key: cord-0908487-gkik7118 authors: Melani, R. D.; Des Soye, B. J.; Kafader, J. O.; Forte, E.; Hollas, M.; Blagovich, V.; Negrao, F.; McGee, J. P.; Drown, B.; Lloyd-Jones, C.; Seckler, H. S.; Camarillo, J. M.; Compton, P. D.; LeDuc, R. D.; Early, B.; Fellers, R. T.; Cho, B.-K.; Baby Mattamana, B.; Goo, Y. A.; Thomas, P. M.; Ash, M. K.; Bhimalli, P. P.; Al-Harthi, L.; Sha, B. E.; Schneider, J. R.; Kelleher, N. L. title: Next-generation Serology by Mass Spectrometry: Readout of the SARS-CoV-2 Antibody Repertoire date: 2021-07-07 journal: medRxiv : the preprint server for health sciences DOI: 10.1101/2021.07.06.21259226 sha: 766c40055333e3032947706f8c89276bae8058bf doc_id: 908487 cord_uid: gkik7118 Methods of antibody detection are used to assess exposure or immunity to a pathogen. Here, we present Ig-MS, a novel serological readout that captures the immunoglobulin (Ig) repertoire at molecular resolution, including entire variable regions in Ig light and heavy chains. Ig-MS uses recent advances in protein mass spectrometry (MS) for multi-parametric readout of antibodies, with new metrics like Ion Titer (IT) and Degree of Clonality (DoC) capturing the heterogeneity and relative abundance of individual clones without sequencing of B cells. We apply Ig-MS to plasma from subjects with severe & mild COVID-19, using the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 as the bait for antibody capture. Importantly, we report a new data type for human serology, with compatibility to any recombinant antigen to gauge our immune responses to vaccination, pathogens, or autoimmune disorders. yield in the optimized elution conditions. Pull-downs from CS1 were used to profile the antibody isotypes (IgA, IgD, IgE, and IgM) and IgG subclasses (IgG1, IgG2, IgG3, and IgG4) being enriched. A specific western blot revealed that IgG1, IgG3, and IgM were the primary isotypes isolated (Supplementary Fig. 2) , similar to what has been reported in the literature 17 . All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 7, 2021. ; https://doi.org/10.1101/2021.07.06.21259226 doi: medRxiv preprint Following antibody enrichment, two distinct workflows were developed to prepare isolated Ig-RBD for Ig-MS analysis (Fig. 1a) . In workflow 1, Ig-RBDs were eluted intact, combined with 100 ng of standard mAb CR3022, and fully reduced/denatured in the presence of a chaotropic agent to liberate HC (48-55 kDa) and LC (22) (23) (24) (25) proteoforms. In workflow 2, patient Ig-RBD were digested with IdeS protease while still attached to the beads, generating F(ab')2 and Fc. In workflow 2, the protease and Fc species (containing HC glycosylation) are washed away. Eluted F(ab') 2 were denatured and reduced to yield the LC and the Fd domain (~25-28 kDa), which contains the N-terminal 220 amino acids of the HC and therefore captures the composition of all three Complementarity-Determining Regions (CDRs). Before the I 2 MS analysis step of Ig-MS, 100 ng of digested and reduced mAb CR3022 were spiked into the sample. To benchmark Ig-MS, we first analyzed mAb CR3022 and annotated the LC and HC proteoforms (Supplementary Fig. 3 ). Subsequently, we estimated the Ig-MS Limit of Detection (LOD) for the HC of NIST mAb (with glycosylation) at 100 nM, whereas the LOD for the LC was well below 10 nM (Supplementary Fig. 4 ). Next, we performed Ig-MS using workflow 1 on CS1, and Supplementary Fig. 5 shows the spectrum obtained for the LC region. Like in Fig. 1c , the highlighted peak at ~24,268 Da represents the mAb CR3022 standard used to calculate Ig-MS metrics, as outlined in Equations 1-3 in the Online Methods. The other peaks with lower mass than the standard LC, ranging from ~22 to 24 kDa, are distinct LC proteoforms originating from B cell clones with different or isobaric CDR sequences in their variable regions. With this first glimpse of an Ig repertoire at the LC and HC levels, the presence of distinct proteoforms shows that single clone resolution is possible, with some present at high titer (i.e., ~500 ng/100 uL plasma). The current dynamic range for detecting All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 7, 2021. ; https://doi.org/10.1101/2021.07.06.21259226 doi: medRxiv preprint different clones that evolve after VDJ recombination is approximately 100 (Supplementary Fig. 5 ). Given that Ig-MS produced a new data type, we created two new metrics. The Ion Titer (IT) is similar to an ELISA titer and uses the intensity of the LC of the standard mAb CR3022 as a reference. It combines intensity of all other LC peaks relative to the mAb CR3022 and ranges from 0 to 100 (Equation 1). The second is the Degree