XA-Novo: high-throughput mass spectrometry-based de novo sequencing technology for monoclonal antibodies and antibody mixtures

Domain: biology
Published: 2026-03-12
Editorial outcome: major_revision

Abstract
Elucidating antibody sequences by mass spectrometry-based de novo sequencing is essential but remains technically challenging. Here we present XA-Novo, an accurate and high-throughput de novo sequencing solution that integrates a single-pot multi-enzymatic gradient digestion method with a beam search-based assembler (Fusion) to reconstruct full-length antibody sequences directly from bottom-up mass spectrometry data. Benchmarking across well-characterized antibodies from multiple species demonstrates that XA-Novo outperforms commercial solutions in identification sensitivity, sequence completeness, and reconstruction accuracy. Furthermore, XA-Novo successfully reconstructs six immunotherapeutic antibodies with unknown sequences, and in vitro/vivo assays validate that these generated antibodies exhibit functionality equivalent to their commercial counterparts. Moreover, XA-Novo achieves over 99.54% accurate sequence coverage in distinguishing mixed COVID-19 neutralizing antibodies, exceeding the performance of current assemblers reported for single-antibody sequencing. Together, XA-Novo establishes a reliable, scalable, and broadly applicable workflow for routine antibody sequencing, thereby accelerating both fundamental antibody research and therapeutic antibody development.

Introduction
Antibodies play a crucial role in host defense against pathogens by recognizing and neutralizing foreign entities, such as pathogenic bacteria and viruses1, 2. In addition to directly neutralizing these pathogens, antibodies coordinate a broader immune response by recruiting effector functions for mechanisms including antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity1. Moreover, antibodies generated in response to an infection can persist in circulation for extended periods, facilitating rapid and robust responses upon re-encountering the same pathogen due to the recall ability of memory B cells3. The multifaceted functionality of antibodies underscores their critical significance across a broad spectrum of disciplines, encompassing immunology, clinical chemistry, biochemistry, therapeutics, and medicine4, 5, 6. Consequently, they have emerged as invaluable tools in numerous research endeavors and practical applications.

The antibody sequence information is crucial for comprehending the structural foundation of antigen-antibody binding, recognition, and interaction7, 8. Additionally, sequence-based recombination, expression, or engineering modifications can be used to achieve constant region substitution of antibodies from different species, thereby altering antibody specificity and enabling the design of diverse antibodies. However, obtaining complete sequences for monoclonal antibodies (mAbs) remains a significant challenging. Traditional hybridoma technology9, 10, which involves antibody screening and sequencing, typically requires three to six months to complete and incurs substantial time and cost. Researchers have made progress in isolating B cells directly from blood or bone marrow, extracting DNA or RNA, and creating sequencing libraries using second-generation sequencing technologies independent of hybridoma method. This advancement has reduced the timeline for obtaining sequences to as short as one month11, 12, 13. Despite these advancements, the time-consuming nature of these methods and the need for complementary information from both genetic and protein levels remain challenges that must be addressed. Additionally, issues such as bacterial contamination or decreased viability during the antibody screening process can render it impossible to obtain sequence information, severely limiting flexibility in antibody screening efforts. Furthermore, since antibodies are secreted into bodily fluids and mucus without a direct connection to their producing B cells, questions arise regarding the quantitative relationship between the secreted antibody pool and its underlying B-cell population—along with potential sampling biases inherent in current antibody sequencing strategies.

Mass spectrometry (MS)-based de novo sequencing of secreted antibodies serves as a valuable complementary approach that can effectively address several challenges faced by conventional strategies, which rely on cloning and sequencing of coding mRNAs14, 15, 16. For example, the MS-based de novo sequencing approach can directly target polypeptide products in biological fluids to acquire antibody sequences. This method has successfully sequenced mAbs from lost hybridoma cell lines, thereby establishing the foundation for the next generation of serological multi-antibody de novo sequencing. Nevertheless, despite these advancements, MS-based de novo sequencing still encounters significant impediments such as large sample requirements, low throughput, unanticipated missing ion information, and difficulties in sequencing and assembly accuracy. During antibody sequencing, the existence of isomers with similar mass (e.g., leucine [Leu] and isoleucine [Ile]) or amino acid combinations that yield identical results (e.g., AG = Q, GG = N) can easily lead to erroneous sequence assembly17, 18, 19. These issues are already encountered when sequencing a single antibody and become even more pronounced during the de novo sequencing of multiple antibodies. Therefore, selecting appropriate peptides from ambiguous sequences and implementing accurate amino acid error correction are essential for developing more precise, efficient high-throughput methods for antibody sequencing.

To address these challenges, we propose XA-Novo, a pipeline that combines a single-pot multi-enzymatic gradient digestion (SP-MEGD) method with a beam search-based Fusion assembler to accurately decipher complete antibody sequences during both the sample preparation and the data analysis phase. We benchmarked XA-Novo using data from multiple known antibodies across various species, which included samples containing only a single antibody as well as mixtures of COVID-19 neutralizing antibodies (NAbs) comprising two or three antibodies per sample. The results demonstrated that XA-Novo outperforms commercial solutions in terms of identification capacity, data completeness, and accuracy of antibody reconstruction. Furthermore, XA-Novo successfully reconstructed six immunotherapeutic antibodies with unknown sequences; in vitro assays confirmed that the generated antibodies exhibit functionality equivalent to their commercial counterparts, thereby further validating the effectiveness of our solution. In conclusion, we have established a precise, robust, high-throughput de novo sequencing solution that advances antibody research by enhancing both the accuracy and efficiency of complex antibody sequencing.

Results
XA-Novo delivers high-throughput, mixture-compatible de novo antibody sequencing by MS-only proteomics

The comprehensive de novo sequencing solution provided by the XA-Novo platform is illustrated in Fig. 1. Specifically, XA-Novo was developed based on SP-MEGD sample preparation for obtaining sufficient peptide-level information, followed by liquid chromatography-mass spectrometry (LC-MS/MS) analysis for generating mass spectra, then employing deep learning-based de novo peptide sequencing to generate peptide reads, and finally using Fusion assembler to assemble these peptides into complete heavy and light chains. The sequencing results obtained through experimental methods were initially validated via intact mass analysis and subsequently confirmed through bioactivity or functional assays. Our mAb sequencing method is capable of functioning with as little as 50 µg of purified mAb. This method is not only applicable to traditional mAb sequencing but also facilitates high-throughput mixture sequencing involving multiple antibodies. Our study provides three key contributions that leverage these advancements, thereby making antibody sequencing at the protein level via MS more attainable:

(1) Extensive experimental results demonstrate the accuracy and robustness of our solution for traditional mAb sequencing, thereby facilitating the acquisition of precise antibody sequence information for routine applications.

(2) XA-Novo enables higher-throughput full-length de novo mAb sequencing within a bottom-up proteomics workflow, while maintaining high accuracy, thereby improving the efficiency of mAb research.

(3) XA-Novo supports the simultaneous analysis of multiple mAbs under identical experimental conditions, reducing sequencing costs and improving resource utilization in large-scale antibody research and development by optimizing enzyme and reagent usage and MS instrument time.

SP-MEGD sample preparation enables sufficient peptide-level information

To tackle the challenge of unexpectedly missing ion information and incomplete sequence coverage, we developed a method termed SP-MEGD. This approach employs a five-protease gradient digestion, with each enzyme utilized in a single pot (Fig. 1a). Samples were collected at two-hour intervals over a total duration of six hours within an integrated reactor. This approach is designed to minimize sample loss and enhance the availability of antibody ion information. During SP-MEGD method development, we optimized and evaluated crucial aspects of antibody preparation using the S2P6 mAb with published sequences20. We prioritized sequence coverage and accuracy as the primary performance metric (refer to Fig. 2a for a detailed explanation). We aimed to identify the best combination of reagents for protein denaturation and reduction, minimizing sample loss compared to the conventional five-step process21. We compared the lysis efficiencies of three compatible lysis buffers: a. 2% DOC + 20 mM TCEP + 200 mM Tris-HCl, pH 8.0; b. 8 M Urea + 20 mM TCEP + 100 mM Tris-HCl, pH 8.0; c. 6 M Gu·HCl + 20 mM DTT. The results showed that the Gu·HCl buffer provided high coverage rates for heavy chains in both high-energy collision dissociation (HCD) and electron-transfer high-energy collision dissociation (EThcD) fragmentation modes when combined with various enzymes - trypsin, chymotrypsin, pepsin, elastase, and Asp-N (Fig. 2b). However, coverage rates for the light chain from the combination of Gu·HCl buffer and Asp-N were not high due to fewer cleavage sites in the tested antibody light chain sequence, resulting in limited peptide information. Therefore, Gu·HCl buffer was selected for lysing antibody samples, considering its overall effectiveness in achieving high coverage.

