21 Tesla Fourier Transform Ion Cyclotron Resonance Mass Spectrometer Greatly Expands Mass Spectrometry Toolbox
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We provide the initial performance evaluation of a 21 Tesla Fourier transform ion cyclotron resonance mass spectrometer operating at the Environmental Molecular Sciences Laboratory at the Pacific Northwest National Laboratory. The spectrometer constructed for the 21T system employs a commercial dual linear ion trap mass spectrometer coupled to a FTICR spectrometer designed and built in-house. Performance gains from moving to higher magnetic field strength are exemplified by the measurement of peptide isotopic fine structure, complex natural organic matter mixtures, and large proteins. Accurate determination of isotopic fine structure was demonstrated for doubly charged Substance P with minimal spectral averaging, and 8158 molecular formulas assigned to Suwannee River Fulvic Acid standard with root-mean-square (RMS) error of 10 ppb. We also demonstrated superior performance for intact proteins; namely, broadband isotopic resolution of the entire charge state distribution of apo-transferrin (78 kDa) and facile isotopic resolution of monoclonal antibody under a variety of acquisition parameters (e.g., 6 s time-domains with absorption mode processing yielded resolution of approximately 1 M at m/z = 2700).
KeywordsFTMS Fourier transform mass spectrometry FTICR High resolution mass spectrometry Monoclonal antibody
Fourier-transform ion cyclotron resonance mass spectrometry (FTICR MS) is the highest performance MS technique, and thus uniquely valued as a research tool for analytical applications requiring ultra-high resolution and mass measurement accuracy [1, 2]. Increasing magnetic field strength enables multiplicative improvements in FTICR MS performance, including improved resolution (for the same acquisition period), acquisition speed, mass measurement accuracy, dynamic range, and resistance to peak coalescence that will have transformative impact in a variety of scientific applications [3, 4]. Unequivocal ability to discern, identify, and quantify diverse molecular species in complex systems will enable researchers to tackle current analytical grand challenges in omics and systems biology, including characterization of intact proteins (i.e., top-down proteomics) and metabolites in complex environmental mixtures (i.e., soil organic matter).
For instance, identification and quantitation of intact molecular structures derived from higher plants and soil microbiome within their native inorganic soil matrix are essential for advancing molecular-scale mechanistic understanding of the role of physical, geochemical, and biological processes below ground, which in turn is central to development of better predictive models of terrestrial carbon cycle  in the face of global and regional climate change . Similarly, advanced MS performance will improve measurements of isotopic patterns in stable-isotope profiling experiments, including protein turnover and metabolic flux measurements, enabling scientists to address the key knowledge gaps in functional understanding of how complex microbiomes influence and are influenced by their environment.
Another area of research that will be significantly impacted by 21T FTICR MS capability is characterization and quantitation of intact proteins, so-called top-down proteomics. Identification of protein variation arising from sequence differences, proteolysis, and post-translational modifications (PTMs) is essential for understanding protein function and identification of novel biomarkers. Critical information, including stoichiometry of PTMs on a single proteoform , or presence of a combination of multiple PTMs on a single proteoform, can only be determined by the top-down approach. Hence, this approach is rapidly becoming an indispensable avenue for (functional) proteomic studies. Recent advances in MS instrumentation, separation, and bioinformatics significantly increased the throughput of top-down proteomics, allowing the identification of hundreds of intact proteoforms [8, 9]. Yet many challenges persist, including effective MS characterization of large proteins on a chromatographic time scale. Because all key measures of FTICR MS performance improve with increased magnetic field strength, a 21T FTICR spectrometer will arguably provide that next level of performance needed to bring top-down proteomics to the mainstream.
Herein, we present the initial performance evaluation of the 21T FTICR MS at the Environmental and Molecular Sciences Laboratory, a US DOE supported national user facility located at the Pacific Northwest National Laboratory. 21T magnetic field strength enables the unprecedented ability to routinely analyze large proteins (>100 kDa), measure isotopic fine structure with high fidelity, and extract more information from complex natural organic matter mixtures. The user facility setting allows access to this highly specialized capability to a range of qualified scientific users.