of Clonality (DoC), a measure of the complexity of all LC clones (proteoforms) in the mixture and ranges from 1 to infinity, with higher numbers reflecting the presence of a more significant number of antibodies in a more complex Ig-MS spectrum. For subject CS1, the calculated IT was 1.30, and the DoC was 3.64. We next sought to compare these Ig patterns and new metrics across patients and perform initial correlations with other COVID-19 antibody tests. We deployed Ig-MS workflows 1 and 2 to survey the Ig population reactive to RBD in a cohort of seven hospitalized patients with severe COVID-19, three outpatients with mild COVID-19 disease, and three healthy people who never had COVID-19 (Supplementary Table 1 ). The standard mAb CR3022 and commercial pooled plasma acquired before November 2019 served as positive and negative controls, respectively. . 2a) ; (2) moderately high IT (14.87) and high DoC (7.25) as observed for the hospitalized patient COVID-19_3 (Fig. 2b) ; and (3) low IT (0.65) and low DoC (3.08) as observed for the outpatient COVID-19_6 (Fig. 2c) . All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 7, 2021. ; https://doi.org/10.1101/2021.07.06.21259226 doi: medRxiv preprint Fig. 2 . Ig-MS readouts from workflow 1 and 2 on a COVID-19 cohort. Results for the LC spectral region from workflow 1 (a-d) and for the LC and Fd fragments from workflow 2 (e-h); ion titers (IT), and degree of clonality (DoC) are shown for two hospitalized patients (a/e and b/f, All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 7, 2021. ; https://doi.org/10.1101/2021.07.06.21259226 doi: medRxiv preprint red) and one outpatient (c/g, purple). The standard mAb (CR3022) that binds SARS-CoV-2-RBD (d/h, blue) was used as positive control (highlighted with gray vertical bar). i, Comparison of Ig-MS patterns between the LC of a single patient obtained with the two workflows. j, IT and DoC values obtained by both workflows of Ig-MS for three outpatients and seven hospitalized patients. Analyses were done in triplicate. Correlation of IT (k) and DoC (l) values from Ig-MS workflows with RBD-binding by ELISA, bioluminescent in vitro diagnostic (Promega), and surrogate virus neutralization. Shown are the Pearson correlation coefficient (r) and P-value (p). The same 13 samples were processed using workflow 2, which generated the LC and Fd for readout by Ig-MS. Supplementary Fig. 9 exhibits the obtained spectra for all samples. The spectra from patients COVID-19_1 (Fig. 2e) , COVID-19_3 (Fig. 2f) , and COVID-19_6 (Fig. 2g) displayed the same immunological response pattern as those obtained with workflow 1, showing the self-consistency of the two workflows. Distinct proteoform masses representing all the LCs and Fds were clearly discernable in both workflows (Fig. 2) . The LC proteoforms have a mass range between 22-25 kDa (Fig. 2 , to the left side of the standard LC highlighted in gray) and comprise the entire length of the LC, including the three CDRs (VL) and conserved region (CL). We can identify κ and λ LCs in the Ig-MS spectra based on the amino acid differences in the conserved regions. In F=18.26, 2 and 33 DF, p<0.001) compared to outpatients, a result corroborated with standard All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 7, 2021. ; https://doi.org/10.1101/2021.07.06.21259226 doi: medRxiv preprint serological tests from previous studies 19, 20 . No statistically significant differences were observed for the DoC metric in these groups. Ion Titers were also calculated exclusively for LC regions and compared between workflow 1 and 2. Although differences occur in the range of ion titers observed between the two workflows, there was a strong correlation (R 2 = 0.88) between them, indicating a systematic bias ( Supplementary Fig. 10) . We believe that this bias arises from the differences in sample and standard preparation. However, the average cross-correlation of the LC from the two studies is 0.78 ±0.04, indicating that the ratios among peaks and the relative proteoform amounts are conserved in both workflows of Ig-MS. With workflow 2, we can analyze Fd proteoforms that include three CDRs from the HC (VH) and the constant region CH1 besides all possible individual variation present in the constant region ( Fig. 2 , to the right side of LC the standard Fd is highlighted in gray). This workflow captures the post-VDJ sequence recombination and dissects the Fc glycosylation pattern away from the intact HC observed in workflow 1, reducing molecular complexity. A general finding in this initial study is a greater degree of heterogeneity in the Fd relative to the LC from