Selectivity over coverage in de novo sequencing of IgGs† †Electronic supplementary information (ESI) available. See DOI: 10.1039/d0sc03438j

Generating protein sequence ladders of the CDR3 variable regions of antibodies facilitates de novo sequencing by mass spectrometry.


Introduction
Antibodies are key molecules of our immune system and therefore of great interest to many researchers, both in academia and industry. [1][2][3][4] The high selectivity of immunoglobulins G (IgGs) for a single antigen has made this class of antibodies of great value in basic research, but even more important in molecular medicine. 5 Therapeutic antibodies reecting those of an individual having survived a severe infection, e.g. by the SARS-CoV-2, Dengue 6 or Ebola virus, 7 can lead to a substantial reduction in the mortality rate of newly infected patients. Shortening the route from the discovery of an antibody to the production of a functional equivalent is therefore of utmost importance, certainly in present times. 8 Structurally, IgGs are made of four disulde-bridged polypeptide chains: two identical light chains (LC) of 25 kDa and two identical heavy chains (HC) of approximately 50 kDa (Fig. 1A). The LC constant domain can be of two types, namely kappa (k) or lambda (l). The HC constant domain, on the other hand, is divided into four subclasses, namely IgG1, IgG2, IgG3, and IgG4, having between 83% and 96% sequence similarity. Only specic stretches of the HC regions differ between the subclasses: mainly the hinge region and the N-terminal side of the HC region below the hinge (Fc). Additionally, IgG subclasses exhibit distinctive disulde bridging with a different number of disulde bonds in the hinge region and different positioning of disuldes between LC and HC. 9,10 Antigen recognition by IgG is mainly determined by three hypervariable regions located in the N-terminal domains of both the LC and HC. Of these so-called complementarity determining regions (CDR1, CDR2 and CDR3), the two CDR3s are oen assumed to be major determinants of antibodybinding specicity. 11,12 Genetic recombination and hypermutation in these CDR regions lead to a plethora of possible human IgG antibodies, whereby it has been postulated that humans can generate about 10 billion unique antibodies, each potentially capable of binding an antigenic determinant (epitope) with high selectivity. 13 This amazingly large repertoire is possible because the CDR sequences are only partly genome-derived. Aer recombination of multiple genome segments into one antibody sequence, socalled V(D)J-recombination, random mutations are introduced in the process of somatic hypermutation. While initially used to increase the spectrum of recognized antigens, these processes subsequently lead to affinity and selectivity enhancements.
A common approach to elucidate the antibody repertoire in a human is to sequence the immunoglobulin mRNAs from memory B cells present in the donor's circulation. 14 The majority of antibodies present in the circulation is however not produced by these circulating B cells, but by long-lived plasma cells that reside in the bone marrow, which are much harder to harvest. The most accessible sequence-containing unit of relevant circulating antibodies is, therefore, the protein itself. 15 Sequencing proteins is nowadays generally performed by mass spectrometry. Characterization of peptides of 5 to 20 amino acids produced by proteolytic cleavage results into correct annotations of the full polypeptide backbone, even for de novo approaches when no DNA or protein sequence is available. [16][17][18][19][20] However, when it comes to antibodies from mixtures such as present in serum, sample complexity rapidly increases, preventing high-condence assignments. 15 One way to overcome this hurdle is to apply top-down proteomics since the hypervariable region of interestthe paired N-terminal domains of the LC and HCis then le intact and can be isolated. 21 The relevant information density increases even further when the constant regions, or at least most of the constant parts containing PTMs such as lysine clipping or glycosylation, are removed.
Here, we hypothesized that the ideal approach for de novo sequencing of antibodies from complex mixtures is a top-down or middle-down mass spectrometry-based method that results in long stretches of straightforward-to-read sequence ladders covering as much of the variable domains as possible, but at least the CDR3. This would resemble a modern, mass spectrometry-based, version of Edman degradation capable of dealing with impurities or complex mixtures and allowing for longer sequence reads. The work presented here is aimed at such a methodology.