Previous studies have demonstrated that limited proteolytic reactions, achieved by reducing enzyme concentration or shortening reaction times, can increase the frequency of missed cleavage sites, producing diverse peptide fragments of varying lengths with overlapping residue extensions that facilitate efficient database-free de novo sequencing22, 23. Therefore, we first evaluated four different enzyme-to-protein ratios (ranging from 1:5 to 1:50) with a fixed 16-hour reaction time to ensure reproducibility. LC-MS/MS analysis showed an increase in unique peptides as the enzyme-to-substrate ratio decreased from 1:5 to 1:50 (Supplementary Fig. 1c-e). The optimal trypsin-to-protein ratios were established at both 1:20 and 1:50, yielding increased peptide counts and peak areas that fulfill the requirements for overlapping segments necessary in downstream assembly. Similar optimizations were carried out for the remaining predicted enzymes, each resulting in its respective optimal enzymatic ratio. To further evaluate the performance of a five-enzyme combination, we employed a commercial antibody (anti-mouse Ly-6G) as a test case. As a result, the ratio of 1:20 demonstrated superior outcomes in terms of peptide counts, sequence coverage, and number of PSMs (Fig. 2c-d). We then investigated the optimal enzymatic digestion time for antibody samples. A comparative analysis was conducted over a range of 2 to 16 hours (overnight), with three technical replicates at each time point. The results indicated that the average coverage of antibody sequences increased from 2 to 6 hours, with the effect observed at 6-hour digestion being comparable to that achieved after overnight digestion at 16 hours. This trend was consistent across both HCD and EThcD fragmentation modes (Fig. 2e). Recognizing the significance of abundant fragment information in supporting peptide assembly in antibody de novo sequencing, we proposed a gradient digestion strategy (SP-MEGD). Samples were collected at the 2nd, 4th, and 6th hours before being pooled to maximize the missed cleavage rates (Fig. 2f). This strategy could provide effective "head-to-tail" evidential information for assembling peptide fragments.

While a sequence coverage exceeding 87% with a confidence level of at least 95% has been achieved using 1:20 single trypsin digestion in gradient digestion (Fig. 2g), significant improvements in peptide counts can be realized by leveraging multiple enzyme combinations. Hence, the final SP-MEGD method employs a five-protease (trypsin, chymotrypsin, pepsin, elastase, and Asp-N) gradient digestion approach, with samples collected every two hours over a total duration of 6 hours. This strategy offers greater diversity in proteolytic peptide length, enhancing the probability of overlapping fragments necessary for de novo assembly and providing flexibility regardless of the protein sequence and properties. We conducted additional experiments to assess the SP-MEGD method using various amounts (50, 100, and 200 µg) of starting materials for antibody samples. Based on PEAKS AB, S2P6 antibodies consistently achieved 100% accuracy in the amino acid sequence within all complementarity-determining region (CDR) at initial quantities of 50, 100, or 200 µg (Supplementary Fig. 2). This was confirmed by comparing the determined sequence to the S2P6 reference sequence, and the measured masses of both light and heavy chains align with theoretical calculations (Fig. 2h). These results demonstrate that our SP-MEGD method is suitable for analyzing antibody products with sample volumes as low as 50 µg, indicating its potential for routine use.

We further compared the SP-MEGD method with the commercial EasyPept-Micro kit (Omicsolution, #OSFP0001) for microscale label-free sample preparation using 25 µg of S2P6 antibody. Given that the EasyPept-Micro kit exclusively employs trypsin for digestion, we initially conducted a comparison of trypsin digestion alone as a benchmark. We evaluated sequence coverage as the primary metric to assess performance. The SP-MEGD method achieved approximately 100% sequence coverage for the heavy chain and 99.5% for the light chain, surpassing results obtained from the EasyPept-Micro kit (Fig. 2i). As the SP-MEGD method is a five-protease gradient digestion approach, we also assessed additional enzymes with complementary specificities (chymotrypsin, pepsin, elastase, and Asp-N) using the commercial EasyPept-Micro kit. The results indicated that, except for chymotrypsin and pepsin, this kit exhibited suboptimal performance in terms of sequence acquisition coverage when employing other enzymes (Fig. 2i, and Supplementary Fig. 1f). Notably, peptide information derived from Asp-N within this kit was substantially lower than that obtained with other enzymes (Student's t-test, p<0.05); this discrepancy may be attributed to compatibility issues. Furthermore, we combined all results from various enzymes within the kit to determine the sequence of S2P6. As a result, both peptides and PSMs obtained through the SP-MEGD method exceeded those acquired via the EasyPept-Micro kit (Fig. 2j, and Supplementary Fig. 1g). Additionally, when the EasyPept-Micro kit was utilized in conjunction with other commercial proteases, the resulting coverage of CDR within HC in S2P6 was consistently demonstrated inferior performance compared to that achieved with SP-MEGD (Fig. 2j). This diminished sequence coverage may hinder effective assembly of peptide information and subsequently affect sequencing accuracy (Fig. 2j). These findings demonstrate that our innovative SP-MEGD strategy is more versatile and efficient compared to the commercially available alternatives, such as the EasyPept-Micro kit.

De novo peptide sequencing with Casanovo

Therefore, we implemented the SP-MEGD strategy for sample preparation and subsequently trained a Casanovo model24, 25 for de novo peptide sequencing of MS/MS spectra. The Casanovo model rapidly converged within the first ten epochs (Supplementary Table 1), exhibiting decreasing training and validation losses alongside increasing amino acid (AA) precision, AA recall, and peptide recall. The best-performing model, selected at epoch 10, achieved validation values of 0.7977 for AA precision, 0.7979 for AA recall, and 0.6006 for peptide recall. We further evaluated the trained Casanovo model on multiple independent external datasets, including eight previously published species-specific datasets and an SP-MEGD-based monoclonal antibody dataset (mAbs-known). Casanovo exhibited strong and consistent performance across diverse species (Supplementary Table 2), with AA precision and recall ranging from 0.7628 to 0.8735, while peptide recall varied from 0.5060 to 0.7430. Performance on the antibody-specific dataset (mAbs-known) also remained robust; it yielded an AA precision of 0.7279, an AA recall of 0.7257, and a peptide recall of 0.5547. These results confirm the reliability and effectiveness of the model in antibody-focused sequencing tasks (Supplementary Table 2). Consequently, the epoch-10 model was selected as the final model used for XA-Novo.

Next, we assessed the assembly potential of the SP-MEGD strategy combined with the Casanovo model utilizing three human COVID-19 neutralizing antibodies, which included SA55, SA5826, and S2P6 (with quantities of 50 µg, 100 µg, and 200 µg), in addition to three murine COVID-19 NAb datasets (85F7, 36H6, and 2B4)27. Combined with five proteases and utilizing HCD-based MS analysis, we collected the following peptide reads (defined as peptides with a score ≥ 50): 12,423 for SA55, 23,998 for SA58, 15,303 for 50 µg S2P6, 15,851 for 100 µg S2P6, 21,967 for 200 µg S2P6, 27,218 for 36H6, 29,993 for 85F7, and finally, 32,862 for 2B4 (Fig. 2k). As illustrated in Supplementary Fig. 2, the sequence coverage achieved 100% for both heavy and light chains across variable and constant domains. Furthermore, the mean depth of coverage within the CDRs, variable domain, and overall chain for both heavy and light chains ranged from 56.12 to 212.44, 76.67 to 216.49, and 107.58 to 283.70, respectively (Fig. 2l, Supplementary Fig. 3, and Supplementary Table 3). Consequently, our pre-trained Casanovo model not only exhibited promising results in predicting peptide sequences and provided sufficient peptide information for complete assembly, but also demonstrated the effectiveness of the SP-MEGD method in yielding comprehensive ion information and complete sequence coverage.

Fusion assembler enables accurate full-length sequence reconstruction

The complete assembly of antibody sequences poses a significant challenge in de novo sequencing. Current algorithms, such as ALPS28 and Stitch15, 29, struggle with accuracy and robustness for routine applications. While commercial software such as PEAKS AB represents advanced solutions in the field, it does not support the reconstruction of multiple monoclonal antibodies from a mixed sample. To overcome these issues, we developed the Fusion assembler, which employs a beam search strategy with a beam size of 5. This approach incorporates a seven-amino-acid sliding window approach and mass-based alignment29 distance metrics, while ensuring signal consistency for template-assisted assembly of mAbs as well as mixtures containing various mAbs. All details about the Fusion assembler can be found in the Supplementary Methods. As illustrated in Fig. 1a, the beam search strategy enhances Fusion to find the correct sequence during the assembly. It extends the contig bidirectionally during CDR retrieval, enabling it to restructure complete and accurate fragments in both heavy and light chains. The Fusion algorithm identifies Leu and Ile residues by integrating template sequence information with experimental evidence and detecting diagnostic w-ions, thus enhancing the precision of isomer discrimination.