the same individual. We next probed the correlation of ITs obtained with the two Ig-MS workflows with titers of anti-RBD antibodies quantified with ELISA 21 and a commercial in vitro diagnostic using bioluminescence. We also determined the correlation between IT and neutralization efficiency obtained with a Spike-specific pseudovirus neutralization test ( Fig. 2k-l, Supplementary Fig. 11 , and Supplementary Table 1 ). Pearson's correlation (r) (Fig. 2k) indicated a significant positive correlation of IT obtained with workflow 2 and the ELISA titers (r=0.68) and a negative correlation with surrogate neutralization (r=-0.66). The same tendency was observed for ITs determined with All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 7, 2021. ; https://doi.org/10.1101/2021.07.06.21259226 doi: medRxiv preprint workflow 1 (r>0.55) but with p-values higher than 0.05. The correlation analysis of the ITs of both workflows with the bioluminescence was low (r≤0.47) and not significant. Furthermore, DoCs from both workflows did not correlate with the other assays (Fig. 2l) , indicating that the number of different Ig signals produced by Ig-MS did not show a correlation with neutralization potential or overall titer in this initial study. For both workflows, there was a significant negative correlation between ITs and days after infection (Supplementary Fig.12) . These results suggest that there is a reduction in the total amount of antibodies over time, but the general degree of complexity in the immune response does not change. Additionally, virus neutralization appears more related to the total amount of antibodies in the plasma than to clonal heterogeneity in the Ig repertoire. Additionally, the average percent of the total fucosylated glycans was 53.9% ±17.7% for the hospitalized group and 95.5% ±0.8% for the uninfected individuals (Supplementary Fig.14) . Similar results were previously reported 23 , and afucosylated IgG induces increased antibodydependent cellular cytotoxicity by rising IgG-Fc receptor IIIa (FcγRIIIa) affinity 24 . Next, we tried to correlate these results to the Ig-MS readouts from workflow 1. Unfortunately, the high mass complexity observed in the HC region of workflow 1 generated from the different amino acid sequences and the multiple glycans structures pushes such analyses outside the scope of this initial study. Antibody titers can be used to indicate the extent of immunity and disease severity for COVID-19 19, 20 . Ig-MS initial results showed that IT from the two workflows were self-consistent and correlated with traditional colorimetric/fluorimetric tests and a surrogate neutralization assay. The DoC metric and patterns of responses did not correlate with these assays, yet their variance in the human population needs to be determined. We observed significant differences in hospitalized patients versus outpatients as well as reduced IT overtime of convalescence, similar to previous reports 25 . With this initial report, Ig-MS proves functional, prompting three general use cases: 1) providing a new longitudinally-stable, multi-parametric correlate of protection, 2) indicating the course or stage of COVID-19 disease as a diagnostic or prognostic indicator, and 3) striating lots of convalescent plasma to quantify protective potential and better control plasma collection campaigns in this or future pandemics 26 . Additionally, antibody amounts and clonal variation can play a complementary role in vaccine campaigns that can strongly correlate to protective immunity (i.e., provide a reliable surrogate to neutralizing titers). These reports quantified responses across over 100 people and thousands of B cells, demonstrating the consistency of the stereotypical immune response, and are a great resource to identify potential therapeutic and prophylactic antibodies. Such convergence can also be detected by Ig-MS because All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 7, 2021. ; https://doi.org/10.1101/2021.07.06.21259226 doi: medRxiv preprint it provides intact mass patterns that reflect the combination of LC and HC CDRs. This is unlike approaches using tryptic digestion of antibodies [8] [9] [10] [11] 29 . Indeed, no technology can sequence whole, endogenous antibodies directly in an Ig repertoire, but >80% sequence coverage by direct fragmentation of LC and HC from monoclonal antibodies is possible today 30 . In summary, we report a new and unique data type for human serology, using COVID-19 cases as the first example. Until now, no serological test was capable of accessing the relative abundance of each antibody generated against a specific