Measurement of Individual Ions Sharply Increases ... - ACS Publications

Subscriber access provided by Iowa State University | Library

Article

Measurement of Individual Ions Sharply Increases the Resolution of Orbitrap Mass Spectra of Proteins Jared Otto Kafader, Rafael D. Melani, Michael W. Senko, Alexander A. Makarov, Neil L Kelleher, and Philip D. Compton Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b04519 • Publication Date (Web): 04 Jan 2019 Downloaded from http://pubs.acs.org on January 6, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

Measurement of Individual Ions Sharply Increases the Resolution of Orbitrap Mass Spectra of Proteins †

†

‡

§

Jared O. Kafader, Rafael D. Melani, Michael W. Senko, Alexander A. Makarov, Neil L. †

†

Kelleher, Philip D. Compton*, †

Departments of Chemistry and Molecular Biosciences, the Chemistry of Life Processes

Institute, the Proteomics Center of Excellence at Northwestern University, Evanston, Illinois 60208, United States ‡

Thermo Fisher Scientific, San Jose, California 95134, United States

§

Thermo Fisher Scientific, Bremen 28199, Germany

*Corresponding author: [email protected]

Abstract: It is well known that with Orbitrap-based FT-MS analysis, longer time domain signals are needed to better resolve species of interest. Unfortunately, increasing the signal acquisition period comes at the expense of increasing ion decay, which lowers signal-to-noise ratios and ultimately limits resolution. This is especially problematic for intact proteins, including antibodies, which demonstrate rapid decay due to larger collisional cross sections which result in more frequent collisions with background gas molecules. Provided here is a method that utilizes numerous low ion count spectra and single ion processing to reconstruct a conventional m/z spectrum. This technique has been applied to proteins varying in molecular weight from 8 to 150 kDa, with 677,000 resolving power achieved for transients of carbonic anhydrase (29 kDa) of only ~250 msec duration. A resolution improvement ranging from 10-20-fold was observed for all proteins, providing isotopic resolution where none was previously present.

ACS Paragon Plus Environment

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Over the decades, mass spectrometry has become an important tool that is used to analyze and solve myriad biological-based questions. During this time, mass spectrometers have seen dramatic improvements in resolution, mass accuracy, and sensitivity, enabling better characterization of larger molecular weight compounds with smaller sample sizes1,2. Higher mass analysis has continued to press into ever-increasing molecular weights, and is now moving into the newer area of native proteomics where proteins and protein complexes with significantly higher molecular weights and accompanied m/z values need to be resolved in a routine manner3,4. Many advances have enabled this climb to higher mass and m/z. Time-of-Flight (TOF) advances to achieve higher mass resolving power include orthogonal acceleration5 which enhanced ion beam collimation, while reflectron6 and multipass7,8 systems greatly increased ion total flight time. Similarly, Fourier Transform Ion Cyclotron Resonance (FT-ICR) has been improved with new cell designs, such as the dynamically harmonized cell9, and the utilization of higher magnetic field (15T and 21T) systems to improve resolving power at higher m/z domains10,11,12,13. For Orbitrap style detectors, changes in analyzer geometry and detection methods have been of focus to increase instrumentation performance. Advances include orbital trapping from Kingdon14 and Knight15 traps to the modern day Orbitrap16 configuration and detection enhancements which include image current detection to allow for continuous ion frequency measurements through time domain signal acquisition17. However, for all Fourier transform based mass spectrometers, resolution is ultimately limited by the decay rate of the time domain signal. The signal decay rate has two primary origins. Collisions with background gas can damp ion motion or cause dissociation of the ions, both which result in reduced signal amplitude. Imperfect electric and/or magnetic fields cause different ions to follow different trajectories, which results in dephasing of the ion cloud and 2 ACS Paragon Plus Environment

Page 2 of 29

Page 3 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


reduced signal amplitude. The imperfect fields can be caused by either geometric constraints, or space charge induced distortions18. Over 20 years ago, in a series of papers from the Smith group, the unique aspects of detecting single ions with FT-ICR were described19,20,21,22. This involved a range of molecules, from 66 kDa bovine serum album carrying 30 charges, all the way to 100 MDa T4 DNA carrying in excess of 30,000 charges20,21. For the T4 DNA, time domain signals of 8 minutes were observed, far exceeding the expectations based on the limitations mentioned above. First, due to the mass of the ion relative to the background gas, collisional effects were insignificant. Second, when considering just a single ion, dephasing of the ion cloud is not possible, so field imperfections are not critical20. This concept for single ion detection was further extended for proteins by collecting successive spectra with moderate ion populations23. Signals of the multiply charged ions that were above a user specified threshold in the individual spectra were summed into a composite spectrum, while all background electronic noise was eliminated. The composite spectrum demonstrated resolved isotopic distributions, which was an achievement previously not possible with larger ion populations due to coulombic restrictions. Similarly, the ability to collect and detect single ion events in an Orbitrap analyzer has been demonstrated using myoglobin with only 20 charges24. Even with the low signal-to-noise ratios generated for these single ions, it was possible to maintain mass precision better than 3 ppm with a resolving power of 45,000, which corresponds to previous experiments25. Since the location of an isolated peak can be determined to a higher degree of precision than the peak width, this led to the concept of creating composite spectra of histogrammed centroids, which