Straightforward-to-read sequence ladders require highly optimized mass spectrometric methods preferentially yielding only one type of fragment pairs (a/x, b/y, or c/z) to avoid spectral congestion and dilution of signal intensity. To this end, electron capture dissociation (ECD), 22 which recently became available for benchtop mass spectrometers, has been shown to offer signicant advantages in obtaining straightforward-to-read sequence ladders. 23,24 Since, ECD has been used to query antibody sequences. It resulted in signicant backbone cleavages in both the LC and HC variable regions of both denatured and native intact antibodies. It also yielded mainly (c/z$) fragment ions. 25,26 While the presence of intrachain disulde bridges complicates the detection of several variable domain regions, it enhances the detection of CDR3 fragments. 25,27 In the present work, we build upon these exciting results and further optimize ECD for the de novo sequencing of IgG CDR3s and their downstream regions, generating very clean, straightforward-to-read sequence-ladders comprised solely of cions (Fig. 1B). Using native conditions in electrospray to ionize , all consisting of two variable regions (colored) with three antigen-binding Complementarity Determining Regions (CDRs) each, and one constant region (black). The first step in the workflow involves the separation by proteolytic cleavage of the IgG CDR-containing F(ab 0 ) 2 from the glycosylated constant Fc using the IdeS enzyme, which cleaves (scissors) just below the disulfides bridges (blue) between the heavy chains (HCs). Besides different numbers of HC-bridging disulfides, IgG 1-4 subclasses also differ by the disulfide bond between the light chain (LC) and the HC: directly above the hinge for IgG1, mediated by a more N-terminal HC cysteine for IgG 2-4. Additionally, the IgG3 hinge region is longer owing to an insertion that is repeated three times, as indicated with a black line above the sequence. (B) The purified F(ab 0 ) 2 portions of all IgG subclasses are subjected to ECD-MS, which preferentially fragments the LC and HC between their disulfide-bridged loops. Clearly separated low m/z c-ions and complementary high m/z bridged z-ions are formed, enabling the straightforward interpretation of fragment ion ladders beneficial for de novo IgG sequencing.
the IgG antibodies, we achieve maximal separation between the precursor and the informative fragment ions. We show that removal of the Fc region also reduces the complexity of the spectrum and simplies the precursor ion selection. We optimize and apply the method using the IgG1 Trastuzumab. Next, in order to investigate in detail whether the method is generically applicable, we apply it to two quartets of IgG covering all human IgG subclasses (i.e. IgG1, IgG2, IgG3 and IgG4). We show that the straightforward sequence reads are obtained for all IgG subclasses. Through structural modeling, we aim to explain and predict the observed fragmentation patterns.
Preparation of intact IgG, F(ab 0 ) 2 and Fab samples for native top-down MS analysis F(ab 0 ) 2 molecules were generated by overnight digestion at 37 C of the IgGs bound to a CaptureSelect FcXL affinity matrix (Thermo Fisher Scientic) using 0.25 U mg À1 FabRICATOR® IdeS (Genovis AB, Llund, Sweden) as described earlier. 31 IgG1 Fab molecules were similarly prepared using 1 U mg À1 FabALACTICA® IgdE (Genovis AB, Llund, Sweden). Prior to native top-down analysis, buffers were exchanged to aqueous 150 mM ammonium acetate (pH 7.5) through six consecutive dilution and concentration steps at 4 C using Amicon® Ultra centrifugal lters with a 10 kDa molecular weight cutoff (Merck KGaA, Darmstadt, Germany). IgG concentrations used for native electrospray ionization mass spectrometry were around 4 mM.

