Multiplexed Middle-Down Mass Spectrometry as a Method for

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Multiplexed Middle-Down Mass Spectrometry Reveals Light and Heavy Chain Connectivity in a Monoclonal Antibody Kristina Srzentic, Konstantin O. Nagornov, Luca Fornelli, Anna A. Lobas, Daniel Ayoub, Anton Kozhinov, Natalia Gasilova, Laure Menin, Alain Beck, Mikhail Gorshkov, Konstantin Aizikov, and Yury O. Tsybin Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b02398 • Publication Date (Web): 25 Sep 2018 Downloaded from http://pubs.acs.org on September 26, 2018

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Analytical Chemistry

Multiplexed Middle-Down Mass Spectrometry Reveals Light and Heavy Chain Connectivity in a Monoclonal Antibody

Kristina Srzentić1$, Konstantin O. Nagornov2, Luca Fornelli1$, Anna A. Lobas3, Daniel Ayoub1, Anton N. Kozhinov2, Natalia Gasilova,1 Laure Menin,1 Alain Beck,4 Mikhail V. Gorshkov3,5, Konstantin Aizikov6 and Yury O. Tsybin2*

1

Ecole Polytechnique Fédérale de Lausanne, 1015 Lausanne, Switzerland

2

Spectroswiss, EPFL Innovation Park, 1015 Lausanne, Switzerland

3

Institute for Energy Problems of Chemical Physics, Russian Academy of

Sciences, 119334 Moscow, Russia 4 5

Centre d’Immunologie Pierre Fabre, 74160 St. Julien-en-Genevois, France Moscow Institute of Physics and Technology (State University), 141707

Dolgoprudny, Moscow Region, Russia 6

Thermo Fisher Scientific GmbH, 28199 Bremen, Germany

$present

address: Northwestern University, 60208 Evanston, IL, USA

* Correspondence should be addressed to Dr. Yury O. Tsybin, Spectroswiss, EPFL Innovation Park, Building I, 1015 Lausanne, Switzerland. E-mail: [email protected]

Running title: Multiplexed middle-down FTMS to reveal chain connectivity in IgG1

ACS Paragon Plus Environment

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Abstract Pairing light and heavy chains in monoclonal antibodies (mAbs) using topdown (TD) or middle-down (MD) mass spectrometry (MS) may complement the sequence information on single chains provided by high-throughput genomic sequencing and bottom-up proteomics, favoring the rational selection of drug candidates. The 50 kDa F(ab) subunits of mAbs are the smallest structural units that contain the required information on chain pairing. These subunits can be enzymatically produced from whole mAbs and interrogated in their intact form by TD/MD MS approaches. However, the high structural complexity of F(ab) subunits requires increased sensitivity of the modern TD/MD MS for a comprehensive structural analysis. To address this and similar challenges, we developed and applied a multiplexed TD/MD MS workflow based on spectral averaging of tandem mass spectra (MS/MS) across multiple liquid chromatography (LC)-MS/MS runs acquired in reduced or full profile mode using an Orbitrap Fourier transform mass spectrometer (FTMS). We first benchmark the workflow using myoglobin as a reference protein and then validate it for the analysis of the 50 kDa F(ab) subunit of a therapeutic mAb, trastuzumab. Obtained results confirm the envisioned benefits in terms of increased signal-to-noise ratio of product ions from utilizing multiple LCMS/MS runs for TD/MD protein analysis using mass spectral averaging. The workflow performance is compared with the earlier introduced multiplexed TD/MD MS workflow based on transient averaging in Orbitrap FTMS. For the latter, we also report on enabling absorption mode FT processing and demonstrate its comparable performance to the enhanced FT (eFT) spectral representation.


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Keywords: top-down mass spectrometry; middle-down mass spectrometry; immunoglobulin G1, IgG1; monoclonal antibody, mAb; chain pairing; Fourier transform mass spectrometry, FTMS; Orbitrap; transient averaging; spectral summation; spectral averaging; absorption mode FT.


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Introduction Top-down (TD) and middle-down (MD) mass spectrometry (MS) approaches are gaining a growing importance in structural characterization of proteins in general and monoclonal antibodies (mAbs) in particular.1-6 For mAbs, TD MS refers to tandem mass spectrometry (MS/MS) of their intact, ~150 kDa, proteoforms, whereas MD MS implies in-solution mAb cleavage (enzymatically or chemically) into a few large, typically of 25-100 kDa, subunits prior to their MS/MS analysis.2,6,7 Despite the recent technical advancements, sensitivity remains a limitation in TD/MD MS-based structural characterization of larger, 50-150 kDa, proteins. Indeed, in MS/MS of proteins the precursor ion current is split into a large number of fragmentation channels, each of which includes a limited ion subpopulation. Therefore, summation or averaging of a certain number of single measurements is typically needed to increase the signal-tonoise ratio (SNR) of product ions to the level required for confident identification. Under a “single measurement” there could be either a mass spectrum, or a time-domain signal (transient). The latter is recorded by data acquisition system in Fourier transform mass spectrometry (FTMS), which is currently the method of choice for TD/MD MS owing to its high resolution and mass accuracy.2,8-11 The most sensitive and accurate approach to FTMS data processing from multiple measurements is to first average all time-domain signals (transients) and then FT the final averaged transient to yield a frequency spectrum, which can be further converted into a mass spectrum.12 An alternative route is to first FT each transient (i.e., at the single microscan level), or FT a transient averaged from a set of microscan-level transients (i.e., at the scan level), and then perform spectral summation (or averaging) of the