antigen. Ig-MS is the first method capable of accessing amounts and relative abundance of antibodies simultaneously using a fundamental advance in MS of individual protein ions 16 to create a unique display of the Ig repertoire of a human being at molecular resolution. Ig-MS successfully captured the clone populations of RBDreactive immunoglobulins and showed promising data on a limited cohort of COVID-19 patients. In the future, a more automated form of Ig-MS will address larger cohorts, use 10-100 fold less All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 7, 2021. ; https://doi.org/10.1101/2021.07.06.21259226 doi: medRxiv preprint sample, and extensively sequence CDR variable regions for comparison with methods for single B cell sequencing like Ig-seq 33 . All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 7, 2021. ; https://doi.org/10.1101/2021.07.06.21259226 doi: medRxiv preprint Disclosures N.L.K., J.O.K., and P.D.C. report a conflict of interest with I 2 MS technology used to readout the Ig profiles, currently being commercialized by Thermo Fisher Scientific. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 7, 2021. were heat-inactivated at 56°C for one hour. Following heat inactivation, samples were diluted at a volume ratio of 1:9 in sample dilution buffer and mixed with a 1:1000 HRP-conjugated RBD All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 7, 2021. ; https://doi.org/10.1101/2021.07.06.21259226 doi: medRxiv preprint solution in HRP dilution buffer at a volume ratio of 1:1 and incubated at 37°C for 30 minutes. Following incubation, samples and kit-provided controls were added to an ACE2-coated 96-well plate, 100 µL/ well in duplicate, and incubated at 37°C for 15 minutes. The plate was then washed four times with 1X wash solution and incubated with 100 µL/well TMB solution in the dark at room temperature for 15 minutes, followed by the addition of 50 µL/well reaction stop solution. The absorbance was then read on a biotek Cytation 3 plate reader at 450 nm. The protocol is The plasmid pCAGGS SARS-CoV-2 RBD comprises an N-terminal signal sequence, amino acids 319-541 of the spike protein from SARS-CoV-2 (the receptor-binding domain, RBD), and a Cterminal 6-His tag 34 . This vector was obtained from BEI Resources (BEI NR-52309) and expressed recombinantly using the Expi293 Expression System as follows: Expi293F™ cell culture (1 L total) was maintained in a 37°C incubator with ≥80% relative humidity, 8% CO2 on an orbital shaker platform and sub-cultured at cell density 3-5 x 10 6 viable cells/mL. One day before transfection the cells were seeded to a final density of 2.5 x 10 6 viable cells/mL and allowed to All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 7, 2021. Purification was performed on AKTAxpress (GE Healthcare Life Science) FPLC purification system. Clarified cell culture supernatant was loaded onto HisTrap FF 5 mL column (GE Healthcare Life Science, Cat #17-5255-01). The column was washed twice, once with binding buffer (10 mM Tris-HCl, 500 mM NaCl, pH 7.4) and then with binding buffer + 12.5 mM imidazole to remove unspecifically bound material. Finally, bound RBD protein was eluted off the column with elution buffer (10 mM Tris-HCl, 500 mM NaCl, 500 mM imidazole, pH 7.4). Column loading, washes, and elution were all performed at a 3 mL/min flow rate. After elution, the collected fraction was buffer exchanged into 1x PBS. Fifty μg of RBD was acetone/TCA precipitated with 8 volumes of cold acetone and one volume of trichloroacetic acid overnight at -20°C. After washing the pellet with ice-cold acetone, the resulting protein pellet was resuspended into 50 μL of 8 M urea in 400 mM ammonium bicarbonate, pH 7.8, reduced with 4 mM dithiothreitol at 50°C All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 7, 2021. The peptides were separated on a 120 min. analytical gradient from 5% ACN/0.1% FA to 40% ACN/0.1% FA. The mass spectrometer was operated in a data-dependent mode. The source voltage was 2.40 kV, and the capillary temperature was 320°C. MS 1 scans were acquired from 300-2000 m/z at 60,000 resolving power and automatic gain control (AGC) set to 3x10 6 charges. The top 20 most abundant precursor ions in each MS 1 scan were selected for fragmentation. Precursors were selected with an isolation width of 2 m/z and fragmented by Higher-energy collisional dissociation (HCD) at 30% normalized collision energy in the HCD cell. Previously selected ions were dynamically excluded from re-selection for 20 seconds. The MS 2 minimum AGC was set to 1x10 3 . All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 7, 2021. Only proteins