Native top-down ECD MS
Top-down MS of native IgG (sub)complexes was performed on an ultra-high mass range (UHMR) Q-Exactive Orbitrap (Thermo Fisher Scientic, Bremen, Germany) using an ECD cell developed by e-MSion. 32 First, IgG, F(ab 0 ) 2 or Fab precursor ions were mass-selected by the quadrupole. The in-source trapping desolvation voltage displays an optimum in terms of ECD at À100 V, likely a consequence of small structural changes and cleavage enhancement occurring as the effective temperature of the ion is raised. 33 The ions were then transmitted into the ECD cell where they were subjected to electron capture dissociation. The small permanent magnets ensure the radial connement of electrons emitted by a cathode while additional electrodes ensure longitudinal connement and efficient ion transfer to the HCD cell. Second, upon transfer from the ECD cell to the HCD cell, post-ECD collisional activation was kept to a minimum (HCD direct eV setting ¼ 1) to avoid (b, y) ion formation and concentrate the fragment signal intensity into the c-and z-ions of interest. Overall, the HCD cell trapping and extraction parameters were optimized for low nitrogen collision gas pressures to achieve efficient detection of the subunits and fragments. All spectra were acquired with the noise threshold parameter set to 3.64. A single charge state of the precursor (21+ for F(ab 0 ) 2 , 13+ for F(ab), and 24+ for intact IgG) was isolated to simplify interpretation of the fragment ion spectra. All the spectra were acquired at a set resolution of 200 000 @ m/z 400.

Data analysis
Processing of the fragmentation spectra involved the conversion of raw les to mzML format by Proteowizard. 34 We used the MSDeisotope python library (Joshua Klein, Boston University CBMS) 35,36 with a minimum_score ¼ 10.0 and mass_error_tolerance ¼ 0.02 to generate a charge deconvoluted spectrum with all the isotopic peaks retained. 35,36 Fragments were assigned by applying LcMsSpectator (Pacic Northwest National Laboratory) 37,38 to the charge deconvoluted spectra generated by MSDeisotope. The accuracy threshold was set to 3 ppm for all assignments following recalibration of the fragment's m/z via shiing by the average error on assignable c-ion fragments. The results were exported as tsv les for further analysis. Sequence assignment accommodated the major ECD ion types (c, z, z$) without considering H 2 O and NH 3 neutral losses, except when explicitly mentioned.

Modelling
The structures of the F(ab 0 ) 2 fragments studied are not available in structural databases. Consequently, homology models of the F(ab 0 ) 2 were built using MODELLER 39,40 similarly to the protocol introduced by Politis and co-workers. 41 For that, we used crystallographic structures with PDB IDs IgG1 1HZH (human), 42 IgG2 1IGT (mouse), 43 IgG3 5A16 (mouse) 44 and IgG4 5DK3 (human) 45 as a template. Renement of the obtained models was also performed in MODELLER using a combination of variable target function method and molecular dynamics with simulated annealing; optimization was repeated for three cycles. All models were assessed using the (negative) Discrete Optimized Protein Energy score. 39,40 Since IgG3 lacks an available F(ab 0 ) 2 structure, the hinge region was built in several steps to avoid knots introduced by MODELLER when more than approximately 15 residues are missing from the template structure. All disulde bridges were checked and manually dened when they were not modelled automatically.
To explore the conformational space of the Fab arm movement, each F(ab 0 ) 2 model was subjected to a rapidly exploring random tree based sampling, which is part of the Integrative Modelling Platform. 46 For that, residues in the upper hinge region were set as exible linker residues, namely: D226-T230 for anti-CD20 IgG1 and D225-T229 for anti-DNP IgG1, E221-K223 for anti-CD20 IgG2 and E220-K222 for anti-DNP IgG2, E221-T232 for anti-CD20 IgG3 and E220-T231 for anti-DNP IgG3, E221-P227 for anti-CD20 IgG4 and E220-P226 for anti-DNP IgG4. The rest of the molecule was connected through inter-chain disuldes and set as a rigid body. Next, the algorithm randomly tested the possibility of rotating an atom of the linker residues in agreement with the torsional space of the corresponding residue. The sampling was performed with the number of iterations and nodes set to 10 000. Subsequently, conformations without steric clashes and overlaps were used as output. High-throughput collision-cross section (CCS) calculations were done using Impact soware 47 using the standard parameters. The obtained CCS values were used for selecting the appropriate set of F(ab 0 ) 2 conformations. Calculation of the center-of-mass (COM) of the light chain, heavy chain and the F(ab 0 ) 2 was performed in Pymol 48 on a sample of 2500 random and 500 most compact conformations. For COM calculations of the IgG3 F(ab 0 ) 2 , we used HC sequences down to the rst two hinge disuldes to avoid the COM offset introduced by the larger hinge region of IgG3, when compared to other IgG subclasses. Next, we calculated the average distances between the COM of the LC or HC and the COM of the F(ab 0 ) 2 for a subset of conformations to assess the degree to which each chain is either inward-or outward-facing. Finally, we used this metric to estimate which chain is likely to be more exposed to the environment when the collapsed gas-phase structures are formed.