3 ACS Paragon Plus Environment


would produce composite peak widths limited only by the precision of the centroids26. This effectively resolves adjacent species not with the separating power of the mass spectrometer, but in time. This is somewhat analogous to the use of time-to-digital (TDC) converters in time-offlight mass spectrometers. TDC’s convert an analog signal from the detector into a single time point with a precision that is much better than the width of the analog signal. Thus, it can provide better resolving power than an analog-to-digital converter used on the same analog signal, but at the sacrifice of dynamic range, since a TDC can only measure one ion at a time27,28. Presented herein is a low ion count collection and processing technique that pushes the boundary of our resolution limitations in Orbitrap systems to isotopically resolve higher molecular weight proteins. We apply the concepts of centroiding and histogramming single ions using a range of proteins to demonstrate optimal conditions to baseline resolve species with partial isotopic resolution or provide isotopic resolution where none was previously present. Experimental Section Sample Preparation and MS Analysis. Ubiquitin (8.7 kDa), myoglobin (16.9 kDa), carbonic anhydrase (29 kDa), enolase (46.5 kDa), and transferrin (80 kDa) obtained from Sigma Aldrich were dissolved in 150 mM ammonium acetate at final concentration of 1mg/mL. Two hundred micrograms of NIST reference antibody (150 kDa) (Material 8671) was submitted to Ndeglycosylation overnight at 37° C using PNGase-F (Promega) following manufacture’s native protocol. All proteins solutions were exchanged to 100 mM ammonium acetate 6 times at 10,000 × g for 10 min into 3, 10, 30, or 100 kDa Amicon Ultra centrifugal filters (Merk Millipore) according to the samples molecular weight. Once desalted, all samples were diluted and electrosprayed under denaturing conditions in a 40% acetonitrile and 0.2% acetic acid solution.


Page 4 of 29

Page 5 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Samples under 100 kDa were diluted to concentrations between 0.05 and 0.4 µM and analyzed by a Q Exactive Plus (Thermo Fisher Scientific) coupled to an Ion Max source (Thermo Fisher Scientific) with a flow rate of 5 µL/min, a spray voltage of 3.7 kV, a sheath gas flow rate of 5, an in-source collision-induced dissociation (SID) value of 0-5 V, and a source temperature of 320° C. To have single or minimal number of ions in each SIM spectrum the voltage on the C-Trap entrance lens was detuned to limit transmission (2.25-5 V). AGC was disabled and the maximum injection time was held constant at 1 ms, with the injection time being decreased to further decrease signal when necessary. Total acquisition time varied based on the molecular weight of the species being investigated and instrument resolution was set 35,000 or 70,000 depending on the sample. Unless otherwise stated, all resolutions listed are relative to m/z 200. Deglycosylated antibody (1 µM) was sprayed through a custom nano-electrospray source at a flowrate of 0.2 µL/min delivered by a syringe pump (Chemyx) into a modified Q Exactive HF (Thermo Fisher Scientific) mass spectrometer29. Instrumental conditions included a spray voltage of 0.8 to 1.6kV, a SID value of 120 V, and a source temperature of 320° C. Survey spectra were acquired at a resolution setting of 15,000 with an acquisition range from m/z 2,400 to 3,800 with settings including an extended trapping value of 2 V, 10 µscans, averaging of 10 spectra, and a 5 ms maximum injection time. Extended trapping allows for a slightly higher trapping efficiency for larger protein species in the HCD cell, while averaging and µscans average spectral measurements on the post- and pre-Fourier transformed signal, respectively. Low ion count spectra were acquired with an isolation window of m/z 2,960 to 2,966 with a total collection time of 13 hours with no extended trapping, an HCD pressure setting of 0 to reduce