Materials and Sample Preparation
Substance P (AnaSpec Inc., Freemont, CA, USA) and human apo-transferrin (Sigma-Aldrich, St. Louis, MO, USA) were used without further purification. Monoclonal antibody (mouse IgG1, intact mAb mass check standard) was purchased from Waters Corporation (Milford, MA, USA). Monoclonal antibody (mAb) was reconstituted in water and divided into 100 μg aliquots at 1 μg/μL. Prior to use, the mAb aliquots were rinsed five times with water using Amicon 0.5 mL centrifugal 30 kDa molecular weight cutoff filters (EMD Millipore, Darmstadt, Germany). Suwannee River Fulvic Acid (SRFA) standard was acquired from the International Humic Substances Society and stored as a 1 mg/mL stock solution. Twenty ppm working solutions of SRFA were prepared by dilution with LCMS grade methanol and analyzed with negative ion mode electrospray ionization (ESI). All peptide and protein samples were prepared at 1 μM in 50:50 acetonitrile:water with 0.1% formic acid and analyzed by direct infusion at 0.5 μL/min for positive ion mode electrospray ionization ESI. Pierce LTQ Velos ESI positive and negative ion calibration solutions and all other chemicals were purchased from Thermo Fisher Scientific (Waltham, MA, USA).
The EMSL 21 Tesla magnet was specified and designed in concert with Agilent Technologies in Yarnton, UK (former Magnex and Varian Magnet Technology Centre manufacturing site). The magnet is a 22-ton, 2.53 m long × 2.44 m wide × 3.36 m high system, with a room temperature bore of 112 mm. The magnet cryostat is a directly pumped, single reservoir system holding approximately 4050 L of liquid helium cryogen that is held at ~55 mbar and 2.2 K, using two Sumitomo dual-stage GM (Gifford-McMahon) type cryo-coolers (Model RDK 408D2) to maintain an inner radiation shield temperature of 8–9 K and an outer radiation shield temperature of 27–28 K. The cryogenics system is a simple, low-loss design that results in a cryogen consumption of ~1 L/d, necessitating a <10% refill of the system only once per year.
The magnet itself features a multi-coil, actively shielded design using both NbTi and Nb3Sn superconducting wire. Compensation and shim coil assemblies are inherent to the magnet/cryostat design; no room-temperature shims are utilized. Fringe magnetic fields of ≤5 Gauss are located at 3.5 m radially and 3.2 m axially. The magnetic field drift rate was measured at 2 ppb/h over a 7 day period 4–5 weeks after installation. The magnetic field homogeneity was measured at 9.2 ppm over a cylinder region of 60 mm diameter by 100 mm length at the same time. Total stored energy in this magnet is approximately 32.7 MJoules.
FTICR Mass Spectrometer
The vacuum chamber that houses the external linear quadrupole ion trap is an extension of the Velos Pro vacuum chamber, and it is pumped by the Velos Pro vacuum system. A 14-way stainless steel block comprises the three stages of ultra-high vacuum, and each stage is pumped by a magnetically shielded turbo molecular pump (EXT556HF; Edwards Vacuum, Tewksbury, MA, USA). Each turbo pump is spaced down from the UHV chamber by some distance to place the turbo pump in areas of fringe magnetic field less than 500 gauss. The titanium flight tube (Sharon Vacuum, Brockton, MA, USA) is 1778 mm flange to flange and allows spacing of the spectrometer back from the face of magnet to accommodate magnetic fringe fields. The three stages of differential pumping reduce pressure from ~2 × 10−5 Torr at the external ion trap to ~1 × 10−10 Torr or less in the analyzer pumping stage with a 24 h bakeout at 120 °C.