Results and discussion
Benchmarking ECD-only on intact IgG1, F(ab 0 ) 2 , and Fab molecules of Trastuzumab At the start of our experiments, we tested the performance of ECD on different preparations of Trastuzumab. A crosscomparison between ECD fragment ion spectra obtained for the 148 kDa intact IgG1, the 98 kDa F(ab 0 ) 2 and the 48 kDa Fab molecules revealed a striking similarity (Fig. S1 and Table S1 †). In line with previously reported data, 27 we observed that most of the abundant fragment ions have masses between 9000 and 16 000 Da, regardless of the Trastuzumab moiety that was used.
Aer careful consideration and examination of the ECD fragmentation spectra, we chose to focus on F(ab 0 ) 2 molecules for the rest of our study for the reasons described below. The ECD fragment ion spectra of intact Trastuzumab were notably more complex to analyze due to spectral congestion by z-ions from the highly conserved Fc region, which overlap with the cions from the region of interest. Additionally, the glycosylation on the Fc dilutes the precursor ion signal over a broader range of m/z values, hampering the use of narrow m/z windows for precursor isolation. Therefore, we chose to disfavor the analysis of intact mAbs.
Although both Fab and F(ab 0 ) 2 preparations yield straightforward sequence ladders corresponding to c-ions from the LC and HC regions of interest, the F(ab 0 ) 2 molecules provide some key advantages. While the IgdE enzyme can reliably produce Fab molecules of IgG1, 49 other IgG subclasses require the use of less specic enzymes such as papain, which result in higher sample heterogeneity. The IdeS enzyme on the other hand, which generates F(ab 0 ) 2 molecules by cleaving at a specic position below the hinge of all IgG subclasses, 50 provides both a reproducible and universal method irrespective of the subclass investigated. The resulting low-heterogeneity samples derived for the F(ab 0 ) 2 preparations enable strict precursor isolation without impacting sensitivity. Additionally, for denatured samples as well as native Fabs, the relatively low m/z of the precursor ions place them in the same m/z window where the ECD fragments are observed. The use of native F(ab 0 ) 2 molecules concentrates the precursor ion intensity on a small number of charge states outside the fragment ion regions, thereby simplifying data analysis. For these reasons, we chose the native F(ab 0 ) 2 molecules as precursors in the remainder of our analysis, although our data conrm that ECD also works reasonably well for both intact mAbs as well as Fab fragments.

Supplemental activation reduces the selectivity of ECD
Combining ECD with supplemental collisional activation can enhance precursor fragmentation. Although this has been demonstrated by Shaw et al. 27 to extend sequence coverage, collisional activation also generates additional b/y-ion series. We argue that the generation of these extra fragment ion series could be disadvantageous for straightforward and condent de novo sequencing of IgGs.
We investigated the effects of supplemental activation by recording EChcD-MS spectra of F(ab 0 ) 2 molecules using different levels of collisional activation. The higher collision voltages used in EChcD indeed introduced other ion types, which increase spectral complexity and make it more difficult to extract unambiguous sequence reads ( Fig. S2 and Table S2 †). Moreover, the slightly improved sequence coverage obtained by EChcD primarily provided information on the highly conserved parts of the antibody sequence, notably the FR3 and constant regions.