ion/background gas collisions in the Orbitrap, a fixed injection time of 1 ms, no spectral averaging of any kind, and a resolution setting of 120,000. Method Development. A workflow with the most important steps of this new method is depicted in Figure 1a. First, a survey spectrum is acquired under standard conditions to identify the species of interest that will be investigated in further detail. The charge state of interest is isolated in the narrowest window possible and the signal is attenuated to collect only a single or few ions in each acquisition event, limiting the overlap of adjacent isotopic peaks. The mass-tocharge centroid of each peak was accurately determined through the standard instrument software. To collect sufficient statistics 1,000 to 150,000 spectra were acquired over the course of minutes to hours. The centroid mass-to-charge ratios were binned in small domains ranging between m/z 0.004 to 0.0001. The bins were used to recreate a composite m/z spectrum. Bin sizes were selected on a per species basis with a size large enough to contain enough single ion signals to provide a Gaussian-like isotope peak shape without further resolution loss. As this is a relatively new approach, algorithms to automatically optimize bin size have not yet been developed or implemented. To ensure only single ion intensities were selected during the binning process, minimum and maximum single ion intensity thresholds were set to exclude arbitrary background electronic noise and multiple overlapping ion peaks. Electronic noise peaks were determined to have an intensity around 25,000-45,000 while the threshold to exclude multiple ion signal intensities was set manually due to variability on both a species and charge state basis. Ion intensities that fell outside these intensity boundaries were excluded from the list of ions utilized to create the composite m/z spectrum. It should be noted that noise peak thresholds will vary from instrument to instrument and are affected by numerous experimental parameters such as ion injection time into the Orbitrap, the quadrupole isolation window, and acquisition time. 6 ACS Paragon Plus Environment

Page 6 of 29

Page 7 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Resolution Calculation. The resolution of a peak can be determined at any mass value through the relationship:

𝑅=𝑘∗

1

Eqn. 1

𝑀

where k is an arbitrary constant16. By a simple ratio, Eqn. 2 can be derived to determine the effective or expected resolution at any m/z value:

𝑅2 = 𝑅1 ∗

𝑀1

Eqn. 2

𝑀2

for a robust comparison between raw and processed spectral resolution. R1 corresponds to a known resolution at a first m/z value, M1, and R2 is the calculated resolution at a second m/z value, M2. The transient duration to obtain a specific resolution is dependent upon the type of Orbitrap analyzer utilized. For instance, a resolution of 140,000 at m/z 200 on a Q Exactive Plus (standard Orbitrap analyzer) requires approximately a 500 ms acquisition time, while to obtain a resolution of 140,000 at m/z 200 on a Q Exactive HF (ultra-high field Orbitrap analyzer) an acquisition time of only 255 ms is needed. In addition, if enhanced Fourier transform is disabled transient times corresponding to a resolution value are doubled. Results and Discussion Fig. 1b shows the difference in obtainable resolution from standard ensemble measurements (top panel) and the utilization of our single ion technique (bottom panel). Fig. 1b illustrates conceptually the premise of our high-resolution technique by using concepts from the aforementioned FT-ICR and TDC TOF single ion techniques. Analyzing and centroiding non7 ACS Paragon Plus Environment


overlapping single ion isotopes simplifies the many limitations of resolving adjacent isotopes. Overlaying multiple centroided ion isotope signals acquired in different scan events limits resolving adjacent isotope peaks to only the mass precision of the single ions collected over the acquisition period. With high mass accuracy on a scan to scan basis, well separated isotope peaks (blue lines in bottom panel) are produced. Figure 2 illustrates the use of this technique on a species that can be easily resolved with a standard instrument, the +9 charge state of ubiquitin. The raw data, acquired at an expected resolution 32,000 (~500 ms transient) for m/z 952.5, is shown in Fig. 2a. Assignment of the intact species is found in the supporting Figure S1. Low ion count data acquired over ~1,700 spectra, corresponding to a collection time of 14 minutes are overlaid on the raw data. The composite single ion spectrum has significantly higher resolution than the raw data with an effective resolution of 318,000 for m/z 952.5, resulting in a 10-fold resolution increase. This technique is not limited by overlapping adjacent peaks and instead is limited by the mass precision of the low signal-to-noise produced by single ions, which is on the order of 1-5 ppm25. The small difference in relative ion isotope distributions between raw and overlaid processed data is attributed to statistics from the collection of random single ion signals from various isotopes and the bin size used to view them. Longer acquisition periods provide more single ion signals that produce a composite m/z that more closely matches the raw data isotopic distribution. An example of this concept is illustrated in the supplemental information (Figure S2). Fig. 2b shows a histogram of the intensities of the signals collected over the 1,700 spectra, composed of 4,597 signals that form two distributions. The distribution at low intensity is attributed to electronic noise. These 748 signals attributed to noise were filtered out and not