with a minimum of two unique peptides above the cutoff were considered identifications. covalently loaded onto Dynabeads® MyOne™ Carboxylic Acid beads (Thermo 65011, Thermo 65012) according to the manufacturer-provided protocol for "Two-Step Coating Procedure using NHS". In brief, for a single preparation, 1.5 mL of bead suspension was dispensed and washed twice with 1.5 mL of 25 mM MES, pH 6.0 for 10 min. at room temperature. Following washes, bead chemistry was activated by suspending the beads into 1.5 mL of freshly-prepared 50 mg/mL N-Hydroxysuccinimide (NHS), mixing in 1.5 mL of freshly-prepared 50 mg/mL 1-Ethyl-3-(3dimethylaminopropyl) carbodiimide (EDC), and incubating for 30 min. at room temperature. Following activation, the beads were again washed twice with 1.5 mL of 25 mM MES, pH 6.0 for 10 min. at room temperature. Activated beads were suspended into 1 mL 25 mM MES, pH 6.0 to which 1.5 mg of RBD protein was added. The sample was mixed and incubated for 30 min. at room temperature. Next, the beads were pulled down, the supernatant was discarded, and loading All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 7, 2021. ; https://doi.org/10.1101/2021.07.06.21259226 doi: medRxiv preprint was quenched by incubating the beads in 1 mL of 50 mM Tris, pH 6.8, for 15 min. at room temperature. After quenching, beads were washed four times in 1 mL 1x PBS + 0.1% human serum albumin (HSA), and finally suspended into 3 mL 1x PBS + 0.1% HSA for storage at 4°C. were assembled by combining 100 µL of patient plasma/serum with 35 µL of RBD-loaded bead suspension and diluting to a volume of 1 mL with 1x TBS. Assembled pull-downs were incubated overnight at 4°C with end-over-end mixing. After this, incubation beads were pulled down, supernatant was removed, and beads were resuspended into 1 mL wash buffer (1x TBS + 0.1% TWEEN + 1% NP-40 + 1% NP-40 substitute). Suspensions were transferred onto a KingFisher Flex for additional 4 washes in 1 mL wash buffer, 2 washes in 1 mL 1x TBS, and a 30 min. incubation in 100 µL 100 mM glycine, pH 11.5 + 0.1% sodium deoxycholate at 37°C to elute antibodies associated with bead-bound RBD. For workflow 2, Ig-pull-downs included an on-bead IdeS digestion of RBD-binding antibodies. This involved an additional incubation step between the first and second 1x TBS washes, during which beads were incubated in 100 µL of 50 mM sodium phosphate + 150 mM sodium chloride (pH 6.6) in the presence of 200 U IdeS enzyme (Promega V751A) for 3.5 h at 37°C. Following elution, 100 ng of mAb CR3022 standard antibody was added to each elution fraction to serve as an internal standard across all samples. For samples including an IdeS digest step, the mAb CR3022 standard underwent an in-solution IdeS digestion according to manufacturer protocols prior to being added to pull-down elution fractions. After supplementation with standard antibody, All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 7, 2021. ; each fraction was combined with 160 µL 8M urea and 25 µL 1M TCEP, mixed, and set to incubate for 1 h at room temperature to facilitate the complete denaturation of the Ig-RBD and permit the complete reduction of all inter-and intrachain disulfide bonds. Following incubation, reduced antibody fragments were cleaned via methanol-chloroform-water precipitation as has been previously described 35, 36 . Fractions of interest were combined with Bolt™ loading buffer (final concentration 1x, Thermo Fisher B0007) and dithiothreitol (DTT, final concentration 100 mM) and incubated at 100°C for 10 min. Boiled samples were loaded onto a 4-12% Bolt™ Bis-Tris Plus polyacrylamide gel, which was run in a Mini Gel Tank (Thermo Fisher A25977) using MES running buffer for 1 h at 120 V. Next, the gel was trimmed, and proteins were transferred to a nitrocellulose membrane using iBlot™ 2 nitrocellulose transfer stacks (Thermo Fisher IB23001) on an iBlot™ 2 Dry Blotting System following manufacturer instructions. The transfer method used was the templated method P3 (20 V for 7 min.). After transfer, the membrane was blocked using 1x TBS + 0.05% TWEEN + 5% milk for 1 h at room temperature with mixing. After 1 h, Goat anti-Human IgG (H+L) HRP conjugate (Thermo Fisher 31410) was added directly to the milk at a 1:20000 dilution and the membrane was moved to 4°C to incubate overnight. The next day, the milk + antibody mixture was discarded, and the membrane was washed 3x in 1x TBS + 0.05% TWEEN for 5 min. at room temperature with mixing. Finally, the blot was imaged on an iBright CL1000 imager (Thermo Fisher) using 800 µL of Immobilon Classico Western HRP substrate (Millipore Sigma