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resulting mass spectra. Mass spectra averaging procedure differs from the summation one by an additional step of summed mass spectrum normalization by the number of mass spectra summed. In the case of LC-MS/MS experiments, the available measurement time is determined by the elution time of a protein from the LC column and by the complexity of the sample – dictating the need to perform MS/MS on different co-eluting precursors in a short period of time. The number of potentially available microscans (single measurements) is thus limited by the total time allocated for the analysis of each selected precursor. For targeted TD/MD MS experiment performed by FTMS, in a single LC-MS/MS run a total of 10-40 microscans are typically acquired across the elution peak of a single protein.2,13 However, to reach the SNR level required to identify low-abundant product ions in convoluted MS/MS spectra, the total number of acquired microscans (transients) is to be significantly increased, preferably more than 10-fold, compared to the single LC-MS/MS datasets. This result can be achieved either using off-line protein purification and subsequent MS/MS experiment from direct infusion of purified proteins,8 or performing multiple consecutive

on-line

LC-MS/MS

experiments

(technical

replicates).2,13

Transients obtained from multiple LC-MS/MS runs can be averaged off-line for a given chromatographic peak to yield the final averaged transient which can be first transformed into a frequency spectrum and then calibrated into a final mass spectrum. When the averaging of N transients is performed, both standard deviation of noise and the noise baseline level of the normalized mass spectrum reduce as √𝑁𝑁, leading to the improved sensitivity and spectral

dynamic range owing to the detection of peaks with lower SNR values. The


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benefits of transient averaging from multiple LC-MS/MS runs for improved TD/MD MS have already been demonstrated for the structural analysis of intact, 150 kDa, IgG1s,2,13 and of their 25 kDa subunits.7 However, transients and their certain FT processing capabilities, such as eFT, are not readily available to the FTMS end-users. Therefore, averaging of mass spectra from multiple consecutive TD/MD MS measurements can be an alternative to transient averaging. Averaging (or summation) of mass spectra across multiple LC-MS/MS experiments for improving the TD/MD MS performance has not been reported yet. Prior to understanding spectral averaging, it is important to define the characteristics of the mass spectra as the single measurement unit: (i) magnitude and absorption mode FT; and (ii) full and reduced profile mass spectra. First, there are different approaches to transient conversion into frequency spectra in modern FTMS, with magnitude mode FT (mFT) and absorption mode FT (aFT) being the most common and fundamental.14 The third approach, known as enhanced FT (eFT) and realized for transient processing on Orbitrap FTMS instruments, uses results from both mFT and aFT processing: the top portion of the peak is a result of the aFT processing and the bottom part is derived from the mFT.15 Owing to the expansion of Orbitrap FTMS, eFT is the most commonly used approach in FTMS nowadays. Second, mass spectra are typically represented in two modes: full profile and reduced profile. The full profile mass spectra contain information on all peaks and all noise components, but have a large file size. To reduce the size of the data files by about an order of magnitude, any peaks below a certain intensity level are removed from the full profile mass spectra to produce the reduced


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profile mass spectra.16 As a result, together with the noise peaks this procedure may also remove the low abundance analyte (product) ions at the single scan level. Therefore, spectral averaging of reduced profile mass spectra is inherently less sensitive than when full profile mass spectra are provided. Here, we develop a TD/MD MS workflow based on spectral averaging (or summation), apply it for the structural characterization of a myoglobin and a 50 kDa F(ab) subunit of trastuzumab and compare its performance to the transient averaging approach. We selected a 50 kDa F(ab) subunit of a mAb as the target protein as the smallest mAb subunit that contains information on the connectivity (pairing) of a light and a heavy chain. More specifically, F(ab) subunit is made of a light chain and an Fd subunit (the N-terminal half of the heavy chain) linked by an inter-molecular disulfide bond. In the past, only MS-based bottom-up proteomics, in a combination with high-throughput genomic sequencing, has been used for the identification of organismproduced mAbs present as complex biomolecular mixtures in body tissues or fluids, e.g., serum.17,18 Genomics data enables the creation of databases containing partial sequence information on separate light and heavy chains of mAbs potentially present in these mixtures, whereas bottom-up proteomics data from the same mixtures provides information on the identity of the light and heavy chains actually present in the samples.17-19 However, the proteolytic reduction of the original proteins (mAbs) into a pool of short peptides in bottom-up proteomics causes the loss of the chain-pairing information and enlarges the list of potential mAb lead candidates, slowing down the discovery of new therapeutic mAbs. The proposed here multiplexed TD/MD MS


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approach aims to overcome the bottom-up proteomics limitation for mAb chain pairing.