Page 8 of 29

Page 9 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


included as part of the composite spectrum. The remaining 3,849 signals are the various isotopes of ubiquitin carrying 9 positive charges which were incorporated into the composite spectrum. Figure 3a shows the non-isotopically resolved spectrum of the +38 charge state of carbonic anhydrase collected with an expected resolution of 17,500 (~250 ms transient) for m/z 785.5. Three distinguishable charge state distributions were observed in the spectrum. Distributions resulting from decarboxylation and water loss were found between m/z 784-785. For construction of the composite spectrum, only the isotopic distribution of the intact acetylated species from m/z 785-786 was processed. A closer look at these assignments is found in the supplemental Figure S3. ~4,000 spectra were acquired over the course of 18 minutes. Fig. 3b shows the intensity histogram of the 6,898 individual signals collected over these spectra. The 307 signals with intensities less than 45,000 are classified as noise and were not included for further processing. Signal intensities between 45,000 and 250,000 correspond to real ions that have decayed during the time domain acquisition period. Ions which decay during the time domain acquisition result in less intense signals than those from ions that last the entire observation period. The non-decayed ions are represented by the Gaussian-like distribution of intensities between 250,000 and 325,000. The time of death of each ion directly relates to the intensity of the resultant peak created in the frequency domain. The clearest feature in these plots corresponds to ions that last the entire detection period, resulting in a collection of ions with similar intensity values with a Gaussian-like distribution between 250,000 and 325,000. To the contrary, the remaining ions undergo collisions that cause decay during the detection period. This decay follows the generic form e ―𝑥, where x is proportional to the collisional cross section of the ion. For ions with low collisional cross sections or an x value close to 0 (where the majority live the entire detection period), the distribution of decayed ions appears flat due to the 9 ACS Paragon Plus Environment


low collisional probability. For larger ions with larger collisional cross sections, the probability of decay early in the transient increases dramatically and creates a decayed ion distribution with higher exponent or x value. Ion signals over an intensity value of 325,000 are attributed to the collection of more than one single ion signal at a certain m/z in a scan event, one or both of which decayed before the end of the transient event. For this reason, they have not been included in the processing. Figure 4 demonstrates the differences in resolution when processing all ion signals versus processing only non-decayed ion signals of the +38 charge state of carbonic anhydrase. Processing all ions (Fig. 4a and b) results in a resolution of 253,000, with clean separation of adjacent isotopes, when this was not achieved in the raw data, and represents a 14x improvement from the expected resolution. Processing only ions that last throughout the entire time domain signal (Fig 4c and d) decreased the full width half max (FWHM) of the isotope peaks from m/z 0.0031 to 0.0023, increasing the resolution from 253,000 to 341,000 corresponding to a further 25% increase. There are two considerations for achieving maximum resolution. First, as demonstrated above, only ion signals that last the entire acquisition event should be processed. Ions above noise threshold that prematurely decay due to collisions with background gas molecules result in lower intensity peaks, which produce centroids with less precision than non-decayed ions. Second, if too many ion signals are observed in each spectrum, the probability of observing adjacent overlapping isotopes increases, which results in centroid values that would lie between the actual isotopes and a sharp decrease in the post-processed resolution.


Page 10 of 29

Page 11 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 5a shows the +55 charge state of enolase with the raw data acquired with an expected resolution of 34,000 (~500 ms transient) for m/z 849.5 and the accompanied composite spectrum. Assignment of the intact species is found in the supporting Figure S4. At this resolution no isotopically resolved peaks are observed in the raw data, but the composite spectrum contains baseline resolved isotopic peaks. The inset contains a close-up of the isotope at m/z 849.530 with a FWHM of m/z 0.0016 and a resolution of 531,000. This result represents a 13-fold resolution increase in comparison to the expected resolution at this m/z. Fig. 5b shows a histogram of signal intensity. Over the 2,700 spectra acquired over 14 minutes, a total of 12,312 signals were recorded. The intensity distribution of enolase is very different in comparison to previous intensity histograms of ubiquitin (Fig. 2b) and carbonic anhydrase (Fig. 3b). Instead of the majority of ion signals lasting the entire acquisition event, most ions decay before the acquisition period is complete. About 1,190 signals below an intensity value of 30,000 are attributed to electronic noise. Above this cutoff most signals have intensity values between 30,000 and 100,000 indicating that the ions decayed in the Orbitrap analyzer during the acquisition event. Only 898 ions appear to survive the entire acquisition event with intensities between 500,000 and 575,000. Unlike FT-ICR MS, single ion signals collected via Orbitrap MS analysis cannot be easily collected at lower kinetic energies. Upon collision with background gas, ion signals are completely lost due to fragmentation, corresponding to lower intensity values than ion signals that last the entire transient event. Because of the extensive decay, and the desire to generate a statistically meaningful isotopic distribution, to produce the higher resolution enolase spectra in Fig. 5a, some decayed ions needed to be included in the processing. Ions that lasted approximately half of the acquisition event with corresponding signal intensities between 350,000 and 575,000 were processed.