Since de novo sequencing benets from non-ambiguous sequence reads, we opted for the use of "pure" ECD (i.e. without HCD) to reduce the fragmentation pattern to the single c-ion type covering the regions of interest. Notably, aer careful analysis of all fragment ions with masses in between 9000 and 16 000 Da, we could conclude that almost all fragment ion intensity originated exclusively from c-ions, with the lowabundant remainder originating from a-ions. Thus, conditions can be employed wherein ECD of F(ab 0 ) 2 molecules purely generates c-ions in this mass window.
Pure ECD produces clean CDR3-spanning c-ion ladders Following our encouraging benchmark data obtained for the F(ab 0 ) 2 molecules of Trastuzumab, we sought to determine whether the observed fragmentation patterns were generic and thus useful for a range of different IgG mAbs. To this end, we characterized two quartets of IgG mAbs, covering the subclasses IgG1, IgG2, IgG3 and IgG4. One of these mAbs targets CD20, the other 2,4-dinitrophenol (DNP). Sequences, as well as theoretical and observed masses, are provided at the end of the ESI in Tables S5 and S6 †.
Application of pure ECD to F(ab 0 ) 2 molecules of all subclasses of the two IgG families consistently yielded ECD fragment ion mass spectra containing pure and easily interpretable c-ion sequence ladders covering CDR3 and FR4 of both the LC and HC (Fig. 2, S3, and Table S3 †). We noticed that the fragment ions originate nearly exclusively from backbone cleavages in a part of the LC and HC sequence that is framed by intramolecular disulde loops, and which span from the beginning of the CDR3 to the constant region. For both LC and HC, the observed backbone cleavages start immediately aer the cysteine residue preceding the CDR3 (LC: Cys-88, HC: Cys-96). This resulted in near-complete sequence coverage of this very important region, with the exception that we observed no cion cleavages at the N-terminal-side of proline residues, in line with earlier reports. [51][52][53] Notably, several of these gaps are covered by the corresponding a-ions, although at relatively lower intensities than the c-ion series.
Ion ladders covering the CDR3 of the LC do not overlap with HC fragment ions in the charge-deconvoluted spectra, making them suitable for direct interpretation. Ion ladders covering the CDR3 of the HC, on the other hand, overlap with fragment ions covering FR4 and the constant region of the LC. Because these regions are highly conserved, a comparison with theoretical spectra from these regions, accounting for the unknown Nterminal part by a variable mass shi, could help disentangle LC and HC fragment ions, making these mass spectra very suitable for de novo sequence assignment.
Exploring fragment complementarity in pure ECD of IgG F(ab 0 ) 2 molecules By using low pressure in the HCD cell and avoiding collisional activation, the ECD fragments of the two IgG quartets nearly exclusively correspond to c-ions (Table S4 †), similarly to our data for Trastuzumab, without the need to invoke small neutral losses such as ammonia or water, or sidechain losses. Apart from c-ions, a minor contribution is being made by a-ions, oen when there is no c-ion fragment.
Although C-terminal z-ions are not apparent within the shown deconvoluted analysis window (9000 < M w < 16 000 Da) of Fig. 2, they were detected completely separated in m/z from the concomitant c-ions (Fig. 3A). This is because the formed zions remain in complex with the remaining F(ab 0 ) 2 precursor, as they are connected through disulde bridges at or near their Ctermini. Charge and mass determination support this conclusion. While the 9-16 kDa c-ion fragments carry approximately 3-8 charges, their concomitant 83-90 kDa bridged z-ion fragments carry approximately 8-17 charges (Fig. 3B). Overall, pure ECD of F(ab 0 ) 2 molecules results specically into a small subset of low-mass c-ions and corresponding high-mass bridged zions.