WBLUC0500). All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 7, 2021. ; https://doi.org/10.1101/2021.07.06.21259226 doi: medRxiv preprint Western blot identification of enriched antibody isotypes. An enrichment was performed as described in the section "Enrichment of RBD-reactive antibodies from patient samples" above to screen for the presence of individual human antibody isotypes (IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, and IgM). In this case, after elution, the fraction was divided into 8 equal parts. Positive controls were assembled for each isotype being examined, with each control consisting of 250 ng of a commercially-obtained purified antibody of that isotype (IgG1: Sigma AG502; IgG2: Sigma I5404; IgG4: Sigma I4640; IgA: Sigma I4036; IgD: Sigma 401164; IgE: Sigma 401152; IgM: Sigma I8260). Negative controls for each isotype were assembled by combining 300 ng of all commercially obtained purified antibody isotypes omitting the specific isotype that the control was. Eluted material, positive, and negative controls were run in polyacrylamide gels, and proteins were transferred to nitrocellulose and blocked as described above. After blocking, each membrane was introduced to a primary antibody that specifically recognized the isotype of the antibodies being examined on that membrane (IgG1: AbCam ab108969; IgG2: AbCam ab134050; IgG3: AbCam ab109761; IgG4: AbCam ab109493; IgA: AbCam ab124716; IgD: AbCam ab124795; IgE: AbCam ab195580; IgM: AbCam ab134159) mixed with 1x TBS + 0.05% TWEEN + 5% milk. Primary antibodies were present at a dilution of 1:10000. Membranes were incubated in primary antibodies overnight at 4°C with mixing. The next day, primary antibody + milk was discarded, and all membranes were washed 3x in 1x TBS + 0.05% TWEEN for 5 min. at room temperature with mixing. After washing, all membranes were submerged into 1x TBS + 0.05% TWEEN + 5% milk to which secondary antibody (Gt-anti-Rb-HRP (Sigma AP307P) had been added at a dilution of 1:5000. Membranes incubated in secondary antibody for 1 h at room temperature with mixing, after which they were washed 3x in 1x TBS + 0.05% TWEEN for 5 min. at room temperature with mixing and imaged as described above. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 7, 2021. ; https://doi.org/10.1101/2021.07.06.21259226 doi: medRxiv preprint Anti-RBD antibody quantification. Titers of antibodies from patient plasma responsive to SARS-CoV-2 RBD were measured using Lumit™ Dx SARS-CoV-2 Immunoassay (Promega VB1080). Plasma from each patient was diluted 1:10 in 1x TBS and incubated at 56°C for 1 hr to inactivate any potential pathogens remaining. Following heat inactivation, samples were used as input and analyzed in triplicate using the assay following manufacturer protocols. Previously, I 2 MS analysis has been demonstrated using an Orbitrap mass analyzer 16, 39, 40 . Briefly, I 2 MS is a novel approach capable of discerning the mass profile of highly complex mixtures not amenable to venerable approaches used in protein mass spectrometry. Rather than measuring in the mass-to-charge (m/z) domain, I 2 MS accurately determines the charge on each individual ion collected through a process called STORI plot analysis. STORI plot analysis, the All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 7, 2021. ; https://doi.org/10.1101/2021.07.06.21259226 doi: medRxiv preprint determination of the slope of individual ion signal accumulation, was completed on each ion as a function of its specific frequency 16 . To simplify this process and reduce file sizes, only timedomain signals at specific frequencies where individual ions occurred, called STORI files, were recorded on an acquisition-to-acquisition basis. Each acquired STORI file was then processed to accurately determine the m/z and charge for every individual ion signal detected. In order to evaluate the lower limits of the GIDI-UP platform, we analyzed NIST antibody standard in a serial dilution series. All samples were infused at 1 µL/min. for 50 min. such that the same number of transients would be acquired for each experiment. Additionally, the injection time was scaled inversely to the sample concentration in order to maintain a constant per-cycle injection of 12.5 pg. For example, the 500 nM run used an injection time of 10 ms, and the 100 nM run used an injection time of 50 ms. were processed to create mass spectra as .mzml files. Briefly, STORI files containing single ion peak information and transient sections are processed using a Short-time Fourier transform (STFT) to assign slopes to single ions 41 . After slope assignment, charges were