As the molecular weight of the investigated species increases, there is a decrease in the number of ions that survive throughout the entire acquisition period. The percent of the total ions processed decreased from over 80% to less than 20% as the molecular weight of the proteins increased from 8.7 kDa to 46.5 kDa. A plot of percent total number of ions processed as a function of species molecular weight can be found in the supporting Figure S5. Increased ion decay and broadening isotopic distributions for larger species result in increased acquisition times required to produce isotopically resolved composite spectra for species over 50 kDa. Although it is impossible to know the exact amount of residual background gas within the Orbitrap analyzer, ion decay can be correlated to the collisional cross-section of the investigated species30. The collisional cross-section of the smaller protein ubiquitin is 1,900 Å2 in the +9 charge state, which is fivefold lower than the cross-section of 10,000 Å2 for the +40 charge state of transferrin25,31,32. Similarly, the collisional cross-section of the humanized NIST monoclonal antibody under native conditions has been determined to be around 7,500 Å2 for the +26 charge state, but the corresponding cross-section for the +49 charge state under denaturing conditions should be significantly larger33. Ion decay and the corresponding longer acquisition times should improve through single ion analysis of lower charge state/native species. However, utilizing lower charge states is accompanied with reduced resolution in the higher m/z domain and lower single ion S/N values, which will decrease the precision of acquired signals over the collection time34. The bottom panel of Figure 6 shows the survey spectrum of the transferrin charge state distribution with various post-translational modifications including glycosylation acquired with a resolution setting of 17,500. The center panel shows the +37 charge state with the raw data and overlaid composite single ion spectra acquired with a resolution setting of 70,000 (~500 ms 12 ACS Paragon Plus Environment

Page 12 of 29

Page 13 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


transient). ~17,000 spectra were acquired over the course of 140 minutes. Although the single ion processing scheme does not result in baseline isotopic resolution, distinct isotopes are visible with a spacing of m/z 0.027. The top panel of Fig. 6 shows a close-up of one of the modifications located at m/z 2,161.7. A FWHM of m/z 0.011 corresponds to a resolution of 197,000 and a 9fold increase in resolution relative to the expected resolution the data was acquired with. Many higher molecular weight species including antibodies are not typically resolved on the isotopic level through Orbitrap analysis due to signal decay over the course of the transient. Higher mass species have only been resolved through unconventional methods such as the utilization of helium as the C-Trap and HCD bath gas. Figure 7a shows a survey spectrum of the denatured antibody following deglycosylation and desalting, acquired at a resolution setting of 15,000 (~30 ms transients). Single ion analysis (Fig. 7b) was performed on the +49 charge state with an isolation window between m/z 2,960 and 2,966 with an instrumental resolution of 120,000 (~250 ms transients). The raw data and composite low ion count spectra were acquired at an expected resolution of 31,000 for m/z 2,963. The larger collisional cross-section of the sample made it necessary to increase the number of low ion count spectra to ~150,000 for a collection time of 13 hours. The processed signals that did not decay through the entire acquisition period provided well resolved isotopic signals with a FWHM of m/z 0.007. This corresponds to a post processed resolution of 423,000 at m/z 2,963, a 14-fold increase over the expected resolution. Although isotopic resolution of a similarly sized antibody species was previously accomplished with a lower resolution of 330,000 for m/z 2,800 on an Orbitrap analyzer, helium was required as the C-Trap and HCD bath gas to minimize signal decay and allow for the acquisition of longer transients35.



Isotopically resolving higher molecular weight species allows for a more robust way to assign overlapping distributions. Overlaying theoretical isotope distributions not only identifies the intact species through the spacing of adjacent isotopes, but multiple distributions can be superimposed on the experimental spectrum to identify adducts and degradation products. Figure 8 illustrates possible NIST antibody species assignments of theoretical distributions overlaid on experimentally observed +49 isotopic distributions determined from processed single ion spectra. Non-isotopically resolved charge state assignments overlaid on the NIST antibody survey scan is found in the supporting information Figure S6. The antibody standard sequence found on the NIST database was utilized in concert with mMass to accurately produce and overlay theoretical distributions with their experimental counterparts36,33. The unmodified theoretical antibody +49 isotopic distribution in Fig. 8a spans about half the FWHM of the experimental distribution. As a result, multiple overlapping species present in the experimental data were assigned which must have lower masses than their unmodified counterpart. This is supported by the non-Gaussian like experimental peak that tails to lower m/z values. Observed data can be explained by water loss distributions arising from the loss of one and two water molecules. The sum of the unmodified and two water loss distributions (yellow trace) is shown in Fig. 8b. There is good agreement between experimental and theoretical distributions, including the tailing feature to lower m/z values. The resolution of the theoretical distributions was increased to match the isotopic resolution of our post processed single ions. The inset in Fig. 8a provides isotopic confirmation with similar isotope spacing of both experimental and theoretical isotopes of m/z 0.02, corresponding to the correct value for the +49 charge state. Although these water loss assignments could be assigned to other modifications with close mass shifts, this example demonstrates the importance of isotopic resolution for the deconvolution and identification of