Experimental methods Chemicals. Water, acetonitrile (ACN), formic acid (FA) and trifluoroethanol (TFE) were obtained in LC-MS purity grade. Water and ACN were purchased from Fluka Analytical (Buchs, Switzerland). FA was obtained from Merck (Zug, Switzerland) and TFE from Acros Organics (Geel, Belgium).

Samples. Horse myoglobin was obtained from Sigma Aldrich (Buchs, Switzerland), therapeutic monoclonal antibody of the IgG1 class, trastuzumab (Herceptin, Genentech) was obtained as the commercially available European Medicines Agency approved version and formulation.

GingisKHAN digestion. The recently introduced GingisKHAN protease has been claimed to demonstrate a particularly high specificity for the cleavage above the hinge region. It is targeted to digest human IgG1 between K223 and T224 producing a homogenous pool of F(ab) and Fc subunits. GingisKHAN (Genovis, Lund, Sweden) digestion of IgG1 was performed in formulation buffer. Two units of GingisKHAN were added to each μg of IgG in Tris-HCl, pH 8, and left to react for 1 hr at 37 °C in presence of 2 mM Cys solution. The reaction was quenched by acidifying the solution to 1 % (v/v) TFA. For analysis, samples were diluted with 0.1 % FA in water to a final protein concentration of 1 μg/μl.


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Liquid chromatography – mass spectrometry. The chromatographic separation of IgG proteolytic fragments was performed using an UltiMate 3000 LC system (Thermo Fisher Scientific, Germering, Germany) under UHPLC conditions. A combination of reversed phase C4 guard-column (Acquity UPLC PrST C4 VanGuard pre-column, 2.1 x 5 mm, particle size 1.7 μm, pore size 300 Å, Waters, Baden-Dättwil, Switzerland) and C4 analytical column (Acquity UPLC PrST C4, 1 x 150 mm, particle size 1.7 μm, pore size 300 Å, Waters) was employed to ensure on-line IgG subunit desalting and separation. For each injection, 1 μg of digestion product was loaded on the column, heated to 60 °C. After initial loading at 5 % solution B (organic phase), a gradient of solution B from 10 to 45 % in 15 minutes was applied at a flow rate of 100 μl/min. Solution A consisted of 0.1% of FA in water, whereas solution B was composed of 99,9 % ACN and 0.1 % FA. The LC column outlet was on-line coupled with the electrospray ionization (ESI) source of the mass spectrometer. MS experiments were performed on an ETD-enabled hybrid linear ion trap highfield Orbitrap FT mass spectrometer (Orbitrap Elite FTMS, Thermo Scientific, Bremen, Germany). Separate LC-MS experiments were dedicated to record broadband mass spectra and ETD tandem mass spectra. All mass spectra were acquired using ion detection in the Orbitrap FTMS, in the m/z ranges 400-2800 and 200-2000 for broadband and tandem mass spectra, respectively. All mass spectrometry aquisitions were performed in „protein mode“: N2 gas pressure in the HCD cell was reduced to reach a pressure in the orbitrap mass analyzer region that is approximately of 0.15E-10 torr higher than the „base pressure“ measured in the same region with the N2 flux completely shut down.7 Additionally, ions were captured and temporarily


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stored in the HCD cell before ion transmission to the Orbitrap analyzer.2,20 Broadband mass spectra were recorded with 15’000 resolution (eFT, at 400 m/z), whereas product ion detection in MS/MS was performed at 120’000 resolution (eFT, at 400 m/z). The target value for the automatic gain control (AGC) was set to one million charges for both MS and MS/MS modes. For ETD experiments, AGC target value for fluoranthene radical anions was set to 7E5 charges, with anion maximum injection time of 100 ms. ETD ion-ion interaction time was set to 10 ms. All Orbitrap FTMS scans were recorded averaging 10 microscans. For F(ab) analysis, in each single LC-MS/MS run up to 100 microscans (10 scans) were averaged, thus for ten consecutive LCMS/MS runs up to 1000 microscans (100 scans) were averaged. Myoglobin elution profile allowed to acquire a twice higher number of scans. Isolation windows for ETD of myoglobin or IgG subunits included multiple charge states per precursor protein (isolation width: 80-200 m/z). To estimate the number of LC-MS/MS runs to acquire for a particular sample, we suggest to evaluate the SNR values for low-abundant isotopic envelopes of product ions in a single LC-MS/MS run, estimate the required SNR increase for confident product ion assignment, and consider that SNR increases as a square root of the number of scans (about 2-fold for 4 LCMS/MS runs, about 3-fold for 9 LC-MS/MS runs, etc.). The next iteration may be then performed, consisting in the evaluation of the SNR values for lowabundant product ions in the averaged tandem mass spectrum and setting objectives for the additional LC-MS/MS runs to acquire. To demonstrate the advantages of the multiplexed approach in this work, up to 10 LC-MS/MS runs were acquired consecutively (technical replicates). The number of LC-


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MS/MS runs was selected based on the prior work on TD/MD MS of whole IgGs and their 25 kDa subunits and following the above considerations.2,7,13

