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High Speed Intact Protein Characterization Using 4X Frequency Multiplication, Ion Trap Harmonization, and 21 Tesla FTICR-MS Jared B. Shaw, Mikhail Gorshkov, Qinghao Wu, and Ljiljana Paša-Toli# Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b04606 • Publication Date (Web): 03 Apr 2018 Downloaded from http://pubs.acs.org on April 4, 2018
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Analytical Chemistry
High Speed Intact Protein Characterization Using 4X Frequency Multiplication, Ion Trap Harmonization, and 21 Tesla FTICR-MS Jared B. Shaw1, Mikhail Gorshkov2,3, Qinghao Wu1, and Ljiljana Paša-Tolić 1* 1
Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory,
3335 Innovation Blvd. Richland, WA 99352, USA 2
V.L. Talrose Institute for Energy Problems of Chemical Physics, Russian Academy of
Sciences, Moscow 119334, Russia 3
Moscow Institute of Physics and Technology (State University), Moscow Region,
Dolgoprudny 141700, Russia
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Abstract Mass spectrometric characterization of large biomolecules, such as intact proteins, requires the specificity afforded by ultra-high resolution mass measurements performed at both the intact mass and product ion levels. Although the performance of time-offlight mass analyzers is steadily increasing, the choice of mass analyzer for large biomolecules (e.g. proteins >50 kDa) is generally limited to the Fourier transform family of mass analyzers, such as Orbitrap and ion cyclotron resonance (FTICR-MS) with the latter providing unmatched mass resolving power and measurement accuracy. Yet, protein analyses using FTMS are largely hindered by the low acquisition rates of ultrahigh resolution spectra. Frequency multiple detection schemes enable FTICR-MS to overcome this fundamental barrier and achieve resolving powers and acquisition speeds 4X greater than the limits imposed by magnetic field strength. Here we expand upon earlier work on the implementation of this technique for biomolecular characterization. We report the coupling of 21T FTICR-MS, 4X frequency multiplication, ion trapping field harmonization technology, and spectral data processing methods to achieve unprecedented acquisition rates and resolving power in mass spectrometry of large intact proteins. Isotopically resolved spectra of multiply charged ubiquitin ions were acquired using detection periods as short as 12 ms. Large proteins such as apotransferrin (MW=78 kDa) and monoclonal antibody (MW=150 kDa) were isotopically resolved with detection periods of 384 ms and 768 ms, respectively. These results illustrate the future capability of accurate characterization of large proteins on timescales compatible with online separations.
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Analytical Chemistry
Introduction The continued expansion of top-down proteomics to enable routine characterization of proteoforms1 of large proteins (e.g. >50 kDa) will require significant advances at every step of the top-down proteomics workflow, including sample preparation, online separations, intact protein mass measurement, and tandem mass spectrometry.2 Large protein intact mass and fragment mass analysis poses significant challenges for mass spectrometry, as ultrahigh resolution and mass accuracy are mandatory for accurate mass determination of the precursors and differentiation of highly convoluted fragment ion isotopic distributions. Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) offers the ultimate in mass resolution and mass measurement accuracy.3 The performance metrics of FTICR-MS, such as resolution and acquisition rate, scale linearly with magnetic field strength.4 For decades, the improvements in these metrics were associated with the progress in magnet technologies.5–7 These efforts culminated recently with two 21T FTICR systems being commissioned, which have shown promising initial results for intact protein characterization.8,9 However, 21T magnetic field strength with low field drift and a large region of homogeneous field suitable for FTICR-MS is at the limits of state-of-the-art superconducting magnet technology. Therefore, further advances in the basic characteristics of this mass-analyzer in the near future will require other means than increasing magnetic field strength. Recent advances in ICR cell design have significantly improved the electric field characteristics that enable operation at greater post excitation radii and significantly extend the period of coherent ion motion. Resolution of 24 million (m/z=609) without apodization has been achieved at 7T with 180 s time-domain acquisitions.10 These results demonstrate the capability of electric field harmonization to dramatically extend the ultimate achievable resolution of FTICR-MS through extended time domain acquisitions. However, these developments do not increase the acquisition rate of highresolution data (i.e. resolution per unit time of acquisition). Detection at multiples of the cyclotron frequency offers an attractive alternative to further technological and cost prohibitive increases in magnetic field strength. Departure from traditional two electrode dipolar detection schemes enables detection of higher order multiples of the ion cyclotron motion.11–18 While the simplest implementation of
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detection at double the cyclotron frequency does not require any change in the ICR cell configuration18,19, modification of the cell electrode geometry to incorporate eight detection electrodes, four electrodes per phase of the detection circuit, enables 4X frequency multiplication. That is, the frequency of ion motion still corresponds to the reduced cyclotron frequency imposed by the magnetic and electric fields within the ICR cell, but the observed ion frequency is quadruple the reduced cyclotron frequency. This enables a 4X reduction in acquisition time, compared to traditional dipolar detection, needed to achieve the same resolution or 4X higher resolution for the same acquisition time. The first demonstration of the 4X configuration for measuring mass spectra of intact proteins was performed on a 10T mass spectrometer, and it demonstrated the feasibility of acquiring
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C-isotopically resolved mass spectra of isolated multiply
charged albumin (MW=64 kDa) at the sub-second acquisition rate.17 Herein, we expand upon those earlier works by combining the recently introduced harmonized “Window Cell” design with multi-electrode ICR cell configuration for 4X frequency multiplication, and applying this 4X-Window Cell technology for mass measurements of large proteins using a recently developed 21T FTICR mass spectrometer.
Experimental Materials and Sample Preparation. Human apo-transferrin and ubiquitin form bovine erythrocytes were purchased from Sigma Aldrich (St. Louis, MO) and used without further processing. Intact mAb Mass Check Standard (monoclonal IgG1) was purchased from Waters Corporation (Minford, MA). The monoclonal IgG1 was further purified by three washes with 10mM ammonium acetate follow by two washes with water using 0.5 mL 30 kDa centrifugal molecular weight cutoff filters (EMD Millipore, Billerica, MA). All other chemicals were purchased from Fisher Scientific (Hampton, NH). Mass Spectrometry. All experiments were performed using an in-house built 21T FTICR mass spectrometer.9 Due to the high ion frequencies resulting from 21T magnetic field and 4X frequency multiplication, ions with m/z 3:1 ratio of the 4X multiple to the 3X multiple. Note that 12 s is the maximum transient duration allowable by the current data station. Resolving power in magnitude mode without apodization was 6,280,000 corresponding to 75% of theoretically expected resolution for an undamped signal. Yet, this resolution is almost three-fold higher than that of 21 Tesla FTICR for the signal of the same acquisition period for the same m/z ion. The extracted transient for the 4X frequency multiple is shown in the inset of Figure 1C. It is evident that even for relatively small molecules, the high kinetic energy of ions at large post excitation radii in high-field FTICR yields conditions for which the achievable transient duration and, thus, the resolution is limited by the UHV conditions. Indeed, the cell prototype under evaluation incorporated PEEK insulators which have a
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Analytical Chemistry
relatively high outgassing rate, thus, limiting the UVH to ~3.5x10-10 Torr. Future designs will utilize alumina which is better suited for UHV. The superior outgassing characteristics of alumina will enable UHV of