Stitch29 is a state-of-the-art public method for antibody sequence assembly; however, its use requires users to select parameters such as peptide cutoff scores, segment orders, and software versions. These choices may necessitate careful consideration and, in some cases, expert knowledge to achieve optimal results. During the first run (Fig. 3a), all assembly results for the light chain exhibited gaps due to the actual length of light chain CDR3 for SA55 and S2P6 antibodies exceeding that of the template. Therefore, we modified the recombined segment order of the light chain to "IGLV*IGLJ IGLC" for these two antibodies. Upon refining the Stitch run to enforce selection of "IGLV*IGLJ IGLC" as the recombined segment order for light chain, nearly all CDRs in human antibody datasets achieved an accuracy improvement to 100%, with one exception being observed in SA55 when processed using Stitch1.4.0 with a 95-peptide cutoff score (Fig. 3b). As a result, there was also an overall enhancement in accuracy for the light chain. In our analysis across five human antibody datasets, varying peptide cutoff score settings substantially influenced both CDR accuracy and overall chain accuracy (Fig. 3b). This phenomenon was similarly noted in three murine antibody datasets (Fig. 3c). Notably, while the actual length of the light chain CDR3 from antibody 85F7 is shorter than that of its corresponding template, all assembled results produced a CDR3 sequence matching that of the template length (see Methods, Supplementary Data 1). On the contrary, Fusion employs an anchor-guided, template-adaptive assembly driven by k-mer overlap evidence and a local beam search toward conserved targets (see the Supplementary Methods, Supplementary Table 4 for more details). It does not require users to pre-specify the orders of recombined segments; instead, it infers CDR boundaries and lengths directly from data using 7-mer anchors flanking each CDR and de novo extension between them. Even when the true CDR is longer or shorter than the template (as in SA55/S2P6-like or 85F7-like scenarios), Fusion yields consistently accurate CDR reconstructions.

To comprehensively assess the assembly performance of Fusion, we conducted a comparative analysis with the best results from Stitch and PEAKS AB software. Across five datasets derived from three human COVID-19 NAbs with known sequences, Stitch exhibited variability in its sequence coverage accuracy. The light chain accurate coverage ranged from 98.61% to 100%, while the heavy chain accurate coverage varied between 97.58% and 99.78% (Fig. 3d). Notably, Stitch was unable to assemble the complete heavy chain of the SA55 antibody, attaining only 76.47% accuracy for the CDR region and 97.58% for the overall chain (Fig. 3d, and Supplementary Fig. 4). Additionally, the light chain of the SA58 antibody exhibited gaps due to the actual length of CDR1 exceeding that of the template, attaining only 85% accuracy for the CDR region and 98.61% for the overall chain (Fig. 3d, and Supplementary Fig. 4). For PEAKS AB, the SA58 light chain achieved 100% accuracy for the CDR region and 99.07% for the whole chain, while the heavy chain showed only 68.97% accuracy in the CDR region, mainly due to 8 amino acid gaps in CDR3. The accuracy in the heavy chain was 97.33% with multiple amino acid gaps and insertions (Fig. 3d, and Supplementary Fig. 4). In contrast, Fusion consistently achieved 100% accurate coverage without any gap and insertion for both heavy and light chains across all datasets, demonstrating superior robustness even when faced with varying sample quantities in the S2P6 antibody datasets (Fig. 3d, and Supplementary Fig. 5). In the context of antibody reverse engineering and re-expression, sequence accuracy thresholds of ≥99% are widely considered essential. Sub-percent errors, particularly those involving insertions or deletions within CDR, can significantly compromise binding affinity or adversely affect product quality30, 31, 32.

We also conducted a comparative analysis using three murine COVID-19 NAb datasets with known variable region sequences (85F7, 36H6, and 2B4)27. Fusion demonstrated superior performance compared to both Stitch and PEAKS AB, achieving 100% accurate coverage of CDR regions for all antibodies (Fig. 3e). In contrast, the performance of Stitch and PEAKS AB showed considerable variability across different antibodies. The accurate coverage of Stitch for light chain variable regions fluctuated between 99.06% and 100%, while for heavy chain variable regions, it ranged from 95.80% to 99.17%. Notably, Stitch was unable to fully assemble the heavy chain CDR3 sequence of the 85F7 antibody due to amino acid gaps (Supplementary Fig. 6). PEAKS AB also displayed inconsistency; its accurate coverage for light chain variable regions varied from 86.73% to 100%, whereas for heavy chains, it ranged from 97.48% to 100%. Furthermore, PEAKS AB failed to assemble the complete heavy chain CDR3 sequence of the 2B4 antibody as well as the light chain CDR3 sequence of the 36H6 antibody due to amino acid errors, gaps, and insertions (Supplementary Fig. 6). These findings suggest that, although the accurate coverage rate may approach 95%, the presence of amino acid errors, gaps, and insertions in the antibody sequence prevents the reliable expression of true protein products, and further demonstrate the reliability of Fusion and de novo sequencing of antibodies based on bottom-up mass spectrometry.

In conclusion, this comparative study underscores that Fusion outperforms both Stitch and PEAKS AB in assembly performance, particularly regarding accuracy and consistency across different samples and critical regions such as CDRs, making it a more reliable tool in our solution for precise antibody sequence analysis and research.

Application of commercial immunotherapeutic monoclonal antibodies with unknown sequence

Cancer immunotherapy is rapidly advancing and has now been recognized as the "fifth pillar" of cancer therapy33. By elucidating the sequence of immune-associated antibodies, it becomes feasible to design more effective targeted antibodies that precisely interact with immune cells within the tumor microenvironment or disrupt mechanisms of tumor-associated immune evasion, thereby enhancing the efficacy of immunotherapy. To demonstrate the effectiveness of our solution (XA-Novo) for therapeutic antibody research, we analyzed six immune-associated antibodies for which sequence information was previously unavailable: anti-CD4, anti-CD8α, anti-CSF1R, anti-IFNAR-1, anti-IFNγR, and anti-LTB4R mAbs . This expanded study served as a large-scale trial to determine whether XA-Novo could achieve results with high confidence comparable to those obtained for COVID-19 NAbs with known sequences. The ultimate goal was to use the deciphered sequences for gene cloning and functional expression of these antibodies.

After de novo peptide sequencing with Casanovo, we collected 39,552 (anti-CD4), 16,856 (anti-CD8α), 33,857 (anti-CSF1R), 37,084 (anti-IFNAR-1), 35,757 (anti-IFNγR), and 25,458 (anti-LTB4R) peptide reads from HCD spectra. The Fusion algorithm successfully assembled these peptides into the complete sequences for both heavy and light chains. The mean depth of coverage in the CDRs, variable domain, and overall chain ranged from 33.69 to 340.52, 73.31 to 425.94, and 128.65 to 356.58, respectively (Supplementary Fig. 7, and Supplementary Table 5).

For comparative analysis, we also executed PEAKS AB to analyze these antibodies (Fig. 4). In contrast to the Fusion assembler, PEAKS AB obtained longer sequences for the heavy chains in anti-CD8α and anti-CSF1R antibodies (Fig. 4d, f). Furthermore, significant differences were observed in the amino acid sequence of these antibodies' heavy chains, especially within the CDR3 region. We further validated this by performing an intact mass analysis. The results indicated that the theoretically calculated mass derived from peaks AB exhibited a significant deviation from the mass measured by MS of the anti-CSF1R (Supplementary Table 6). In contrast, the sequence assembled by Fusion was more closely aligned with the MS-measured mass of the source antibody (Supplementary Table 6). This finding suggests that Fusion holds promise for accurately characterizing the actual antibody sequences. The results of intact mass analysis also confirm that "VTAASGPEKVTTITLTPKVT" represents an insertion within the sequence generated by PEAKS AB, which will be deleted as a refined sequence during subsequent recombinant expression. Additionally, the ninth amino acid in the CDR3 region of the anti-CSF1R heavy chain was identified as isoleucine (Ile) by PEAKS AB and leucine (Leu) by Fusion (Fig. 4f). Similarly, in the light chain of the anti-CD8α antibody, although the calculated mass was consistent in both methods, we observed a different presence of amino acid combinations (AG = Q) in the two methods (Fig. 4d). Therefore, further validation using biochemical methods is necessary to confirm the accuracy of the sequences generated by the Fusion assembler.