Data acqusition and signal processing. The mass spectra (*.raw) were obtained in either full or reduced profile mode in separate data sets via standard software interface (Tune, Thermo Fisher Scientific). In parallel to mass spectra acquisition for both data sets, the transients in MIDAS file format (*.dat)21 were acquired using an advanced feature added by the vendor to the commercial data acquisition software (Thermo Fisher Scientific), as employed previously.2 Further, the given number of mass spectra were averaged and renormalized to the base peak within each single LC-MS/MS run via standard data analysis software (Xcalibur, Thermo Fisher Scientific). The averaged mass spectra from multiple separate LC-MS/MS runs were produced using MS File Reader (Thermo Fisher Scientific) and Peak-by-Peak software for FTMS data processing (Spectroswiss, Lausanne, Switzerland). The averaging of the corresponding transients (in *.dat format) was performed using Peakby-Peak. Transients obtained in this manner were submitted for absorption mode Fourier transform (aFT) signal processing performed using a dedicated algorithm,13

implemented

as

a

part

of

AutoVectis

software

suite

(Spectroswiss). The final mass spectra for both spectral and transient averaging approaches were converted into mzXML or MGF open file formats using Peak-by-Peak for downstream data analysis. In the second approach to transient averaging, transients were averaged directly at Thermo Fisher Scientific and processed using enhanced Fourier transform (eFT) algorithm to yield mass spectra in *.raw format.2,13,15


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Data analysis. Deconvolution of intact protein mass spectra was performed using Protein Deconvolution software (Thermo Fisher Scientific). Peak picking and deconvolution of tandem mass spectra were performed using MASH Suite software with the following parameters.22 For both myoglobin and F(ab) analysis, signal-to-noise ratio (SNR) threshold, as estimated and provided by MASH Suite software, was set to 5 for both transient and full profile mode spectral averaging. The SNR level of 5 was selected based on an extensive data analysis which demonstrated an increased confidence of our product ion assignment, including improved P-score values.23 For example, reducing SNR threshold to 3 increased sequence coverage, but also provided a higher Pscore, indicating the reduced confidence of product ion assignment. For spectral averaging of the LC-MS/MS runs acquired in the reduced profile mode, we found SNR threshold of 0.2 (similarly, estimated and provided by MASH Suite software) as an optimum value. The specified threshold level reflects the nature of the reduced profile mass spectra – whereas the noise is already removed by pre-processing in each scan, averaging of many scans builds up a certain noise level.24 Therefore, further noise thresholding, albeit not as pronounced as for the full profile data, is beneficial for product ion assignment. For product ion assignment, ETD mass spectra of myoglobin and F(ab) subunit were matched against their respective custom databases (containing the primary sequences of proteins in question). In case of the branched (zHC + zLC) ions analysis, a list of theoretical fragments (neutral mass) was calculated using an in-house Python script based on the Pyteomics library.25 The internal


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disulfide bonds as well as the interchain disulfide bonds were considered preserved. All probable pairs of heavy and light chains were considered; fragmentation N-terminal to proline or inside the cysteine loops was excluded from the calculations. A maximum of one cleavage site in each chain was allowed. A minimum matching score of 80%, as estimated by MASH Suite, between theoretical and experimental isotopic distibutions was typically used for filtering product ions. When considering branched product ions, which demonstrate lower abundance and thus statistics of isotopic envelope presentation, we also considered selected cases with the matching score reduced down to 70 %. Manual validation was performed for each assigned product ion.

Results and discussion Spectral averaging in FTMS. Spectral averaging of N mass spectra reduces the standard deviation of noise as √𝑁𝑁 for either aFT or mFT spectral

representation modes (Figure S1, Supporting Information). However, the noise baseline (mean of noise) for mass spectra in the mFT mode is found at about the same level for a single scan mass spectrum and the final averaged one. On

contrary, averaging of mass spectra in the aFT mode lowers the baseline level as √𝑁𝑁, providing improved sensitivity and dynamic range. The reason for that difference is simple. All components of mass spectra in the mFT mode, as well

as the mean of noise level, have positive values, whereas the aFT mass spectra contain both positive and negative components and the mean of noise is typically around zero. Spectral averaging of mass spectra in the eFT mode behaves comparably to the mFT processing, as expected from the eFT


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definition (Figure S2, Supporting Information). Results reported in Figures S1 and S2 further confirm the expected advantages from transient averaging of all scans (followed by aFT or eFT processing) compared to single-scan transient FT processing and spectral averaging of eFT or mFT mass spectra. It should be noted that spectral averaging of mass spectra each represented in the aFT mode would provide results similar to transient averaging of all scans (followed by aFT processing). However, up to date true aFT signal processing of transients acquired on Orbitrap FTMS has not been reported. To summarize, spectral averaging of full profile mass spectra is preferred over the reduced profile one. If available, transient averaging should be performed, followed by FT processing. Averaging of mass spectra represented in the aFT mode is advantageous compared to eFT and mFT modes. If aFT full profile mass spectra are available, their averaging should be comparable in performance to the transient averaging of all scans followed by aFT or eFT processing. In the following, we will benchmark these diverse data averaging procedures for improving the performance of TD/MD MS.