Our laboratory has recently developed and implemented a harmonized ICR cell that utilizes external shimming to closely approximate an ideal quadrupolar trapping electric field (i.e., the window cell) . The window cell consists of two coaxially nested cylinders. The outer cylinder is formed by 19 stacked ring electrodes (each 4.8 mm wide, 0.9 mm spacing) used for external shimming. The inner cylinder is split into four sectors used for dipolar excitation and detection and has a 46 mm inner diameter and 49 mm outer diameter. Each sector has five rectangular openings (43.7 mm long, 2.8 mm wide, 3.4 mm separation), or windows, which allow penetration of external shimming electric field into the trapping volume of the ICR cell. The gap between the four excite and detect electrodes is equal to the spacing between adjacent windows in the electrodes (i.e., 3.4 mm). The trapping volume is enclosed axially by two 42 mm diameter end caps with 6 mm inner diameter apertures. The inner surface of the end caps are spherical (76.3 mm radius) and approximate the ideal hyperbolic boundary. The spacing between the end caps is 69 mm, or 1.5 times the trapping volume inner diameter.
Instrument Control, Data Acquisition, and Data Processing
Modifications to the Velos Pro tune software enabled integration of DC power supplies and the FT acquisition computer from an LTQ-FT Ultra (ThermoFisher Scientific, San Jose, CA, USA). Time-dependent control of the DC offsets for the external ion trap, lenses, all quadrupole ion guides, and the ICR cell was integrated into the Velos Pro tune software and enabled automated tuning of ion transfer to the ICR cell. All experiments were performed with 1 V applied to the endcaps, 289 V on the front and back shim electrodes, and −38 V on the center shim electrode. Stored waveform inverse Fourier transform (SWIFT) excite waveforms were generated by the FT acquisition computer and amplified by a linear power amplifier (T&C Power Conversion, Inc., Rochester, NY, USA). For magnitude mode spectra, all time-domain data processing was performed on the FT acquisition computer, and spectra were stored as .raw files. For absorption mode spectra, time-domain data were saved as .dat files and processed off-line using Autophaser [16, 17]. Absorption mode resolution enhancement of greater than 2× reported here is a result of the use of different apodization routines during calculation of magnitude mode and absorption mode spectra. Generation of theoretical isotopic distributions and protein spectral deconvolution using the Xtract algorithm was performed using Thermo Xcalibur 2.2 (ThermoFisher Scientific, San Jose, CA, USA).
For the analysis of SRFA, peak picking in the range 200 < m/z <1000 was performed using the ICIS algorithm within Thermo Xcalibur, and peaks with S/N > 3 were considered for molecular formula assignment. Molecular formula assignment was performed using an in-house refactored version of Compound Identification Algorithm (CIA) developed by Kujawinski and Behn  and modified by Minor et al. . Previously reported molecular formula assignments by Stenson et al.  from SRFA samples were used as internal calibration targets with initial matching tolerance of 3 ppm resulting with 125 calibration peaks with mean post-calibration mass measurement accuracy of 0.00 ppm and standard deviation of 0.05. Based on this assessment, a full CIA database search with 0.5 ppm mass tolerance was performed limiting non-oxygen hetero-atom counts to 0–1 for N and P and 0–2 for S. The database was searched starting from m/z = 200 with molecular formula matches propagated using CH2, H2, and O building blocks. In the case of multiple candidates, the formula with the lowest N+S+P count was selected. If this criterion did not bring resolution, formula with smaller absolute mass error was selected. The matching formulas from this search were filtered using 3-sigma rule from calculated mean mass measurement accuracy.
Results and Discussion
The performance metrics of the FTICR MS directly relate to magnetic field strength. Linear improvements in resolution and acquisition rate and quadratic improvements in mass measurement accuracy, dynamic range, and charge capacity are expected when moving to ever increasing magnetic fields. The transformative effects of higher magnetic field strength are most evident during the analysis of complex mixtures, such as natural organic matter. For instance, ultra-high resolution and mass accuracy, charge capacity, and large dynamic range are essential for observation of isotopic fine structure and unambiguous speciation of aerosols or soil organic matter via accurate molecular formula assignment. Additionally, higher magnetic field strength provides significant advances in the sensitivity and throughput for characterization and quantitation of large intact proteins (i.e., >100 kDa) enabling physiologically relevant studies of microbes and higher eukaryotes.