Page 14 of 29

Page 15 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


overlapping species. Isotopically resolving proteins including antibodies has a multitude of biopharma applications including quality control, robust characterization of new drugs, and a more complete understanding of how antibody variations affect drug delivery. Conclusion Extending previous approaches that utilize single ion measurements to the Orbitrap mass analyzer, we demonstrated the capability to isotopically resolve proteins that routinely cannot be resolved using Orbitrap based mass spectrometers operated in an ensemble mode. This analysis has been applied to various molecular weight species ranging from 8.7-150 kDa, with the possibility for future expansion over the 150 kDa regime. This technique is compatible with low sample concentrations, and with limited computational efforts, resolution can be enhanced 1020-fold. Limitations for this technique exist including significant ion decay for species over 40 kDa, which results in hours of total acquisition time to produce a composite mass spectrum with a statistically meaningful isotopic distribution. Acknowledgements This work was completed with support from Thermo Fisher Scientific.



List of Figures Figure 1. (a) Low ion count technique workflow scheme including data acquisition and postacquisition processing steps to produce higher resolution spectra. (b) Conceptual differences in obtainable resolution of ensemble vs centroided single ion spectra. The dashed red trace lines indicate isotopes that are non-resolved. Figure 2. (a) Raw data (black trace) and subsequent increase in resolution from acquired and processed single ions (red trace) of the +9 charge state of ubiquitin (ID: P0CH28, MW: ~8.7 kDa). (b) Intensity histogram of individual ions collected to produce the red trace higher resolution spectra. The blue dashed line indicates the separation of low intensity noise peaks and higher intensity real ions used for spectral processing. Figure 3. Survey spectrum of (a) the +38 charge state of carbonic anhydrase (ID: P00921, MW: ~29 kDa) and (b) intensity histogram of individual ions collected to produce higher resolution spectra. Ion intensities from lowest to highest (separated by blue dashed lines) are labeled as noise peaks, real ions that decayed, and real ions that did not decay throughout the transient event, respectively. Ions encompassed by the blue and green boxes represent ions processed to produce color coded resolution variations in Figure 4. Figure 4. Differences in enhanced resolution of processing single ions that have decayed and not decayed (a and b) verses ions that have not decayed (c and d) throughout the transient event. (b) and (d) are zoomed in images of isotopes of the +38 charge state of carbonic anhydrase. Figure 5. (a) Raw data (black trace) and subsequent increase in resolution from acquired and processed single ions (red trace) of the +55 charge state of enolase (ID: P00925, MW: ~46.5kDa). The inset provides a closer look at a typical isotope found in the charge state. (b) 16 ACS Paragon Plus Environment

Page 16 of 29

Page 17 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Intensity histogram of individual ions collected to produce the red trace higher resolution spectra. The blue dashed line indicates the separation of ions intensities to include noise peaks, real ions that decayed, and real ions that did not decay throughout the transient event. Figure 6. Survey spectrum of multiple charge states of transferrin (~80 kDa) to reveal various modifications (bottom panel). The middle panel shows further analysis of the +37 charge state with the raw data illustrated with the black trace and the higher resolution processed single ion spectra illustrated with the red trace. The top panel is a closer look at a typically resolved isotope. Figure 7. (a) Survey spectrum of the NIST antibody sample (~150 kDa) and (b) further analysis of the +49 charge state. Raw data (black trace) and higher resolution processed single ion spectra (red trace) are shown above with investigation of a typical isotope shown in the inset. Figure 8. Comparison of experimental and theoretically determined NIST antibody (Ab)33 isotopic distributions of the Ab +49 charge state. Theoretically determined intact species (red trace) and possible water loss products (green and black trace) are overlaid on the experimental spectrum (blue trace) in (a), while the sum of these assigned distributions (yellow trace) is overlaid on the experimental spectrum in (b).



Figure 1. Kafader et al. 18 ACS Paragon Plus Environment

Page 18 of 29

Page 19 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 2. Kafader et al.



Figure 3. Kafader et al.