Top-down/middle-down protein analysis workflow with data multiplexing. A generic pipeline for top-down/middle-down protein analysis using data multiplexing as suggested herein is shown in Figure 1. The structural analysis of mAbs targeting heavy and light chain connectivity problem is taken as an example. The enzymatic procedure entails the use of the novel KGP protease (commercially available as GingisKHAN).26 This protease is selective towards the immunoglobulin G1 (IgG1) class and, more importantly, it cleaves in a highly specific and reproducible manner demonstrating primary structure


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specificity uniquely to the …K-T... bond above the hinge region. The total ion chromatogram (TIC) of a KGP-digested IgG1 typically shows baselineseparated ~50 kDa Fc and ~50 kDa F(ab) subunits, Figure 1.26,27 After a single rapid digestion step, a number of consecutive LC-MS/MS runs, for example ten, are performed. The gas-phase fragmentation of a F(ab) subunit backbone generates a series of “branched product ions”.28 These branched product ions allow for chain pairing by containing a C-terminal part of a light chain and a C-terminal part of an Fd subunit connected via an inter-molecular disulfide bond. Electron transfer dissociation (ETD) can be employed for generating extensive backbone cleavages on one hand and keeping the inter-chain S-S bond intact on the other hand.2,29-32 When branched product ions are not formed, canonical c/z-type ions upon N-Cα bond cleavage in a F(ab) protein backbone are produced in a concert with the inter-chain S-S bond “reduction” by ETD. The interchain S-S bond dissociation upon electron transfer (or capture) is believed to leave one cysteine residue in an even-electron thiol form (or a cyclic sulfide)33 and another one in a radical form.29,30 Other MS/MS methods, e.g., higher energy collisional dissociation (HCD)34 and ultraviolet photo-dissociation (UVPD),3 can be performed instead or in conjunction with ETD.35-37 The spectral and the transient averaging approaches are organized as described above.

Method development for analysis of myoglobin. The proposed workflow was first tested on a small intact protein, ~17 kDa myoglobin (Figures S3-S5, Supporting Information). As expected, using data averaging from several LCMS/MS runs the SNR values of product ions improve and the experimental


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distributions of isotopic peak intensities approach more closely the theoretical ones. However, given relatively simple ETD fragmentation patterns of myoglobin in TD MS, the sequence coverages obtained with spectral and transient averaging for the employed numbers of scans are comparable.

Enzymatic production of F(ab) subunits. Broadband mass spectra of trastuzumab subunits confirm the specific nature of GingisKHAN enzyme (Figure S6, Supporting Information).27,38 In case of F(ab) subunits, a “wide“ isolation window of 200 m/z was centered at a charge state 38+ and comprised six isotopically unresolved charge states of F(ab) aimed for MS/MS (Figure S6 top panel). An expanded view into a broadband mass spectrum of the Fc subunit eluting prior to F(ab) shows the characteristic glycoform distribution in the 33-35+ charge states (Figure S6 bottom panel). For the Fc subunit an ETD event was not triggered, as the Fc portion of the antibody does not carry information on chain connectivity. On the other hand, the workflow described herein should be also advantageous for the analysis of Fc proteoforms, or glycoforms.

Tandem mass spectrometry of F(ab) subunit. The total number of potential fragmentation channels in ETD MS/MS of a ~50 kDa F(ab) subunit is high and includes not only the canonical N- and C-terminal-containing product ions, but also internal product ions and the branched product ions. As a result, tandem mass spectra are convoluted, Figure 2. Transient averaging from 10 LC-MS/MS runs expectedly improves a single LC-MS/MS run data quality for MS/MS analysis of a F(ab) subunit of trastuzumab (Figure S7,


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Supporting Information). Low abundant product ions may become visible and more confidently identifiable due to up to 3-fold noise reduction and improved isotopic envelope statistics. The equidistant peaks in the ETD mass spectrum shown in Figure 2 are believed to be due to the interference of the overlapping isotopic envelopes of the highly charged large product ions. Furthermore, transforming full profile into reduced profile mode mass spectra decreases the possibility to observe in the averaged mass spectrum the very low abundant product ions. Figure S8 (Supporting Information) reports several examples of low-abundant product ions as they appear in mass spectra obtained by summing either reduced or full profile mode mass spectra (or by averaging the corresponding transients followed by aFT processing). These examples show that the isotopic distributions resulting from the sum of reduced profile mass spectra may deviate from the theoretical distributions, potentially leading to the loss of a product ion match, whereas this may not be the case when full profile mode mass spectra are employed. Results reported in Figure S9 (Supporting Information) serve to benchmark the performance of the aFT spectral representation mode by its comparison to the common eFT mass spectra representation. Results presented here suggest that the aFT processing of transients provides comparable results to the eFT processing in regard to the peak intensity and resolution. On one hand, these results demonstrate, for the first time, that aFT processing can be performed on Orbitrap transients. On the other hand, they open up a possibility for a general user to operate with transients and match the performance of eFT processing, the latter being vendor-specific and not available to the general public.