Supplementary Figure S1 shows the observed resolution (in magnitude mode) for time domain acquisitions ranging from 24 ms to 6.144 s using a calibration mixture. Instrument manufacturers commonly specify resolution at m/z = 200, 400, and/or 1000; therefore we have included the observed resolution at m/z 195, 393, 524, and 1022 for easy comparison. Additionally, Figure S1 illustrates the high linearity of resolution as a function of acquisition time achieved with a relatively high AGC target of one million. With internal calibration, mass accuracy of 0.02 ppm or less is typically observed for a wide range of AGC targets from 5E4 to 3E6.
Isotopic Fine Structure for Complex Mixture Analysis
Intact Protein Analysis
The utility of top-down proteomics is rapidly expanding because of recent wide availability of commercial high resolution and mass measurement accuracy instrumentation, such as time of flight and Orbitrap mass analyzers. However, routine analysis is generally limited to intact proteins with molecular mass less than approximately 30 kDa. A number of factors contribute to this limitation, including sample preparation and inefficient on-line separations, but poor sensitivity for high resolution mass analysis is likely one of the current major limitations. Moving to higher magnetic field strength in FTICR enables greater charge capacity and reduces the acquisition time (i.e., ensures greater resolution per unit time) needed to accurately measure the broad isotopic distributions of large proteins. To the best of our knowledge, there has been only one report of isotopic resolution and accurate mass determination for a protein with a molecular mass greater than 100 kDa using instrumentation other than FTICR MS . This can be attributed to the limited resolution of time of flight mass analyzers (generally <100,000) and the high energy injection of ions into Orbitrap mass analyzers . 21T magnetic field strength and the supporting instrumentation described in this report have enabled routine analysis of large proteins, including facile isotopic resolution of intact monoclonal antibodies (Mr ~150 kDa).
High resolution and mass measurement accuracy is vital to the success of tandem MS of intact proteins as well. The Velos Pro provides CID capabilities. Additionally, we have implemented beam-type CID in the external quadrupole ion trap and ultraviolet photodissociation (UVPD) within the ICR cell similar to what has been described for lower field instruments . Modifications are under way that will incorporate a 90° bend after the external quadrupole ion trap. This will enable UVPD to be performed within the external quadrupole ion trap as previously described .
Initial performance characterization of the 21 T FTICR system at the Environmental Molecular Sciences Laboratory at the Pacific Northwest National Laboratory demonstrates enormous potential for future application to the extremely complex molecular mixtures and systems frequently encountered in environmental, biological, atmospheric, and energy research. The transformative effects of higher magnetic field strength are realized in the ability to acquire high S/N and resolution spectra of the entire charge state distribution of a 78 kDa protein (apo-transferrin), facile isotopic resolution of the proteoforms of a 150 kDa monoclonal antibody, precise measurement of the isotopic fine structure of the peptide Substance P, and the assignment of 8158 molecular formulas in a natural organic matter mixture (SRFA) with mass measurement accuracy within 0.1 ppm. Hence, as hypothesized, FTICR at 21T offers the unprecedented ability to routinely analyze large proteins (>100 kDa), measure isotopic fine structure with high fidelity, and extract more information from complex natural organic matter mixtures. This highly specialized capability is located within the user facility and thus available to the researchers worldwide.
This research is part of the “High Resolution and Mass Accuracy Capability” development project at EMSL, a DOE Office of Science User Facility sponsored by the Office of Biological and Environmental Research and located at the Pacific Northwest National Laboratory in Richland, WA. The authors thank David P. A. Kilgour, Spencer A. Prost, Kenneth J. Auberry, Randolph V. Norheim, Gordon A. Anderson, Jean H. Futrell, James R. Ewing, and Mark R. Townsend for expert help with bioinformatic analyses, electronics, data acquisition workflows, and mechanical design and fabrication throughout this project.
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