Page 20 of 29

Page 21 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60





Page 22 of 29

Page 23 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60





Page 24 of 29

Page 25 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60




Supporting Information Available: Contents present information pertaining to justification of species assignment and determined statistical trends for this method. Isotopically resolved experimental species are assigned via theoretically determined isotopic distribution comparison. Evidence of relative differences in isotope distributions based on the number of single ion signals acquired and the percentage of total ion signals processed as a function of species molecular weight are also presented. References

Toby, T. K.; Fornelli, L.; Kelleher, N. L., Progress in Top-Down Proteomics and the Analysis of Proteoforms. Annu. Rev. Anal. Chem. 2016, 9, 499-519. 2 Scigelova, M.; Hornshaw, M.; Giannakopulos, A.; Makarov, A., Fourier transform mass spectrometry. Mol. Cell. Proteomics 2011, 10, 009431-009450. 3 Skinner, O. S.; Haverland, N. A.; Fornelli, L.; Melani, R. D.; Do Vale, L. H. F.; Seckler, H. S.; Doubleday, P.F.; Schachner, L. F.; Srzentić, K.; Kelleher, N. L.; Compton, P. D., Top-down characterization of endogenous protein complexes with native proteomics. Nat. Chem. Biol. 2017, 14, 36. 4 Fort, K. L.; van de Waterbeemd, M.; Boll, D.; Reinhardt-Szyba, M.; Belov, M. E.; Sasaki, E.; Zschoche, R.; Hilvert, D.; Makarov, A. A.; Heck, A. J. R., Expanding the structural analysis capabilities on an Orbitrap-based mass spectrometer for large macromolecular complexes. Analyst 2018, 143, 100-105. 5 Xian, F.; Hendrickson, C. L.; Marshall, A. G., High Resolution Mass Spectrometry. Anal. Chem. 2012, 84, 708−719 6 Mamyrin, B. A., Time-of-flight mass spectrometry (concepts, achievements, and prospects). Int. J. Mass Spectrom. 2001, 206, 251−266. 7 Pringle, S. D.; Giles, K.; Wildgoose, J. L.; Williams, J. P.; Slade, S. E.; Thalassinos, K.; Bateman, R. H.; Bowers, M. T.; Scrivens, J. H., An investigation of the mobility separation of some peptide and protein ions using a new hybrid quadrupole/travelling wave IMS/oa-ToF instrument. Int. J. Mass Spectrom. 2007, 261, 1−12. 8 Toyoda, M.; Okumura, D.; Ishihara, M.; Katakuse, I., Multi‐turn time‐of‐flight mass spectrometers with electrostatic sectors. J. Mass Spectrom. 2003, 38, 1125−1142. 9 Kostyukevich, Y.I., Vladimirov, G.N., Nikolaev, E.N., Dynamically Harmonized FT-ICR Cell with Specially Shaped Electrodes for Compensation of Inhomogeneity of the Magnetic Field. Computer Simulations of the Electric Field and Ion Motion Dynamics. J. Am. Soc. Mass Spectrom. 2012, 23, 2198-2207. 10 Chen, L.; Wang, T.-C. L.; Ricca, T. L.; Marshall, A. G., Phase-modulated stored waveform inverse Fourier transform excitation for trapped ion mass spectrometry. Anal. Chem. 1987, 59, 449−454. 11 Guan, S.; Marshall, A. G., Stored waveform inverse Fourier transform (SWIFT) ion excitation in trapped-ion mass spectometry: Theory and applications. Int. J. Mass Spectrom. Ion Processes 1996, 157/158, 5−37. 12 Marshall, A. G.; Wang, T.-C. L.; Ricca, T. L., Tailored excitation for Fourier transform ion cyclotron mass spectrometry. J. Am. Chem. Soc. 1985, 107, 7893−7897. 1