The disulde links present in the IgG subclasses direct specic ECD fragmentation The intact IgGs, but also the F(ab 0 ) 2 and Fab molecules, contain hundreds of amino acid residues, which could all theoretically be cleaved by ECD, potentially leading to hundreds of c-and zions. Still, we observe that, very selectively and reproducibly, only a few dozen specic fragment ions are preferentially formed. To understand this distinct fragmentation pattern, observed in all ECD spectra of F(ab 0 ) 2 molecules from the different IgG subclasses, we next looked into their (distinct) structural features. Since exact structural models of the studied F(ab 0 ) 2 molecules are not available, we performed homology modelling, whereby available IgG structures were used as templates for the structural reconstruction of the target F(ab 0 ) 2 molecules. The high quality of the resulting F(ab 0 ) 2 structural models, as displayed in Fig. 4 and S5, † can be assessed from their Discrete Optimized Protein Energy (DOPE) proles (Fig. S4 †). An overview of the tools used for the structural analysis of the here studied IgG F(ab 0 ) 2 molecules is provided in Fig. S6. † The disulde bridges are among the most distinctive structural features that differentiate one IgG subclass from another, and not surprisingly these linkages affect the observed ECD cleavages in intact IgG and F(ab 0 ) 2 molecules most notably. 25 In Fig. 4A (anti-CD20) and Fig. S5A † (anti-DNP), the distinctive morphologies of the IgG subclasses are readily distinguishable. Differences are primarily in the distribution of the disulde bridges and the length of the hinge regions, a notable distinctive feature in between the IgG subclasses.
To assess the impact of the disulde linkages on the ECD, we rst assessed the number of disulde bond cleavages required to produce N-terminal fragments of either the LC or HC. Overlaid with the c-ion patterns produced by ECD of F(ab 0 ) 2 molecules, the resulting disulde proles nicely explain nearly all detected ECD fragments for both the LCs and HCs (Fig. 4B and C for anti-CD20 IgGs and Fig. S5B and C † for anti-DNP IgGs). In other words, only backbone cleavages in the regions that are not part of an intra-or interchain disulde loop result in separation and detection of free N-terminal ions.
Since the LCs of all subclasses within a given IgG quartet are identical, it is not surprising that they yield identical ECD fragments with comparable abundances (Fig. 2 and S5B †). The ECD patterns of HCs differ between the IgG subclasses ( Fig. 2 and S5C †), but this is readily explained by the location of the LC-HC interchain disulde bridges. In the HC sequences of IgG2-4, the interchain link is mediated by Cys-136, which is in the middle part of the sequence. For the IgG1 HC on the other hand, the interchain link is mediated by the more C-terminal Cys-225. As a result, a larger part of the IgG1 HC sequence is free from disulde looping, and therefore susceptible to ECD.
Probing distinctive structural dynamics of F(ab 0 ) 2 originating from different IgG subclasses In addition to the striking disulde-directed fragmentation, we noticed that the total ion intensity of HC fragments is higher for IgG1 compared to other subclasses, with IgG2 displaying the lowest fraction of HC fragment ion intensity ( Fig. 4D and S5D †). We hypothesized that a possible explanation could involve differences in the conformations and structural dynamics of the IgG molecules. 33,41,54,55 In the absence of extensive collisional activation, low energy (<1 eV) electron capture dissociation is known to result in the preferential release of fragments from the surface-exposed regions of native proteins. 17,[45][46][47] The rationale is that exposed domains and chains have fewer non-covalent interactions upon transition to the gas phase, and therefore more readily release backbone fragments upon electron attachment. Thus, we set out to probe the structural features of F(ab 0 ) 2 molecules using modelling.
It has recently been reported that IgG molecules undergo a substantial structural collapse upon transfer to the gas phase during electrospray ionization. 41,54 In this structural rearrangement, the high exibility of the hinge region enables one or more Fab arms to clasp the IgG Fc region. 41 When processed by the IdeS enzyme, the remaining F(ab 0 ) 2 portion of IgG retains its hinge region and, hence, F(ab 0 ) 2 should also be able to adopt a similar more compact structure upon transition to the gas phase. Although we are not able to directly predict the exact collapsed state of IgG F(ab 0 ) 2 molecules, we can assess their conformational space prior to the collapse for which we used rapidly exploring random tree based conformational sampling from the Integrative Modelling Platform. 46 First, we sampled the motions of the Fab arms (n ¼ 2500, Fig. 4F and S5F †). The differences in conformational dynamics between the different IgG subclasses can be assessed by displaying the center-of-mass (COM) of both chains of a Fab arm for a conformation sample. 55 In order to infer the relative position of LC and HC in the putative F(ab 0 ) 2 gas-phase conformations, we calculated the average distances between the COM of each chain and the COM of the molecule (Fig. S7 †). Second, we considered the 500 most compact F(ab 0 ) 2 conformations as determined from their collision cross-sections. We then assumed that the most exposed chains, as inferred from the COMs, are less prone to end up buried within the collapsed structures and will, therefore, be more likely to capture electrons and release backbone fragments. Finally, we compared all subclasses for anti-CD20 IgG ( Fig. 4E and F) and anti-DNP IgG (Fig. S5E and F †).