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Sequence coverage of F(ab) subunits. When a single LC-MS/MS run is considered, the fragmentation maps obtained from ETD mass spectra of F(ab) resulting from spectral (full profile, eFT) or transient averaging (aFT) are very similar (9-10 % sequence coverage for each chain), see Figure S10, Supporting Information. Interestingly, most of the matched product ions in the fragmentation maps are c-type ions. Presumably, that is due to the disulfide bonding network pattern and the charge location in the F(ab) subunits. The inter-molecular disulfide bridge linking light chain and a Fd subunit (the Nterminal part of the heavy chain) is located at the C-terminus of each subunit and involves the last residue of the light chain and the third to last residue of the Fd subunit.38 The results of spectral averaging for eFT mass spectra acquired in 10 LC MS/MS runs and represented in reduced profile (Figure 3) or in full profile (Figure 4) modes demonstrate an important sequence coverage increase compared to a single LC-MS/MS run. In the case of data acquired in full profile mode, sequence coverage reaches almost 19 and 30% for light chain and Fd subunit, respectively. Overall, summing reduced profile spectra rather than full profile ones decreases the total final sequence coverage by about 10-15%. The difference between reduced and full profile final sequence coverage is a function of the number of scans averaged. Therefore, if more LC-MS/MS runs are considered, this difference will increase proportionally. The mass spectra obtained by transient averaging produced a slightly higher sequence coverage for the F(ab) subunit (Figure S11, Supporting Information). Considering the minimal difference between the spectral and


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transient averaging methods for what concerns the canonical c- and z-type ions, we can conclude that the spectral averaging approach works well, particularly when full profile mode is employed. Nevertheless, the transient averaging provides more confident identification of product ions compared to spectral averaging in both the full and reduced profile modes.

F(ab) subunit structure analysis using branched product ions. The branched product ions are defined as product ions comprising a portion of the heavy chain (zFd product ions of Fd subunit) and a portion of the light chain (zLc product ions) that are linked by the above mentioned inter-molecular disulfide bond. The c-type ions can be excluded as they cannot lead to the branched product ion formation. Remarkably, the total theoretical number of zFd+zLc ions for F(ab) is about 7’500. An unambiguous assignment of the branched product ions based solely on the MS/MS data is not always possible. About 16 % of product ions share their molecular formulas being structural isomers. The vast majority of structural isomers belong to the branched ion category, whereas only 4 of the canonical product ions (in this case, 4 c-ions) contribute to the ambiguity in the assignment (Table S1 and Figure S12, Supporting Information). The summed tandem mass spectra of a F(ab) subunit were used for matching a list of potential branched ions to the experimental data. This attempt demonstrated that ETD indeed forms branched z-type ions, as depicted in the right panels of Figures 3 and 4, as well as in Table S2 (Supporting Information). About 50% of the matched branched product ions are located in those regions of the sequences of each chain that seem to be


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poorly covered in the fragmentation maps including only c- and z-type ions, particularly the C-terminus of both light chain and Fd subunit. Notably, some very large branched product ions (>15 kDa) were also matched. The transient averaging method exhibits an advantage in increasing the final SNR of tandem mass spectra, as it allows to increase the sequence coverage from branched product ions from the 8-9 % obtained by reduced profile spectral averaging to about 12 % for full profile spectral averaging and further to about 15 % for transient averaging with eFT signal processing (Figures S10 and S11, Supporting Information). A comparative product ion analysis performed on both terminalcontaining (i.e., almost uniquely N-terminal c-ions) and branched product ions reveals that the favored hot-spots for backbone cleavages (i.e., the bonds whose rupture produces the most abundant fragments) are found within disulfide-free loops (Figure S13, Supporting Information). The current explanation is that in this case the generation of a product ion requires only one bond to be cleaved, as opposed to the case of disulfide-protected regions within which two cleavages are needed.2 Although the presented here assignment strategy is more sophisticated than the traditional one that accounts only for the non-branched product ions, it still does not include the assignment of internal product ions (produced within a single protein, or a single chain), the number of which can be high, as well as y-ions.39,40

Extension of the multiplexed LC-MS/MS approach. Data analysis performed here confirmed that increase in sensitivity, or SNR values, scales comparably for spectral and transient averaging as a function of a number of scans in


20

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TD/MD FTMS. As expected, the most confident and extensive protein sequence coverage is obtained with transient averaging, followed by spectral averaging of mass spectra acquired in the full profile mode and then by spectral averaging of mass spectra acquired in the reduced profile mode, Table 1. Identification of the interchain S-S bond location and degree of its scrambling is one of the important metrics of the mAb structural analysis methods.38,41 Results summarized in Table 1 illustrate the importance of increasing the SNR values of product ions for enabling detection of branched product ions, which contain a preserved interchain S-S bond. Therefore, the total amount of the branched product ions is minor compared to the canonical ones already for the most common IgG1 structure. In case of scrambling of interchain S-S bonds, it is unlikely that the presented here approach would be particularly beneficial compared to the current ones.38,41 From the light and heavy chain pairing perspective, primarily large branched product ions, with cleavages in the variable domains of both chains (specifically within their CDRs), are the most informative. Therefore, scrambling of the S-S bonds, especially to the cysteine residues of the C-terminal intrachain S-S bond of any chain, should not influence the outcome of the chain pairing approach presented here. Based on the fundamental nature of the transient and spectral averaging procedures (SNR dependence on the number of spectra/transients averaged), the approaches presented here for ETD MS/MS and a given model of Orbitrap FTMS (Orbitrap Elite) are directly applicable for protein structure analysis using other MS/MS methods and FTMS instruments. However,