Subsequently, the sequences generated by Fusion were cloned and transfected into cells for recombinant expression, followed by affinity purification. The recombinant antibodies and matched commercial antibodies were then administered to C57BL/6J mice (n = 3 per group, Fig. 4a). The third day post-injection revealed effective clearance of T-cells in both the spleen and blood. In contrast to control mice, which showed no depletion of T-cells, the XA-Novo-generated antibody demonstrated clearance efficiencies of 99.65% (anti-CD4) and 99.71% (anti-CD8α) in the spleen, as well as 99.78% (anti-CD4) and 99.72% (anti-CD8α) in the blood (Fig. 4c-e, Supplementary Fig. 8, and Supplementary Fig. 9). These results were consistent across all animals, as illustrated in Supplementary Fig. 9. This highlights the reproducibility of clearance efficiency observed in each animal's blood and spleen, confirming that the effects of XA-Novo-generated antibodies corresponded with those of commercial antibodies. We further investigated the role of Ile and Leu in the heavy chain CDR3 region of anti-CSF1R through recombinant expression (Fig. 4f, and Supplementary Fig. 10). The results demonstrated that the anti-CSF1R antibody containing Leu at the ninth amino acid position in the heavy chain CDR3 region exhibited enhanced macrophage depletion in the spleen (Fig. 4g, XA-Novo (125L): 65.50%; XA-Novo (125I): 40.94%), highlighting the critical role of Ile and Leu identification in the CDR region and confirming the effectiveness of our method for the Ile/Leu differentiation. Furthermore, we observed that the clearance efficiency of antibodies generated by XA-Novo (L) in peripheral blood was slightly lower than that of antibodies produced by XA-Novo (I) (Fig. 4g, and Supplementary Fig. 10). This discrepancy may be attributed to the relatively low content of macrophages in peripheral blood, which is susceptible to dynamic fluctuations within the circulatory system34. Consequently, this makes it challenging to accurately assess the clearance effect of antibodies on tissue macrophages. The spleen is a crucial immune organ in mice, containing a substantial population of T cells, B cells, and macrophages35. Therefore, utilizing spleen results as primary conclusions while considering peripheral blood findings for dynamic monitoring will facilitate a more comprehensive understanding of the clearance mechanisms involved. Furthermore, the flow cytometry and Biacore analyses of the anti-LTB4R, anti-IFNAR-1, and anti-IFNγR antibodies further corroborated the accuracy of the sequences analyzed by Fusion (Supplementary Fig. 11-13). These discoveries provide biological validation for our solution, underscoring its effectiveness in accurately reconstructing sequences for unknown antibodies and its potential impact on antibody research and development.

Application of a Monoclonal Antibody Mixture for COVID-19

The COVID-19 pandemic, which is caused by the SARS-CoV-2 virus, continues to spread. NAbs have emerged as pivotal players in preventing and treating COVID-1936, 37, 38. However, the persistent emergence of new variants has resulted in widespread evasion of NAbs, presenting significant challenges to NAb-based therapeutics26. There is a high demand for the clinical development of NAb drugs resistant to future variants39, 40. Rapidly determining the amino acid sequence of antibodies is critical for NAb drug discovery, enabling sequence-based recombination or engineering modifications that can alter antibody specificity and facilitate the design of diverse antibodies.

To address this challenge, we next evaluated whether our sequencing platform could accurately reconstruct antibody sequences from mixtures, thereby mimicking the complexity encountered in therapeutic antibody discovery. In a mixture containing two antibodies, S2P6 and SA55, both reached an impressive sequence accuracy of 100%, with complete CDR region fidelity observed in both heavy and light chains (Supplementary Fig. 14a). Notably, XA-Novo demonstrated consistent differentiation of I-L with 100% accuracy across all antibodies, irrespective of whether the mixture contained two or three antibodies. When analyzing a more complex combination comprising S2P6, SA58, and SA55 antibodies (Fig. 5b), S2P6 continued to exhibit full sequence coverage with 100% accuracy across both chains and all CDR regions. For SA55 and SA58, while all CDR regions still displayed perfect accuracy at 100% in both heavy and light chains, there was a slight reduction in overall accuracy—99.78% for the heavy chain of SA55 and 99.54% for the light chain of SA58. Taking the results of other assemblers in traditional single-antibody sequencing as a baseline (Fig. 3), Fusion's overall performance in antibody mixture sequence assembly remains superior to other assemblers, which are limited to sequencing single antibodies. These small discrepancies are attributed to shared template-derived positions: the last amino acid in the heavy chain FR4 region (TLVTVSSASTKGPS) for SA58, and the 154th amino acid in the constant region of the light chain (VRQWKVDNALQSGN) for all three antibodies. Fusion's assembler tolerates these minor deviations as they occur in structurally conserved regions with negligible impact on functional accuracy. Nevertheless, incorporating intact mass measurements for each antibody allows complete correction of these minor inconsistencies, yielding fully accurate sequences across all components. We further evaluated antibody mixtures derived from mouse species that included three distinct antibodies: 85F7, 36H6, and 2B4. Remarkably, our approach achieved 100% sequence accuracy across all three antibodies, with complete fidelity in both heavy and light chains, including all CDR regions. These results were consistent regardless of whether the mixture contained two or three antibodies (Fig. 5c, and Supplementary Fig. 14b-d), and the reconstructed sequences were identical to those obtained using traditional single-antibody sequencing workflows. Collectively, these mixture experiments show that accurate sequence reconstruction is maintained as mixture complexity increases. The reconstructed sequences are consistent with those derived from conventional single-antibody workflows, indicating the platform’s utility for simultaneous analysis of multiple antibodies and enabling high-confidence antibody characterization.

Web-based tools for antibody de novo sequencing analysis

Lastly, we have developed a user-friendly, web-based tool for analyzing antibody de novo sequencing (Fig. 6). This tool leverages advanced deep learning models for peptide sequencing and incorporates a beam search-based Fusion assembler that we previously established. This resource is readily accessible at https://xa-novo.com/.

Researchers can perform their analyses with relative ease, eliminating the need for complex configurations or specialized hardware requirements. The only prerequisite is to provide a link to the mass spectrometry data along with related species information regarding the detected antibody to the XA-Novo platform.

Methods
Ethical Statement

Animal experiments were carried out in accordance with the approval of the Institutional Animal Care and Use Committee at Xiamen University (XMULAC20230310) and by the Guide for the Care and Use of Laboratory Animals.

Antibody reagents and production

The anti-mouse CD4 (clone# GK1.5), anti-mouse CD8α (clone# 2.43), anti-mouse CSF1R (clone# AFS98), anti-mouse IFNAR-1(clone# MAR1-5A3), and anti-mouse IFNγR (also known as IFNGR; clone# GR-20) mAbs were purchased from BioXCell (BE0003-1, BE0061, BE0213, BE0241, and BE0029). The anti-mouse Ly-6G (clone# 1A8) mAb was purchased from Leinco (L280-25mg). The anti-human LTB4R (Mouse, clone# 7B1) mAb was purchased from Thermo Fisher (MA1-20232). Recombinant human COVID-19 NAbs (S2P6, SA55, and SA58, in human IgG1 backbone) were expressed by the ExpiCHO-S™ suspension expression system (Thermo Fisher Cat#A29133) and subsequently purified using MabSelect SuRe resin (Cytiva). The amino acid sequences of these mAbs in our expression plasmids are shown in Supplementary Table 7 and Supplementary Table 8. Of note, compared with the published sequence, the SA58 construct used in this study contained an I51V mutation in the H-chain and a D154S mutation in the L-chain (as indicated in Supplementary Table 7). Three murine COVID-19 NAbs (85F7, 36H6, and 2B4) were purified from ascitic fluid derived from hybridoma cells using MabSelect SuRe resin. The variable regions of these mouse mAbs were sequenced via RT-PCR using mRNA isolated from the corresponding hybridoma cells and were presented in Supplementary Table 8.

Sample preparation based on the SP-MEGD method

Initially, the lysis buffer [6 M guanidine hydrochloride (Gu·HCl), 20 mM dithiothreitol (DTT), 100 mM Tris, pH 8.5] was added to 200 μg of purified mAb at an approximate ratio of 1:1 (microliters of lysis buffer to micrograms of protein). The mixture was denatured and reduced at 60℃ for 30 min, followed by alkylation with 40 mM iodoacetamide (IAA) in the dark for 30 min at room temperature. The alkylated antibody samples were transferred through a 10 kDa filter (Millipore, USA) at 4 °C into a solution containing 0.8 M urea, 50 mM Tris-HCl, adjusted to pH 8.0, to eliminate interfering substances that could affect enzymatic hydrolysis. Proteases were then added at a mass ratio of 1:20, specifically trypsin, chymotrypsin, pepsin (with the solution's pH pre-adjusted to below 3), elastase, and aspartic acid protease. A separate reaction vessel was utilized for each enzyme and incubated at 37°C for a total duration of six hours, with samples collected at two-hour intervals. After digestion, the reaction mixture was quenched with 10% trichloroacetic acid (TFA) for 30 min at 37°C. The supernatant was desalted by Sep-Pak C18 Vac cartridges (Waters) following the instructions before being lyophilized to dryness or stored at −20°C or dissolved in 0.1% FA for LC−MS/MS analysis.