Page 26 of 29

Page 27 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Schaub, T.M., Hendrickson, C.L., Horning, S., Quinn, J. P., Senko, M. W., Marshall, A. G., HighPerformance Mass Spectrometry: Fourier Transform Ion Cyclotron Resonance at 14.5 Tesla. Anal. Chem. 2008, 80, 3985-3990. 14 Kingdon, K. H., A Method for the Neutralization of Electron Space Charge by Positive Ionization at Very Low Gas Pressures. Phys. Rev. 1923, 21, 408-418. 15 Knight, R. D., Storage of ions from laser‐produced plasmas. Appl. Phys. Lett. 1981, 38, 221-223. 16 Makarov, A., Electrostatic Axially Harmonic Orbital Trapping: A High-Performance Technique of Mass Analysis. Anal. Chem. 2000, 72, 1156-1162. 17 Marshall, A. G.; Hendrickson, C. L.; Jackson, G. S., Fourier Transform Ion Cyclotron Resonance Mass Spectrometry. In Encyclopedia of Analytical Chemistry, 2006 (eds R. A. Meyers and O. D. Sparkman). doi:10.1002/9780470027318.a6006m. 18 N. Nikolaev, E.; N. Vladimirov, G.; Jertz, R.; Baykut, G., From Supercomputer Modeling to Highest Mass Resolution in FT-ICR. Mass Spectrom. 2013, 2, S0010. 19 Smith, R. D.; Cheng, X.; Brace, J. E.; Hofstadler, S. A.; Anderson, G. A., Trapping, detection and reaction of very large single molecular ions by mass spectrometry. Nature 1994, 369, 137. 20 Chen, R.; Cheng, X.; Mitchell, D. W.; Hofstadler, S. A.; Wu, Q.; Rockwood, A. L.; Sherman, M. G.; Smith, R. D., Trapping, Detection, and Mass Determination of Coliphage T4 DNA Ions by Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry. Anal. Chem. 1995, 67, 11591163. 21 Bruce, J. E.; Cheng, X.; Bakhtiar, R.; Wu, Q.; Hofstadler, S. A.; Anderson, G. A.; Smith, R. D., Trapping, Detection, and Mass Measurement of Individual Ions in a Fourier Transform Ion Cyclotron Resonance Mass Spectrometer. J. Am. Chem. Soc. 1994, 116, 7839-7847. 22 Chen, R.; Wu, Q.; Mitchell, D. W.; Hofstadler, S. A.; Rockwood, A. L.; Smith, R. D., Direct Charge Number and Molecular Weight Determination of Large Individual Ions by Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry. Anal. Chem. 1994, 66, 3964-3969. 23 Bruce, J. E.; Anderson, G. A.; Udseth, H. R.; Smith, R. D., Large Molecule Characterization Based upon Individual Ion Detection with Electrospray Ionization-FTICR Mass Spectrometry. Anal. Chem. 1998, 70, 519-525. 24 Makarov, A.; Denisov, E.; Lange, O.; Horning, S., Dynamic Range of Mass Accuracy in LTQ Orbitrap Hybrid Mass Spectrometer. J. Am. Soc. Mass Spectrom. 2006, 17, 977-982. 25 Makarov, A.; Denisov, E., Dynamics of Ions of Intact Proteins in the Orbitrap Mass Analyzer. J. Am. Soc. Mass Spectrom. 2009, 20, 1486-1495. 26 Makarov, A. Method of generating a mass spectrum having improved resolving power; U.S. Patent 9043164; May 26, 2015. 27 Waters LCT Premier--Enhanced Spectral Resolution for Improved Analytical Specificity; APNT1545457; Milford, MA, 2007. 28 Agilent Technologies, Time-of-Flight Mass Spectrometry; 5990-9207EN; Santa Clara, CA, 2011. 29 Belov, M. E.; Damoc, E.; Denisov, E.; Compton, P. D.; Horning, S.; Makarov, A. A.; Kelleher, N. L., From Protein Complexes to Subunit Backbone Fragments: A Multi-stage Approach to Native Mass Spectrometry. Anal. Chem. 2013, 85, 11163-11173. 30 Sanders, J. D.; Grinfeld, D.; Aizikov, K.; Makarov, A.; Holden, D. D.; Brodbelt, J. S., Determination of Collision Cross-Sections of Protein Ions in an Orbitrap Mass Analyzer. Anal. Chem. 2018, 90, 58965902. 31 Covey, T.; Douglas, D. J., Collision cross sections for protein ions. J. Am. Soc. Mass Spectrom. 1993, 4, 616-623. 32 Javahery, G.; Thomson, B., A segmented radiofrequency-only quadrupole collision cell for measurements of ion collision cross section on a triple quadrupole mass spectrometer. J. Am. Soc. Mass Spectrom. 1997, 8, 697-702. 33 Campuzano, I. D. G.; Larriba, C.; Bagal, D.; Schnier, P. D., Ion Mobility and Mass Spectrometry Measurements of the Humanized IgGk NIST Monoclonal Antibody. In State-of-the-Art and Emerging 13



Technologies for Therapeutic Monoclonal Antibody Characterization Volume 3. Defining the Next Generation of Analytical and Biophysical Techniques, American Chemical Society: 2015; Vol. 1202, pp 75-112. 34 Zubarev, R. A.; Makarov, A., Orbitrap Mass Spectrometry. Anal. Chem. 2013, 85, 5288-5296. 35 Shaw, J. B.; Brodbelt, J. S., Extending the Isotopically Resolved Mass Range of Orbitrap Mass Spectrometers. Anal. Chem. 2013, 85, 8313-8318. 36 Strohalm, M.; Kavan, D.; Novák, P.; Volný, M.; Havlíček, V., mMass 3: A Cross-Platform Software Environment for Precise Analysis of Mass Spectrometric Data. Anal. Chem. 2010, 82, 4648-4651.


Page 28 of 29

Page 29 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table of Contents Only


Measurement of Individual Ions Sharply Increases ... - ACS Publications

Recommend Documents