21


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whereas technically the SNR dependence will remain valid, the analytical benefits are to be benchmarked for specific methods and instruments. For example, transferring the developed here approach of spectral averaging to HCD MS/MS on a Q Exactive HF Orbitrap FTMS confirms a comparable, and well-pronounced, advantage of the multiplexing LC-MS/MS for sequencing of a F(ab) subunit of trastuzumab (Figures S14 and S15, Table S3, Supporting Information). Despite the fact that HCD is believed not to yield substantial sequence coverage on large proteins, such as F(ab) subunit, the obtained results provide sequence coverage with canonical b/y ions exceeding 20% for the light chain and 24% for the Fd subunit of the F(ab). Therefore, the potential HCD MS/MS application for pairing light and heavy chains in mAbs may be investigated. Similar analytical benefits may be expected for the MS/MS methods more recently applied for top-down FTMS, such as UVPD and in-beam ECD.4244

Finally, the proposed here workflow welcomes further approaches to

increase ETD MS/MS sensitivity, for example by an additional energetic excitation of intermediate radical species, which may reduce the number of the LC-MS/MS runs required to achieve the same level of performance.35,45

Conclusions The developed workflow for top-down/middle-down FTMS has demonstrated the envisioned advantages for targeted protein structure analysis when spectral data from multiple LC-MS/MS experiments (technical replicates) can be made available. The obtained increase in protein sequence coverage is more pronounced for larger (50 kDa versus 17 kDa) proteins which exhibit


22

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particularly dense fragmentation patterns. A recently characterized enzyme, GingisKHAN, validated its single-cleavage site specificity to yield 50 kDa F(ab) subunits of IgG1. Another recent IgG-structure specific enzyme, IgdE, could be potentially used instead of the GingisKHAN.38 Within the frame of this work, both spectral and transient averaging capabilities, accompanied by the allied data processing, have been implemented as stand-alone software tools. Importantly, off-line absorption mode FT (aFT) signal processing has been successfully applied here for the first time on transients acquired from Orbitrap FTMS. The resolution and sensitivity performances of mass spectra produced via aFT and standard commercial enhanced FT (eFT) signal processing approach appear to be comparable. The described methodology applied here for the 50 kDa F(ab) subunit analysis showed final levels of spectral SNR that allowed the identification of low abundant product ions, including those that can lead to the confirmation of the cysteines involved in inter-molecular disulfide bridges. This information is fundamental to reveal light and heavy chain pairing in antibodies. Therefore, the methodology presented here can be used as a template for future antibody-based vaccine and therapeutics discovery research studies requiring the identification of selected IgGs from complex antibody mixtures derived from natural sources. The general character of the described multiplexing TD/MD workflow enables its application for improved structural analysis of other proteins, for example in protein structural biology.46


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Acknowledgements We thank Genovis for generously providing the GingisKHAN enzyme. We express our gratitude to Thermo Fisher Scientific Inc. for providing access under license to Orbitrap Elite FTMS transient signals and related functionality enabled by the developers’ kit. We are grateful for financial support through the European Research Council (ERC Starting Grant 280271 to YOT). MVG and AAL also thank the Russian Foundation for Basic Research (Swiss-Russia project #16-54-21006 to MVG).

Supporting Information. Comparison of spectral and transient averaging for TD/MD MS. Intact myoglobin and F(ab) mass measurements. Orbitrap Elite and Q Exactive HF FTMS tandem mass spectra and fragmentation maps of myoglobin and F(ab) subunit of trastuzumab. Results of product ion abundance analysis for ETD MS/MS and HCD MS/MS of F(ab) subunit. (pdf)

Financial conflict of Interest. Dr. Aizikov is an employee of Thermo Fisher Scientific, which manufactures Orbitrap FTMS instruments. Dr. Tsybin, Dr. Nagornov and Dr. Kozhinov are employees

of

Spectroswiss,

which

develops

Peak-by-Peak

software.

Computational tools described in this work are available as either opensource, freeware or commercially so a financial conflict of interest is declared.