LC-MS/MS analysis

The digested peptides were separated through reversed-phase chromatography on a Vanquish™ Neo UHPLC (column packed with PepMap™ 1C00 C18, 75 μm×50 cm, 2 μm, Thermo Fisher Scientific, USA) coupled to an Orbitrap Eclipse mass spectrometer. Samples were eluted over a 70-min gradient from 0 to 35% solvent B (0.1% formic acid in 80% acetonitrile) at a flow rate of 300 nL/min. Solvent A was 0.1% formic acid in water. Full MS1 scans were acquired over a range of m/z 350−2000 with a resolution of 120, 000. MS1 scans were obtained with a standard automatic gain control (AGC) target and a maximum injection time of 100 ms. The precursors were fragmented by stepped HCD as well as EThcD. The stepped HCD fragmentation included steps of 27%, 35%, and 40% normalized collision energies (NCE). EThcD fragmentation was performed with calibrated charge-dependent electron-transfer dissociation (ETD) parameters and 30% NCE supplemental activation. For both fragmentation types, MS2 scans were acquired at a 30,000 resolution, a 5E4 AGC target, and a 250 ms maximum injection time. Other method details are presented in the Supplementary Information. In total, thirteen bottom-up mass spectrometry datasets were generated for this study, based on traditional mAb sequencing (Supplementary Table 7 and Supplementary Table 8) or mAb mixture sequencing (Supplementary Table 9).

Deep learning model for de novo peptide sequencing of MS/MS spectra

Casanovo24 uses a transformer architecture to treat de novo peptide sequencing as a sequence-to-sequence translation task, translating from the series of peaks in an MS2 spectrum to a series of amino acids. The model consists of an encoder and a decoder, each based on transformer architecture. The encoder is responsible for learning an in‐context representation of the input MS2 spectrum, while the decoder predicts the subsequent amino acid in the peptide sequence based on the given spectrum representation and previously predicted amino acids. Both the encoder and decoder are composed of nine layers with eight heads per layer, featuring a hidden dimension of 512 and a feedforward dimension of 1024. Similar to other deep learning models, Casanovo predicts a peptide sequence one amino acid at a time, using beam search decoding to identify the predicted peptide sequence that achieves the highest score25. Notably, no post‐processing step is implemented to enforce alignment between the predicted peptide mass and observed precursor mass; however, there exists an optional filter that penalizes predictions deviating from precursor mass tolerance by assigning them negative scores. We trained the Casanovo model (v3.2.0) for 20 epochs with its pre-defined parameters on the MassIVE KB v1 dataset41, which encompasses over 2.1 million precursors from 19,610 proteins. This dataset was meticulously assembled from more than 31 TB of human data, sourced from 227 public proteomics datasets. Notably, it maintains rigorous false discovery rate controls, ensuring a comprehensive and robust training environment for our model. After filtering PSMs with unrelated variable modifications, we obtained a total of 1,909,041 PSMs corresponding to 1,376,990 unique peptides for the training set and 38,937 PSMs corresponding to 28,100 unique peptides for the validation set. It is important to note that no common peptides were shared between these split datasets. We employ a training-validation split of 98:2, with the 98% training proportion aligning with the practices reported by Beslic et al.42 in their recent comprehensive evaluation of peptide de novo sequencing tools for monoclonal antibody assembly. During training, 10 ppm for the precursor mass tolerance and 0.02 Da for the fragment mass tolerance were specified. Carbamidomethylation of cysteine (C + 57.02 Da) was set as a fixed modification, while oxidation of methionine (M + 15.99 Da), deamidation of asparagine, glutamine (N + 0.98 Da and Q + 0.98 Da), and Pyroglutamate formation from glutamine (Q − 17.03 Da) were set as variable modifications.

To further validate the generalizability of our model, we conducted extensive benchmarking on independent external datasets:

(A) Nine-species benchmark dataset (Wen et al., 202343; originally by DeepNovo44):

This dataset was initially introduced by DeepNovo and subsequently revisited in detail by Wen et al. (2023), who systematically reprocessed the data with stringent PSM-level false discovery rate (FDR ≤1%) controls using the Tide and Percolator search engines. Wen et al. specifically highlighted: "Finally, because some of the single-species datasets are markedly larger than others, we produced a more balanced version of the dataset". We utilized this carefully balanced version of the eight-species benchmark dataset to ensure a fair, unbiased, and comprehensive evaluation across different species while explicitly excluding the human dataset (PXD004424) from performance interpretation due to its overlap with our training set.

(B) mAbs-known dataset (internally generated):

This dataset comprises monoclonal antibodies with determined sequences that were generated in this study (SA55, 50 µg S2P6, 100 µg S2P6, 200 µg S2P6, 85F7, 36H6, 2B4). Each spectrum was annotated using the PEAKS DB database search engine to provide high-confidence peptide sequence labels, enabling a direct evaluation of Casanovo's performance on antibody-specific spectra relevant to our application.

In addition to the training-validation split, we conducted a 50-fold peptide-level cross-validation on the same dataset to evaluate the stability of model performance. Each fold was trained from scratch, and we observed consistent results across all folds (Supplementary Fig. 15), with an AA precision of 0.7969 ± 0.0054, AA recall of 0.7953 ± 0.0058, and peptide recall of 0.5928 ± 0.0060. These results were consistent with those obtained from our initial 98:2 split, further reinforcing the robustness of the model.

Furthermore, to assess the generalizability of the de novo peptide sequencing model used in XA-Novo and to exclude the possibility that its performance arises from a favorable train–validation split, we evaluated all 50 cross-validation models on independent external datasets. Across these datasets, which span different species and antibody repertoires, performance remained consistent with that observed on the internal data (Supplementary Fig. 15), indicating that the results are not driven by any particular data partition.

Each instrument vendor employs its proprietary file formats to store results from MS/MS experiments, requiring conversion to open-format files for compatibility with the pre-trained Casanovo model. We used ProteoWizard software45 to reformat the raw MS/MS data files of each antibody dataset into Mascot Generic Format (MGF). MGF files store the m/z and intensity pairs of multiple mass spectra within a single text format. Afterward, Casanovo deciphers amino acid sequences by interpreting the mass variations observed in the MS/MS peaks. Once peptide sequencing is completed, we reformat the output to conform to the PEAKS X+ style, which is compatible with downstream assembly tools such as Stitch 1.4, Stitch 1.5, and Fusion. Both sequence-level and amino acid-level scores are normalized on a scale ranging from 0 to 100. An example of an annotated MGF file entry for our trained Casanovo model is as follows:

“BEGIN IONS

TITLE=My spectrum title

PEPMASS=602.2881

CHARGE=2+

RTINSECONDS=985.44604

SEQ=HQ(-17.03)GVM(+15.99)VGC(+57.02)GQKQ(+.98)MVN(+.98)K

(without SEQ entry for de novo mode)

84.08081817626953 25805.216796875

87.74003601074219 69811.5234375

...

END IONS”

Sequence assembly

De novo peptides were then used by the Fusion assembler to automatically reconstruct the complete sequences of mAb and mixtures of various antibodies. Human or mouse germline antibody sequences from IMGT are employed as templates to guide the assembly of de novo peptide reads.

Intact mass verification of the antibody light and heavy chains

One hundred micrograms of each antibody were reduced with 50 mM DTT and treated with 2 µL PNGase F (Promega, USA) at 37°C for over 16h with stirring before being transferred to a new vial for LC-MS analysis. More experimental conditions are given in the Supplementary Methods.

LC-MS analysis of intact light and heavy chains

Intact LCs and HCs were analyzed using an Orbitrap Eclipse mass spectrometer (Thermo Fisher Scientific, USA) coupled to a Vanquish™ Flex UHPLC (Thermo Fisher Scientific, USA). All details can be found in the Supplementary Methods.

Data analysis

During the development and optimization of the SP-MEGD method, for bottom-up analysis, the raw files obtained for digesting peptides of each antibody were searched with PEAKS AB (v.3.0) using the parameters described in the Supplementary Methods. The de novo sequencing analysis of mAbs and their mixtures in our solution was conducted utilizing the XA-Novo platform (https://xa-novo.com/). The intact mass spectra were processed and deconvoluted using BioPharma Finder 5.2 software (Thermo Fisher Scientific, USA), subsequently enabling the comparison of mass information for light and heavy chains.