Our modelling data suggest that the LCs and HCs are nearly equally solvent-exposed in the collapsed structures of IgG1 F(ab 0 ) 2 , resulting in comparable abundances of the LC and HC ECD fragments. IgG4 F(ab 0 ) 2 exhibit slightly less exposed HCs which seemingly result in a higher fraction of LC fragments. Notably, the relative rigidity 55 and predominant presence of LCs on the surface of collapsed IgG2 F(ab 0 ) 2 structures correlate pleasingly well with the prevalence of LC fragments over HC fragments, which explains the lower abundance of HC fragments compared to the other subclasses. Owing to its larger and more exible hinge region, F(ab 0 ) 2 of IgG3 shows the widest range of Fab arm motions and the broadest distribution of collision cross-sections (Fig. S8 †). It is likely that the high exibility of IgG3 F(ab 0 ) 2 leads to dramatic structural rearrangements upon transfer to the gas phase, which can affect fragment separation upon ECD. Summarizing the modelling data, in IgG2, IgG3 and IgG4, the LC is more exposed than the HC while, in contrast, IgG1 displays nearly equal surface occupation for the LC and HC when sampling the 500 most compact F(ab 0 ) 2 conformations. Notably, our analysis supports that the predominant presence of the LC on the surface of the collapsed IgG2 F(ab 0 ) 2 structures and relative rigidity of the overall structure hamper the formation of abundant HC fragments compared to the other subclasses of both anti-CD20 and anti-DNP IgG.

Conclusions
Here, we explored the benets of electron capture dissociation in attempting to sequence antibodies. Differentiating our approach from earlier attempts, 21,27,56 we aimed deliberately for a very restricted, albeit targeted, sequence coverage, spanning just the important hypervariable CDR3 of both the light and heavy chain (LC and HC) of the mAbs. We optimized our method by rst getting rid of the sequence-wise less informative constant regions (Fc) by using the IdeS protease, to obtain F(ab 0 ) 2 fragments. Next, we conducted ECD at minimal collisional activation and low pressure, allowing us to get exclusively straightforward-to-read amino acid sequence ladders of c-ions spanning the CDR3s. The ladders we obtained provide easily interpretable sequence reads covering the CDR3 of both chains, whereby we seemingly only observe a gap in the sequence when there is a proline present. We argue that this optimized method can be very powerful and benecial for the de novo sequencing of human antibodies.
Next, we sought to explain the observed highly specic fragmentation behavior by studying whether it was generic. Therefore, we investigated two quartets of mAbs (one directed against CD20 and one against DNP) being expressed in all their subclass variants; IgG1, IgG2, IgG3 and IgG4. On the one hand, the ECD data of these eight mAbs showed extremely conserved fragmentation of their CDR3s with the exclusive formation of cions in the low mass region (9000-16 000 Da). On the other hand, specic features were observed in between the different subclasses, which could be largely explained by differences in the disulde bridges within these molecules, and by their distinctive structural features in the context of the gas-phase transition.
Through this work, we improve our understanding of the observed very specic fragmentation in the CDR3 regions of all different subclasses of the IgGs. The models generated based on this data provide a rst step in predicting the fragmentation patterns observed in ECD of IgGs. Such a spectrum prediction tool would greatly facilitate de novo sequencing of antibodies.

Conflicts of interest
There are no conicts to declare.