immonium ions which are not typically observed by ion trap collision induced dissociation (CID). Photodissociation efficiencies of approximately 100% and MS/MS (tandem mass spectrometry) efficiencies of greater than 60% were observed for both multiply and singly protonated peptides. In general, higher sequence coverage of peptides was obtained using IRMPD over CID. Further, greater than 90% of the product ion current in the IRMPD mass spectra of doubly charged peptide ions was composed of singly charged product ions compared to the CID mass spectra in which the abundances of the multiply and singly charged product ions were equally divided. Highly charged primary product ions also underwent efficient photodissociation to yield singly charged secondary product ions, thus simplifying the IRMPD product ion mass spectra.

Multiplexed four-channel rectilinear ion trap mass spectrometer.

A four-channel multiplexed mass spectrometer with rectilinear ion trap (RIT) mass analyzers was designed, constructed, and characterized. The system consists of four parallel atmospheric pressure ion (API) sources, four RIT mass analyzers, four sets of ion optical elements, and four conversion dynode detectors. The complete instrument is housed in a single vacuum manifold with a common vacuum system. It has a relatively small footprint, and costs and complexity were minimized and controls simplified by sharing the electronics and control modules among different channels. Each channel of the instrument can be operated in either positive or negative ion mode with a choice of ionization methods to improve the information content from an experiment. Also, the instrument is equipped with simultaneous data acquisition capabilities from all four channels, but the use of a common RF electronics system limits the degree to which the analyzer channels can be scanned independently. The instrument was characterized over the mass/charge range of 150 to 1300 Th. Mass misassignments in different ion traps because of machining and assembly tolerances were avoided by the application of supplementary direct current signals to each mass analyzer to correct mass offsets. A multiplexed automatic gain control (AGC) scheme was developed to control the ion population in each of the traps independently. These two features allow tandem mass spectrometry to be performed with an isolation window of 1 Th so trapping identical ions in all four channels. There are two principal modes of operation. In one, the same sample is analyzed in all four channels using different ionization methods to increase the information content of the analysis. In the other mode of operation, different samples are analyzed in all four channels with the same ionization method, so providing higher throughput. These capabilities were demonstrated by examining lipids produced by Escherichia coli and complex mixtures containing drugs of abuse.

A proteomics grade electron transfer dissociation-enabled hybrid linear ion trap-orbitrap mass spectrometer.

Here we detail the modification of a quadrupole linear ion trap-orbitrap hybrid (QLT-orbitrap) mass spectrometer to accommodate a negative chemical ionization (NCI) source. The NCI source is used to produce fluoranthene radical anions for imparting electron transfer dissociation (ETD). The anion beam is stable, robust, and intense so that a sufficient amount of reagents can be injected into the QLT in only 4-8 ms. Following ion/ion reaction in the QLT, ETD product ions are mass-to-charge (m/z) analyzed in either the QLT (for speed and sensitivity) or the orbitrap (for mass resolution and accuracy). Here we describe the physical layout of this device, parametric optimization of anion transport, an evaluation of relevant ETD figures of merit, and the application of this instrument to protein sequence analysis. Described proteomic applications include complex peptide mixture analysis, post-translational modification (PTM) site identification, isotope-encoded quantitation, large peptide characterization, and intact protein analysis. From these experiments, we conclude the ETD-enabled orbitrap will provide the proteomic field with several new opportunities and represents an advance in protein sequence analysis technologies.

Rectilinear ion trap mass spectrometer with atmospheric pressure interface and electrospray ionization source.

A rectilinear ion trap (RIT) mass analyzer was incorporated into a mass spectrometer fitted with an electrospray ionization source and an atmospheric pressure interface. The RIT mass spectrometer, which was assembled in two different configurations, was used for the study of biological compounds, for which performance data are given. A variety of techniques, including the use of a balanced rf, elevated background gas pressure, automatic gain control, and resonance ejection waveforms with dynamically adjusted amplitude, were applied to enhance performance. The capabilities of the instrument were characterized using proteins, peptides, and pharmaceutical drugs. Unit resolution and an accuracy of better than m/z 0.2 was achieved for mass-to-charge (m/z) ratios up to 2000 Th at a scan rate of approximately 3000 amu/(charge.s) while reduced scan rates gave greater resolution and peak widths of less than m/z 0.5 over the same range. The mass discrimination in trapping externally generated ions was characterized over the range m/z 190-2000 and an optimized low mass cutoff value of m/z 120-140 was found to give equal trapping efficiencies over the entire range. The radial detection efficiency was measured as a function of m/z ratio and found to rise from 35% at low m/z values to more than 90% for ions of m/z 1800. The way in which the ion trapping capacity depends on the dc trapping potential was investigated by measuring the mass shift due to space charge effects, and it was shown that low trapping potentials minimize space charge effects by increasing the useful volume of the device. The collision-induced dissociation (CID) capabilities of the RIT instrument were evaluated by measuring isolation efficiency as a function of mass resolution as well as measuring peptide CID efficiencies. Overall CID efficiencies of more than 60% were easily reached, while isolation of an ion with unit resolution at m/z 524 was achieved with high rejection (>95%) of the adjacent ions. The overall analytical capabilities of the ESI-RIT instrument were demonstrated with the analysis of a mixture of pharmaceutical compounds using multiple-stage mass spectrometry.

Ion trap mass spectrometry: a personal perspective.

This paper is a personal perspective of the commercial development of the three-dimensional quadrupole ion trap mass spectrometer. Early ion trap invention and development which dates back to 1953, is described. The development of the ion trap is traced through three ages with the last age being where commercial development takes place. Key technical breakthroughs in ion trap technology and commercialization are presented and described up to the present time.