Expression and purification of anti-CD4, anti-CD8α, and anti-CSF1R antibodies

The mAb sequences obtained from our solution were utilized to generate antibodies in ExpiCHO-S cells following the manufacturer's instructions. The proteins were purified from culture supernatants collected on day 7 after transfection using a Protein A column (Cytiva). Elution of the protein from the column was carried out with 70 mM citric acid monohydrate and 20 mM sodium phosphate dibasic dodecahydrate. The eluted fractions were pooled and concentrated using the Protein Quantification kit (Thermo Fisher Cat#23225).

Functional validation of anti-CD4, anti-CD8α, and anti-CSF1R antibodies

Three groups of 8-week-old female C57BL/6J mice were administered intraperitoneally with either 200 μg of an XA-Novo-generated antibody (n = 3 per group), 200 μL of a commercially available antibody (n = 3 per group), or 200 μL of phosphate-buffered saline (PBS) (n = 6 per group). The objective was to evaluate the effective clearance rates of CD8+ and CD4+ T cells in their peripheral blood mononuclear cells (PBMCs) and spleens (Supplementary Fig. 9). All groups of mice were matched for age. Similarly, four additional groups of 8-week-old female C57BL/6J mice were administered intraperitoneally with either 200 μg of XA-Novo-generated antibody (125I, n = 4 per group), 200 µg of XA-Novo-generated antibody (125L, n = 3 per group), 200 μg of a commercially available CSF1R antibody (n = 2 per group), or 200 μL of PBS buffer (n = 3 per group). The aim was to assess the effective clearance rate of macrophages in their PBMCs and spleens (Supplementary Fig. 10). The mice received antibody or PBS buffer injections on the first day. On the third day post-injection, single cells were isolated from peripheral blood or spleen tissue and resuspended in an appropriate volume of PBS buffer. Cell counting was performed, followed by treatment with a blocking buffer (Purified Rat Anti-Mouse CD16/CD32, Mouse BD Fc Block™, BD, Cat. No. 553141) to reduce nonspecific antibody binding. Subsequently, the cell suspension was incubated with fluorescently labeled antibodies: anti-CD4 (PerCP-Cy5.5-anti-CD4), anti-CD8α (FITC-anti-CD8α), and anti-CSF1R (PE-anti-F4/80). After incubation, the cells were washed with PBS buffer to remove unbound antibodies. The labeled cell suspension was then processed using a flow cytometer to measure the fluorescence intensity of the labeled dyes. Utilizing this data, CD4+ T cells, CD8+ T cells, and macrophages were sorted. Finally, FlowJo (BD Biosciences, version 10.10) was used to analyze the data and assess the quantity, activity, and expression of CD4+ T cells, CD8+ T cells, and macrophages. All details can be found in the Supplementary Methods.

Animal research

All animal experiments were approved by the Institutional Animal Care and Use Committee of Xiamen University (protocol number: XMULAC20230310) and conducted in accordance with the Guide for the Care and Use of Laboratory Animals. Female C57BL/6J mice were used in this study. Female mice were selected to minimize inter-individual variability associated with stress and aggression, which are more commonly observed among male littermates, and to ensure greater behavioral consistency across experimental groups. A total of 36 eight-week-old mice were included. Animals were housed under a standard 12‑h light/dark cycle (lights on at 07:00) with ad libitum access to standard chow and water. All procedures were designed to minimize animal suffering, and the number of animals used was reduced to the minimum required to achieve statistically robust conclusions. No wild animals or field-collected biological samples were used in this study.

Assembly of Stitch for known monoclonal antibodies

Stitch has recently emerged as a state-of-the-art public method for assembling antibody sequences. Users are required to define three main parameters: the cutoff score for peptide reads, the placement cutoff score, and the recombined segment orders. The typical recombined segment orders are IGHV * IGHJ IGHC for the heavy chain and IGLV IGLJ IGLC for the light chain. The asterisk (*) represents a gap between adjacent segments, which is extended by overhanging peptide reads. We have chosen cutoff scores of 95, 90, 85, and 50 for peptide reads. Scores of 95, 90, and 85 are based on the default parameters from Stitch's paper or batch file examples, while 50 is used as the threshold in Fusion. We conducted tests using both the stable version of Stitch 1.4.0 and the latest version of Stitch 1.5.0 with these peptide cutoff scores, common recombined segment orders, and other default parameters.

Evaluation metrics

The accepted evaluation metrics in de novo peptide sequencing include amino acid-level precision (the proportion of correctly predicted amino acids among all predicted amino acids), amino acid-level recall (the proportion of correctly predicted amino acids relative to the total number of ground-truth amino acids), and peptide-level recall (the proportion of correctly predicted complete peptide sequences compared to the total number of ground-truth peptide sequences).

Antibody sequence assembly was evaluated based on three key metrics: coverage, accuracy, and accurate coverage (Fig. 2a). Sequencing coverage was calculated as the percentage of amino acids of the antibody sequence that were covered by the assembled contig. Sequencing accuracy was calculated as the percentage of all annotated sequence calls that were labeled correctly. To comprehensively evaluate the performance of sequence assembly, we employed the metric of accurate coverage, defined as the percentage of amino acids of the antibody sequence that were correctly covered by the assembled contig, equal to the product of coverage and accuracy. Since Fusion achieved 100% sequence coverage for all antibodies, in this context, accurate coverage is equivalent to accuracy.

For the alignment between assembled sequences and actual antibody sequences, we utilized the Needleman-Wunsch algorithm46 in conjunction with the BLOSUM60 matrix to achieve the most intuitive results.

Data Availability

The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the partner repository iProX under the dataset identifier PXD060500 (https://proteomecentral.proteomexchange.org/cgi/GetDataset?ID=PXD060500). The weights of the Casanovo model utilized in XA-Novo and the split datasets for training Casanovo are both available on Zenodo [https://zenodo.org/records/17266057] and [https://zenodo.org/records/18627093], respectively. Additionally, the revised nine-species benchmark dataset by Wen et al. is available on Zenodo [https://zenodo.org/records/13653420]. Unless otherwise stated, all data supporting the results of this study can be found in the article, supplementary, and source data files. Source data are provided with this paper.

Code Availability

Result files and code to reproduce the results in this study are available on GitHub [https://github.com/biocc/SP-MEGD_Fusion]. An executable version of the code and the computational environment used in this study are also available as a Code Ocean capsule [https://codeocean.com/capsule/4653442/tree].