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(22) Cai, W.; Guner, H.; Gregorich, Z. R.; Chen, A. J.; Ayaz-Guner, S.; Peng, Y.; Valeja, S. G.; Liu, X.; Ge, Y. Molecular & Cellular Proteomics 2016, 15, 703714. (23) Fellers, R. T.; Greer, J. B.; Early, B. P.; Yu, X.; LeDuc, R. D.; Kelleher, N. L.; Thomas, P. M. Proteomics 2015, 15, 1235-1238. (24) Schuhmann, K.; Thomas, H.; Ackerman, J. M.; Nagornov, K. O.; Tsybin, Y. O.; Shevchenko, A. Analytical Chemistry 2017, 89, 7046-7052. (25) Goloborodko, A.; Levitsky, L.; Ivanov, M.; Gorshkov, M. Journal of The American Society for Mass Spectrometry 2013, 24, 301-304. (26) Moelleken, J.; Endesfelder, M.; Gassner, C.; Lingke, S.; Tomaschek, S.; Tyshchuk, O.; Lorenz, S.; Reiff, U.; Mølhøj, M. mAbs 2017, 9, 1076-1087. (27) Sjögren, J.; Andersson, L.; Mejàre, M.; Olsson, F. In Bacterial Pathogenesis: Methods and Protocols, Nordenfelt, P.; Collin, M., Eds.; Springer New York: New York, NY, 2017, pp 319-329. (28) Ayoub, D.; Fornelli, L.; Srzentic, K.; Beck, A.; Tsybin, Y. O. In Proceedings of the 62nd American Society for Mass Spectrometry Conference on Mass Spectrometry and Allied Topics: Baltimore, MD, USA, June 2014. (29) Zubarev, R. A.; Kruger, N. A.; Fridriksson, E. K.; Lewis, M. A.; Horn, D. M.; Carpenter, B. K.; McLafferty, F. W. Journal of the American Chemical Society 1999, 121, 2857-2862. (30) Tureček, F.; Julian, R. R. Chemical Reviews 2013, 113, 6691-6733. (31) Ganisl, B.; Breuker, K. ChemistryOpen 2012, 1, 260-268. (32) Zhurov, K. O.; Fornelli, L.; Wodrich, M. D.; Laskay, U. A.; Tsybin, Y. O. Chemical Society Reviews 2013, 42, 5014-5030.


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Figure captions.

Table 1. Summary of a sequence coverage obtained with transient or spectral averaging from one or 10 LC-MS/MS runs from a F(ab) subunit of trastuzumab. Lc is the light chain; Fd is the subunit belonging to the heavy chain.

Figure 1. Schematics of the proposed MS-based workflow for topdown/middle-down structural analysis of F(ab) subunits from IgG1s. Sample preparation entails a rapid digestion of IgG1s with a GingisKHAN protease followed by multiple consecutive LC-MS/MS runs with ETD. The MS/MS results for a given precursor ion are obtained either as mass spectra that are first averaged within a single LC-MS/MS run and from there are subjected to spectral averaging across multiple LC-MS/MS runs; or alternatively are represented as the unprocessed data (transients) which are averaged in a time-domain across multiple LC-MS/MS runs prior to FT. Analysis of the final mass spectra, obtained via spectral or transient averaging, yields information on protein sequence and modifications.

Figure 2. An expanded view of an ETD mass spectrum of a F(ab) subunit of trastuzumab. Indicated are the assigned c- and branched zLc+zFd- product ions after absorption mode FT of an averaged transient from 10 LC-MS/MS runs. The equidistant peaks visible in the ETD mass spectrum are potentially due to the interference of the overlapping isotopic envelopes of the highly charged large product ions.


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Figure 3. ETD MS/MS fragmentation maps of a F(ab) subunit of trastuzumab produced by GingisKHAN digestion, obtained after spectral averaging of 10 LC-MS/MS runs recorded in reduced profile mode. Fragmentation maps are shown for (top panels) light chain and (bottom panels) Fd subunit sequences both when the inter-molecular disulfide bridge between the light and heavy chains of IgG is cleaved (left panels) or preserved (right panels). ETD product ions (c- and z- type as well as branched zLc+zFd ions) are color coded according to the respective color legends. Cysteines forming disulfide bonds are highlighted in orange. CDRs are highlighted in grey. Resulting sequence coverage is indicated below each chain in each panel.

Figure 4. ETD MS/MS fragmentation maps of a F(ab) subunit of trastuzumab produced by GingisKHAN digestion, obtained after spectral averaging of 10 LC-MS/MS runs recorded in full profile mode. Fragmentation maps are shown for (top panels) light chain and (bottom panels) Fd subunit sequences. The annotation rules are as in Figure 3.


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Table 1.

Sequence coverage, % Data averaging method

Cumulative

# LC

Interchain S-S bond cleaved

Interchain S-S bond preserved

sequence coverage, %

runs Lc

Fd

Lc

Fd

Lc

Fd

Spectral, full

1

8.9

9.4

N/A

N/A

8.9

9.4

Transient

1

8.9

9.8

N/A

N/A

8.9

9.8

Spectral, reduced

10

15.4

25.4

8.5

9.4

20.6

30.0

Spectral, full

10

18.7

29.9

12.2

12.05

27.6

37.9

Transient

10

22.0

30.3

15.0

15.6

34.0

41.9


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Figure 1.


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Figure 2.


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Figure 3.


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Figure 4.


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TOC figure 80x38mm (300 x 300 DPI)


Multiplexed Middle-Down Mass Spectrometry as a Method for

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