assigned to individual ion using an iterative voting algorithm, and spectra were generated using a Normal kernel density estimate (KDE) and exported as either profile or centroided .mzml files. Coefficients. Ig-MS Ion Titers were calculated using a custom script, in which .mzml I 2 MS files were centroided and spectra divided into regions; the Light Chain (LC) region, the regions of the Standard peaks, and either the Fd fragment or Heavy Chain (HC) regions for samples that were All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 7, 2021. ; https://doi.org/10.1101/2021.07.06.21259226 doi: medRxiv preprint reduced with and without IdeS digestion, respectively. An average noise was calculated over these regions, and the sum was subtracted from the intensity for the region's total 42 . The titers were obtained by determining the ratio between the sum of the standard peak regions and the regions for the LC and Fd/HC regions, divided by the value of the spike-in standard, as shown in Equation The degree of clonality (DoC) was calculated by centroiding profile spectra for a given mass window corresponding to the LC region. Firstly, the highest centroid peak is determined and using an averagine distribution, a window is created around the peak 43 Spectral correlation coefficients were calculated for two spectra by taking centroided spectra and creating a padded array of equal length for each. Each peak in each centroided spectra was fitted to a Normal KDE and summed into the padded arrays, yielding two gaussian fitted spectra of equal length with indexes corresponding to the same mass. Calculating the Cosine Similarity using Equation 3, yielded the spectral correlation coefficients of the two spectra. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 7, 2021. IgG Antibody glycan analysis. Reduced IgG purified from plasma were separated on an SDS-PAGE gel and the band correspondent to the heavy chain was cut and subjected to in-gel digestion as follows: gel bands were washed in 100 mM Ammonium Bicarbonate (AmBic)/Acetonitrile (ACN) and reduced with 10 mM dithiothreitol at room temperature for 45 min. Cysteines were alkylated with 50 mM iodoacetamide in the dark for 45 min. at room temperature. Finally, gel bands were washed in 100 mM AmBic/ACN prior to adding 600 ng Lys-C for overnight incubation at room temperature. Following digest, supernatants containing peptides were transferred into new tubes. Gel pieces were washed at room temperature for 10 min. with gentle shaking, in 50% ACN/5% FA, and supernatants were combined with peptide solutions. This wash was repeated each by 80% ACN/5% FA, and 100% ACN, and all supernatants was saved. Pooled supernatants were then subject to speedvac drying. After lyophilization, peptides were reconstituted with 5% ACN/0.1% FA in water. Peptides were analyzed by LC-MS/MS using a Dionex UltiMate 3000 Rapid Separation nanoLC and a Q Exactive™ HF Hybrid Quadrupole-Orbitrap™ Mass Spectrometer (Thermo Fisher Scientific Inc). The peptide samples were loaded onto the trap column, which was 150 μm x 3 cm in-house packed with 3 µm C18 beads. The analytical column was a 75 µm x 10.5 cm PicoChip column packed with 3 µm C18 beads (New Objective). The flow rate was kept at 300 nL/min. Solvent A was 0.1% FA in water and Solvent B was 0.1% FA in ACN. The peptide was separated on a 60 min. analytical gradient from 5% to 50% of Solvent B. The mass spectrometer All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 7, 2021. ; https://doi.org/10.1101/2021.07.06.21259226 doi: medRxiv preprint was operated in the Full MS scan. The source voltage was 2.30 ~ 2.50 kV and the capillary temperature 320°C. Full MS scans were acquired from 400-2000 m/z at 60,000 resolving power and automatic gain control (AGC) set to 3x10 6 . MS data were processed using Skyline (Version 20.2). The integration and correction for the chromatographic peaks of 18 glycans were performed manually. Three of the most intense precursor ions of each glycan were selected, summed, and exported as the quantitative value of the corresponding glycan. Total ion intensities were used to generate the plots. Statistical analysis. ANOVA statistics were calculated using SAS (SAS Institute, Cary, NC). Person's correlations were calculated using the function "cor.test" and method "pearson" on RStudio v3.1.1073. Violin and dot plots were generated with GraphPad Prism 9.0.2 and that same software was used for calculating the statistical significance by one-way ANOVA with Tukey's multiple comparison test (* p<0.05; ** p<0.01). Data and software availability. Processed datasets utilized for the IgMS analyses can be found on the MassiVE repository, MSV000087529, after puplication. Custom compiled code used to process and create I 2 MS files is already available 16 . Additional desired software and data that support the findings of this study are available from the corresponding authors upon request. Protein Production Core Facility. The reagent was produced under HHSN272201400008C and obtained through BEI Resources, NIAID, NIH: Vector pCAGGS Containing the SARS-Related Coronavirus 2, Wuhan-Hu-1 Spike Glycoprotein Receptor-Binding Domain (RBD), NR-52309. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 7, 2021. ; https://doi.org/10.1101/2021.07.06.21259226 doi: medRxiv preprint IgG glycosylation and bottom-up proteomics were measured by the Northwestern Proteomics Core Facility. A pneumonia outbreak associated with a new coronavirus of probable bat origin Comorbidities and the risk of severe or fatal outcomes associated with coronavirus disease 2019: A systematic review and meta-analysis Provisional Mortality Data -United States Antibody resistance of SARS-CoV-2 variants B.1.351 and B.1.1.7 SARS-CoV-2 infection induces sustained humoral immune responses in convalescent patients following symptomatic COVID-19 Longitudinal Isolation of Potent Near-Germline SARS-CoV-2-Neutralizing Antibodies from COVID-19 Patients Isolation of potent SARS-CoV-2 neutralizing antibodies and protection from disease in a small animal model Molecular deconvolution of the monoclonal antibodies that comprise the polyclonal serum response The promise and challenge of high-throughput sequencing of the antibody repertoire A proteomics approach for the identification and cloning of monoclonal antibodies from serum Evolution of immune responses to SARS-CoV-2 in mild-moderate COVID-19 Molecular-level analysis of the serum antibody repertoire in young adults before and after seasonal influenza vaccination Proteoform: a single term describing protein complexity Analysis of Monoclonal Antibodies in Human Serum as a Model for Clinical Monoclonal Gammopathy by Use of 21 Tesla FT-ICR Top-Down and Middle-Down MS/MS Multiplexed mass spectrometry of individual ions improves measurement of proteoforms and their complexes A serological assay to detect SARS-CoV-2 seroconversion in humans Phenotyping Polyclonal Kappa and Lambda Light Chain Molecular Mass Distributions in Patient Serum Using Mass Spectrometry Sex, age, and hospitalization drive antibody responses in a COVID-19 convalescent plasma donor population Magnitude and Kinetics of Anti-Severe Acute Respiratory Syndrome Coronavirus 2 Antibody Responses and Their Relationship to Disease Severity A serological assay to detect SARS-CoV-2 seroconversion in humans Enhanced binding of antibodies generated during chronic HIV infection to mucus component MUC16 Afucosylated IgG characterizes enveloped viral responses and correlates with COVID-19 severity Unique carbohydrate-carbohydrate interactions are required for high affinity binding between FcγRIII and antibodies lacking core fucose Neutralizing antibody titres in SARS-CoV-2 infections Convalescent Plasma Therapy for COVID-19: A Graphical Mosaic of the Worldwide Evidence Deep Sequencing of B Cell Receptor Repertoires From COVID-19 Convergent antibody responses to SARS-CoV-2 in convalescent individuals De Novo MS/MS Sequencing of Native Human Antibodies Accurate Sequence Analysis of a Monoclonal Antibody by Top-Down and Middle-Down Orbitrap Mass Spectrometry Applying Multiple Ion Activation Techniques Potent neutralizing antibodies against multiple epitopes on SARS-CoV-2 spike Antibody titers against SARS-CoV-2 decline, but do not disappear for several months The Pipeline Repertoire for Ig-Seq Analysis SARS-CoV-2 Seroconversion in Humans: A Detailed Protocol for a Serological Assay, Antigen Production, and Test Setup A comprehensive pipeline for translational top-down proteomics from a single blood draw A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids An informatic framework for decoding protein complexes by topdown mass spectrometry Novel Interface for High-Throughput Analysis of Biotherapeutics by Electrospray Mass Spectrometry Individual Ion Mass Spectrometry Enhances the Sensitivity and Sequence Coverage of Top-Down Mass Spectrometry Isotopic Resolution of Protein Complexes up to 466 kDa Using Individual Ion Mass Spectrometry STORI Plots Enable Accurate Tracking of Individual Ion Signals Automated reduction and interpretation of high resolution electrospray mass spectra of large molecules Determination of monoisotopic masses and ion populations for large biomolecules from resolved isotopic distributions