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The authors of “XA-Novo: an accurate and high-throughput mass spectrometry-based de novo sequencing technology for monoclonal antibodies and antibody mixtures” have submitted a robust and detailed manuscript about their new technology – dubbed XA-Novo – that aims to improve sequencing accuracy, confidence, and simplicity of monoclonal human and murine antibodies. In this work, Xiong, et al, details a thorough investigation, involving sample preparation assay development, mass spectrometry method optimization, a detailed comparison of their proposed sequencing method vs. current standard methods, and an in vivo evaluation, demonstrating their new method of sequence determination can have real-life impact on future preclinical work.\nOverall, I would recommend this manuscript be approved for publication with minor revisions at Nature Communications due to the significance of potential impact of this work and the robustness of the data provided and discussed.\nI would like to suggest the following minor revisions to the authors for their manuscript:\n1. I would like to see a different citation for line 51/52 (ref.7). I understand this citation is from the authors previous work, but it is a pre-print citation and is not from a peer-reviewed publication, and I would like to see a more robust publication cited that discusses the importance of antibody sequence details here. The current ref. 7 can be cited later when the pre-print is referenced specifically.\n2. Line 200: the sentence says “when employing trypsin, the SP-MEGD method achieved approximately 100% sequence coverage…” – I am wondering if “trypsin” is supposed to be Guanidine HCl + 20 mM DTT, as the figure appears to be a comparison of lysis methods, and it was described earlier that the SP-MEGD method contains 5 enzymes of trypsin, chymotrypsin, pepsin, elastase, and Asp-N, not just trypsin alone. If this statement is accurate then please add some clarification.\n3. The in vivo validation of this method really helps to show the strength and promise of this process. The n value listed for the animal studies is 2/3, yet only one example of each flow cytometry CD8/CD4 is shown (Fig S8). I think it works well for this figure as a representative comparison, but I would like to see the same analysis shown for the other mice used in this study to add confidence in the representation. Potentially another supplemental figure with similar analysis from the blood and spleen from all mice.\n4. In lines ~241 and ~343, you describe how your system is superior at differentiating Ile/Leu compared to other methods, specifically PEAKS AB. However, in line ~364, you mention that the accuracy of the light chain declined due to Falcons challenges in distinguishing between amino acids I and L in the mixture. Because of the discrepancy here, I would like to see this discussed a bit more within the text near line 364 where the challenge is observed – why do you suspect there are difficulties differentiating between these AAs here when there wasn’t previously, are there still improvements that need to be made to improve this differentiation that is supposed to be superior, etc.\n5. Line 364 – I think there might be an error in the text and/or figure. Text says “…accuracy of the overall light chain declined slightly to 99.78% due to challenges…” in reference to SA55 – in the figure, the decrease is listed under the SA55 Heavy Chain where the I/L differentiation challenge was, not the light chain, and it is also listed as 99.56%, not 99.78% as described in the text.\n6. In figure 5, there is a spelling error in b: the “LC Chain” is spelled as “LC Chian” for both S2P6, SA55, and SA58.\n7. I would like if figure 5 was a bit more clear about which HC CDR/HC Chain/LC CDR/LC Chain corresponds to which antibody. I assume it corresponds top to bottom as it is written from left to right in the title of that section (i.e., “S2P6-SA55-SA58-Mix”, then the S2P6 analysis is the first set, SA55 analysis is the second set, etc…). It would be much more clear to the reader if the specific antibody was written to the right of it’s respective section to add clarity and easy comprehension.\n8. Line 380 – thus far, the authors have done a fantastic job identifying and explaining all deviations that the XA-Novo program has in it’s sequencing compared to the RefSeq results. However, there appears to be a deviation in the final mouse species with three antibodies (85F7, 36H6, 2B4) group – in fig 5c, there seems to be a difference in LC VJ for 8547 (an N/D deviation) that is not called out in the text, as well as one in the HC VDJ of 36H6, a Q/S deviation. I would like if the authors continued to speak on the deviations presented for this group as well.\nMethod Questions:\n9. Line 630 – Please specify the age and sex of the black mice used for this model.\n10. Line 631 – Please note why the number of mice was not consistent between the groups (some had n=3, some had n=2/3; why did some only have 2). Please also note the specific cohort of mice that only had 2 replicates.\n11. Line 636 – Please add more details regarding the third day post-injection for the anti-CSF1R antibody. After the injection with antibodies, were the mice euthanized and dissected to retrieve the spleen tissue? If so, how long was the antibody allowed the circulate the body before euthanasia, and how was euthanasia performed (CO2 asphyxiation, cervical dislocation, etc…). Was blood taken while mice were alive (and if so, which method: tail vein, retro-orbital, etc), or as part of the dissection via heart pull, etc. I feel there are quite a few critical details regarding the methods used to perform the mouse work missing in the methods.\n12. Are there any animal compliance/welfare committees and protocols (such as IACUC or equivalent) that was used for this work, and if so please list them and the institutions that they are monitored by. If not, please explain why this work was performed in the absence of an IACUC (or equivalent) welfare protocol.\n13. Additionally, line 635 – “single cells were isolated from peripheral blood or…” – please describe the method used to isolate the single cells. This can be added to supplemental methods.\n14. Line 637 – please identify which blocking buffer you used to reduce nonspecific antibody binding (ex. BSA, tween, commercially-purchased mixture, etc?).\n15. Line 638 – please identify the fluorophore the antibodies were labeled with that were used for flow cytometry detection.\n16. Can you please identify what process was used to confirm the CD4/CD8 gating used in the blood and spleen analysis is accurate (i.e., how you identified each section as being CD4 or being CD8, etc).\nOnce again, thank you to the authors for submitting a well-prepared and exciting manuscript. I look forward to seeing any revisions and edits made as a part of the review process.", "reviewer_2": "The manuscript entitled: “XA-Novo: an accurate and high-throughput mass spectrometry-based de novo sequencing technology for monoclonal antibodies and antibody mixtures” presents a mass spectrometry and computational strategy for the characterization of monoclonal antibodies. The authors apply their methods in different contexts and compare elements of their methods to previously proposed approaches.\nGeneral Comments :\nWhile the manuscript is generally well written, the novelty and improvements over current methods appear limited. The impact of the study is therefore also limited. Furthermore, the computational methods are not described in enough detail to fully evaluate their validity. Most of them are also not benchmarked or validated sufficiently to convince the reader of their benefits. Finally, some experiments appear to not be replicated.\nMajor Comments :\n1. Even if the authors are using a pre-trained version Casanovo. A benchmarking of the usage of Casanovo is needed.\nFurthermore, validation of its results should be provided using a dataset with known ground truth. However, the authors provided no results regarding its accuracy, specificity and sensitivity, despite performing a data split in training and validation set. On this topic, cross-validation was not used to assess the performances of Casanovo, which is odd. The split in the data of only 2% for validation could lead to thinking that the tool has great performances simply by chance.\n2. The features given to Casanovo need to be described. Was the data processed in any way prior to be used as input for training and testing?\n3. Were the experiments of Figure 2 b, c, d, h, and i replicated? Statistical significance should be assessed for these results. The authors make claims without any statistical analysis, such as in line 207 where they claim results are \"significantly lower\".\n4. There seems to have little to no gains of Fusion over PEAKS AB, in Figure 3d. The actual values should be given, or the scale of y-axes should be modified such that the results can be compared. Marginal gains are observed in Figure 3e.\n5. The number of sequences analyzed seems too small to arrive to any conclusions about the overall improved performances of Fusion over PEAKS AB and Stitch.\n6. No benchmarking with Stitch and PEAKS AB is provided in Figure 5. This should be done.\n7. The deposited mass spectrometry data is inaccessible since no reviewer credentials were provided.", "reviewer_3": "The manuscript addresses a very exciting topic and it seemed at first read that the paper presents new experiments and analyses, but we see major points that need to be addressed.\nThe first is that the submitted manuscript seems to be a direct descendant of an already publicized preprint (doi: 10.1101/2024.08.09.607415), which of course in itself is exactly the point of having a preprint. The issues lies in the fact that this submitted manuscript refers to the preprint as if it is a separate publication. We do not think these two manuscripts can be regarded as two separate manuscripts, as the submitted manuscript reuses the data from the preprint without proper disclosure. Figure 3 seems to use the same data as figure 7 in the preprint, figure 4 seems to use the same data as figure 4 in the preprint, figure S3 seems to use the same data as figure 5 in the preprint, and figure S9 seems to use the same data as figure 5 in the preprint. Additionally, parts of the preprint text have been copied verbatim into the submitted manuscript, with the first example being the first paragraph of the introduction. Even the stated goals of both the submitted manuscript and the preprint are extremely similar, while we find it hard to believe that both studies are the first to “enhance the throughput of full-length de novo” MS based sequencing. We do not see any possibility to keep the reference to the original preprint in the submitted manuscript. As such we would suggest to remove this reference and merge the last remaining parts of the original preprint into the manuscript as well: a description of “Fusion” and the isoleucine/leucine disambiguation.\nMany of the references seem to be wrong in several ways. Quite some of the references seem to be using the wrong number, a non-exhaustive list is: 7, 8, 9, 10, 11, 12, 15, 16, and 17. Some references do not follow the referring guideline of pointing to the first study to introduce a concept, a non- exhaustive list of examples: when introducing Stitch, and Needleman-Wunsch alignment. Some important algorithms are missing references entirely: Levenshtein distance and Casanovo. This makes the work look sloppy and not well edited by the senior contributors.\nThere is no description of the differences in the first stages between the proposed algorithm “Fusion” and the earlier reported “Stitch”. From a cursory reading it looks like both do exactly the same and are even using the same “mass-based alignment”. We urge the authors to extend their description of the “Fusion” algorithm to include the differences between this and the competitors. If on the other hand the stages are the same, either a rewrite or reuse of the existing “Stitch” code, this must be properly described and referenced in the manuscript.\nThere are three different alignment algorithms in use in the manuscript with no explanation on the reasoning behind the choice and why this is different for each step. In order of increased complexity, Levenshtein distance is used for the graph creation, Needleman-Wunsch for final analysis, and mass-based alignment for the peptide placement. We would argue that Levenshtein distance, while advantageous for its simplicity in implementation and runtime cost, is the worst metric of the three to actually build the graph. We urge the authors to reconsider this choice or at the very least give a thorough and detailed explanation why this algorithm was chosen. If Levenshtein distance was chosen for performance reasons a thorough analysis of the errors made by this alignment in comparison to the other two should be included.\nOne of the three major goals claimed in the paper is the ability to sequence multiple sequences at once. This is only shown for a mixture of monoclonal antibodies, that importantly are very distantly related at the sequence level. Claiming to be able to improve throughput with this as evidence is a stretch, because this will only improve throughput in comparatively easy cases, such as the one tested here by the authors. Importantly, the authors do not address the issue of sequencing directly from serum or any other natural sources of antibodies.\nIn many places it is unclear what data was run through the algorithms. As two separate de novo peptide sequencing programs are employed throughout the paper (Casanovo and PEAKS) it should be clear which one is actually used for each of the analyses.\nLastly, to enable the broader scientific community to learn from these efforts, the “Fusion” assembler should be made open source as is strongly encouraged by the Nature policies: https://www.springernature.com/gp/open-science/code-policy.\nOther minor points (line numbers in brackets):\n• (25) “a beam search-based Fusion assembler” reword\n• (39) With this wording it looks like only the Y shaped immunoglobulins have any influence while of course all other shapes and sizes have parts to play (which is also true for other shapes and sizes of IgG)\n• (95) “more than two or three” make this ‘more than two’ or ‘two or three’\n• (155) Sentence is unclear.\n• (165) “Consequently” change introduction word\n• (207) from the graphs it looks like the EasyPeptMicro kit actually did very well\n• (209) cost effectiveness cannot be claimed unless an analysis of the costs is made\n• (217) Casanovo gives peptides in a score range of -1 to 1, so a cutoff of 50 is an uncommon choice, unless the cutoff score from “Stitch” was meant\n• (231) “Even advanced software” this seems unnecessarily harsh towards the software mentioned in the previous sentence, which do outperform this “advanced” one in some areas\n• (252) a cutoff score of 95 seems very high\n• (308) what software was used for the peptide reads detection?\n• (318) “However” makes it looks like this sentence is related to the previous one, while intact mass analysis cannot help with I/L detection\n• (321) “undoubtedly” weird word choice\n• (416-419) this so called optimization does reduce machinetime not humantime spent analysing the data, inherently increasing the complexity of the data does increase the time spent on analysis\n• (424) double period\n• (427-429) this is a promising future outlook but needs at least some direction of how the authors envision to be able to reach this future\n• (547) pepsin is only active in low pH solutions while all other proteases are only active in high pH solutions, the addition of pepsin therefore needs some additional explanation\n• (573) reword sentence\n• (580) this is a highly suspicious split for training and validation, commonly split are 80:20 or if more robust training is required 50:50\n• (667) Needleman-Wunsch is always global (the local variant is called Smith-Waterman)\n• (674) the authors should provide the global identifier (PR…) as well as ensure the data is made public", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Response: We sincerely thank the editor and reviewers for their thorough and constructive evaluation of our manuscript titled “XA-Novo: An accurate and high-throughput mass spectrometry-based de novo sequencing technology for monoclonal antibodies and antibody mixtures.” We sincerely appreciate the positive feedback provided, and we have carefully addressed all comments point by point. We have revised the manuscript to clarify any ambiguities, incorporate additional data, and enhance the overall statement. All modifications made in the manuscript are marked. Below, we provide detailed responses to each comment (our replies are highlighted in blue).\nWe have strived to respond respectfully and comprehensively while implementing all requested revisions."}, {"round": 2, "reviewer_comments": {"reviewer_1": "I am happy with the changes the authors made based off of my original suggestions and questions. The authors have made significant changes to the citations, text, and methods based off my clarifying comments. The authors have also modified figures based off the suggestions, which is very appreciated, and I find the modified figures acceptable. I think these changes have made the manuscript much more robust, clear, and easy to comprehend. I have no additional comments and will defer to the comments of the other three reviewers for the combined final acceptance decision.", "reviewer_2": "I still have two major concerns about this study.\nI do not understand why the authors are not performing a cross-validation on their 98%–2% split to ensure that the performances they are seeing are not just the result of sheer luck. It is a simple implementation that would go a long way toward showing the impact of the work.\nSpeaking of impact, as I mentioned before, the improvement over PEAKS AB and Stitch is marginal and therefore not particularly impactful or significant.", "reviewer_3": "We want to thank the authors for their extensive efforts, the revised paper has become a lot more solid. We appreciate all revisions, but there is one main question which we feel is not answered to satisfaction.\nAdditionally some small textual comments are provided focussed on the newly added text.\n1) The applied textual changes and the removal of the citation of the preprint have made the manuscript a lot clearer.\n2) We are sorry to hear of the issues with the used software and sorry to have to point out one more error. There is at least one study that occurs twice in the bibliography.\n3) Thanks for the clarification provided in the response. From reading the code it becomes clear that Stitch is used for the entire \"Stage 1\", this is not clear from the description as provided in the supplemental methods. The description in the supplemental method should be revised to faithfully represent the method as used by the authors, as was asked in the original review.\nAdditionally, the question asked to add a description of the difference between the newly introduced Fusion and Stitch, is still lacking. One of the most obvious differences is in the approach of both pieces of software. Fusion is a single python script that gives a single answer without any estimates of confidence or context of the answer, the user should just trust what the computer tells them. While Stitch is designed to give an answer but with the context of all the input data that lead to that answer. This makes it easy for humans to disagree with the automated analysis and refine the answer based on human intuition. For any piece of software these two strategies have to be weighed against each other and choosing one over the other does not make the software necessarily 'better'. Therefore, lines 274-291 should be improved to be more of a scientific investigation of which of these strategies Fusion adapts versus Stitch.\n4) Thanks for the additional background. From reading the code and the description it is clear that Levenshtein distance is used is the last stage of Fusion. So there are no other metrics used downstream. As stated in the manuscript \"precise amino acid error correction is crucial\" (lines 87-89) which we agree with and the use of Levenshtein distance is not congruent with this statement. We also see that Fusion is, in its current form, useful as evidenced by the plentiful examples in the paper.\n5) Thanks for the response. The additional background and changes to the manuscript have cleared up this question.\n6) Thanks for making it open source. It might be good however to translate the Chinese comments into English. Additionally, check if this is indeed the latest version of the code as there are no references to satellite ions in the listed code.\nAdditional questions:\n7) It might be good to emphasize what the \"single pot\" in SP-MEGD means. From a cursory reading it could be understood as digesting the mAb with multiple proteases in a single vessle, while from the plots and Casanovo output files it is clear that the use of a single pot for the entire duration of the digestion is meant here, but each protease is used in a separate vessel.\n8) (864) Sharing model weights via Google drive is not a long term solution, it would be better to look into entities that are set up for long term storage of scientific data like Zenodo.\n9) No mention is made in the entire manuscript or the code on the method in which the isoleucine leucine disambiguation is being done. Only line 273 hints to the use of w-ions. If these ions are actually used it would be useful to provide at least one sentence on how they are used. Additionally, it would be good to check if indeed the latest version of Fusion is uploaded as no use of satellite ions can be found in the current code.\n10) Line 826, there are three main parameters: peptide cutoff score, placement cutoff score, and recombined segment orders.\n11) Line 450 and onwards. This tool is clearly a way of sending data to the group to manually run the data through the code. This would produce undue pressure on the team if the tool would find good use by other labs. Are there plans in place to either fully automate this system or otherwise will this tool stop working when the current PhD students leave the lab.\n12) (215) \"Consequently\" change introduction word", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Response: We sincerely thank the editor and reviewers for their thorough and constructive evaluation of our manuscript titled “XA-Novo: An accurate and high-throughput mass spectrometry-based de novo sequencing technology for monoclonal antibodies and antibody mixtures.” We sincerely appreciate the positive feedback provided, and we have carefully addressed all comments point by point. We have revised the manuscript to clarify any ambiguities, incorporate additional data, and enhance the overall statement. All modifications made in the manuscript are marked. Below, we provide detailed responses to each comment (our replies are highlighted in blue).\nWe have strived to respond respectfully and comprehensively while implementing all requested revisions."}, {"round": 3, "reviewer_comments": {"reviewer_2": "The authors have addressed all of my comments.", "reviewer_3": "While we still have concerns regarding the (incremental) novelty we thank the authors for the extensive reply to the posted questions. All questions have been satisfactorily answered with satisfactory changes to the manuscript. The listed changes that provide more insight into the process of Fusion sounds like a very helpful change for users and the use of different alignment algorithms for the final assembly is very good to see. It is good to see that the 98:2 split, while maybe not a common split, gives robust results.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Response: We sincerely thank the Reviewer for the thorough re-evaluation of our manuscript and for the constructive feedback. We appreciate the recognition that all questions have been satisfactorily addressed and that the revisions provide clearer insight into the Fusion process. We are particularly grateful for the positive comments regarding the inclusion of alternative alignment algorithms and the robustness of the 98:2 split strategy."}]

Decision letter