Abstract Comprehensive analysis of intact sialylated N-glycopeptides remains challenging because of their low abundance, extensive structural heterogeneity, and limited peptide backbone fragmentation during tandem mass spectrometry. Here, we present an integrated workflow for high-confidence identification of intact sialylated N-glycopeptides that combines selective TiO₂ enrichment, dual LC-MS/MS analysis of intact and deglycosylated glycopeptides, and the GPMAW glyco-search platform based on high-accuracy mass mapping. Unlike most conventional glycoproteomics search engines, GPMAW uses experimentally identified deglycopeptides to constrain glycan assignment before matching intact glycopeptide precursor masses to candidate glycan compositions. Identifications were validated using diagnostic oxonium ions, glycopeptide-associated Y-ion fragments, and an experimentally derived glycopeptide score. In addition, GPMAW integrates an interactive spectrum annotation interface that enables rapid manual validation of candidate identifications through visualization of annotated Y-ion series, oxonium ions, and peptide fragments, allowing individual assignments to be readily accepted or rejected. The workflow was optimized using bovine fetuin, validated on standard glycoproteins, and applied to depleted human plasma, where more than 2800 unique intact sialylated N-glycopeptides were identified across hundreds of glycosites and glycoproteins. Moreover, more than 1000 unique N-glycopeptides were identified from only 1 μL of plasma. Comparative analysis demonstrated that GPMAW glyco-search identified more confidently assigned intact sialylated N-glycopeptides than three widely used N-glycoproteomics search engines while maintaining high reproducibility and low false-positive rates following manual validation. Together, this workflow provides a robust, flexible, and accessible platform for large-scale, high-confidence characterization of intact N-glycopeptides and establishes experimentally constrained glycan composition assignment combined with interactive spectrum validation as an effective strategy for reducing ambiguity in N-glycoproteomics. Highlights The program “GPMAW glyco-search” enables high-accuracy mass mapping for confident identification of intact N-glycopeptides. Integrated workflow combining TiO₂ enrichment, dual LC-MS/MS of intact and deglycosylated glycopeptides and GPMAW glyco-search for intact sialylated N-glycopeptides. Optimized TiO₂ enrichment provides >95% selective enrichment of sialylated N-glycopeptides from complex biological samples. Interactive spectrum annotation and Y-ion-based scoring enable rapid manual validation and high-confidence glycopeptide identification. GPMAW glyco-search confidently identified more intact sialylated N-linked glycopeptides compared to three established glycoproteomics search engines.

Host cell protein (HCP) analysis by liquid chromatography-mass spectrometry (LC-MS) enables identification and quantification of individual impurities in biopharmaceutical products, supporting risk-based impurity assessment. However, unlike enzyme-linked immunosorbent assay (ELISA) methods, systematic evaluation of method-specific HCP coverage for LC-MS assays remain poorly defined. In particular, limited attention has been given to proteins that are inherently undetectable under specific analytical conditions, potentially creating blind spots in impurity characterization. Here, we present DARK-COV, a transparent and generalizable in silico pipeline for assessing theoretical HCP coverage of LC-MS-based impurity assays. The pipeline combines physicochemical filtering of in silico-digested proteomes-based on peptide hydrophobicity, charge state, and mass-to-charge ratio-with assay-specific LC-MS parameters to predict which peptides and proteins can be detected. Proteins predicted to yield fewer than two detectable peptides were classified as Dark Host Cell Proteins. The pipeline was benchmarked against a large-scale experimental dataset derived from more than 15,000 LC-MS analyses curated in HCPedia™, a comprehensive database of empirically observed HCP peptides generated using the same LC-MS workflow. As a proof-of-concept demonstration, DARK-COV was applied to four industrially relevant expression systems-Chinese Hamster Ovary (CHO) cells, Escherichia coli, Human Embryonic Kidney (HEK) cells, and Spodoptera frugiperda (Sf9) cells-revealing that only 1.0-5.2% of proteins across these proteomes were predicted to be theoretically undetectable under idealized in silico conditions. These predictions present an upper bound on detectability and do not account for matrix effects, ion suppression, or concentration-dependent detectability in real samples. Comparison with experimental observations across > 15,000 LC-MS datasets demonstrated strong concordance with DARK-COV predictions, supporting the classification of proteins that are likely to fall outside the detectable range of the LC-MS assay. Functional annotation and comparison against known high-risk HCPs indicated that the majority of Dark Host Cell Proteins are low-molecular-weight proteins with limited relevance to biopharmaceutical impurity risk. Overall, this pipeline provides a reproducible, method-specific approach for quantifying theoretical coverage and comparing predictions with experimental observations in LC-MS assays. By identifying analytical blind spots, the strategy supports risk-based evaluation of undetected HCPs and offers a rational foundation for regulatory justification of the LC-MS-based HCP impurity analyses described in USP Chapter 1132.1.

Supplemental Table 2: EC50 (µm) of the drugs tested on cells dissociated from patients tumors expressing (middle column) DNAJB1::PRKACA or (right column) ATP1B1::PRKACA.

Kallistatin is a serine protease inhibitor (serpin) that specifically inhibits tissue kallikrein, a key enzyme involved in kinin generation and vascular homeostasis. While serpins are known to polymerize under certain conditions, the structural properties of human kallistatin-particularly its glycosylation profile, tissue distribution, and polymerization potential-remain poorly defined. In this study, we generated a panel of kallistatin-specific monoclonal antibodies and developed a sensitive sandwich ELISA that enables reliable quantification of kallistatin in both plasma and tissue-conditioned media. The generated antibodies facilitated both quantitative and qualitative analyses, allowing detailed characterization of kallistatin's structural features, glycosylation profile, and localization in vascular tissue from patients with abdominal aortic aneurysm. Using mass-spectrometry, we characterized the glycosylation sites of kallistatin, providing experimental confirmation of the putative glycosylation at Asn 33. Biochemical analyses revealed that kallistatin can polymerize and is susceptible to structural rearrangements typical of serpins. Deglycosylation markedly increased polymer formation demonstrating that glycosylation plays a critical stabilizing role in preventing polymerization. Functional assays using a fluorogenic tissue kallikrein substrate showed that polymerized kallistatin loses inhibitory activity, whereas deglycosylated kallistatin retains normal function. This indicates that glycosylation primarily supports structural stability rather than directly modulating inhibitory capacity. These findings provide new insights into kallistatin's structural features, including its glycosylation, stability, and polymerization behavior, and establish essential tools for further exploring its physiological and pathological roles.

Supplemental Table 1 Levels of PKA subunits between FLC and adjacent normal tissue as assessed by mass spectrometry (peptides) or transcriptome. With trypsin digestion a unique peptide was not detected at the fusion junction between DNAJB1 and PRKACA

The Spatial transcriptomics data from figure 7 with the cell by cell data for the transcripts of Cyp3A, Muc13 and Col11A1

The native structure of natalizumab, an IgG4/κ α4β5 integrin monoclonal therapeutic antibody used for treatment of multiple sclerosis, was determined by a combination of chemical cross-linking, mass spectrometry, biophysical, and immunochemical methods. This revealed a compact, closed, m-shaped ellipsoidal structure with the paratopes at each end. Physicochemical stress, in the form of elevated temperature and lowered pH, induced an open, Y-shaped conformation of the antibody. This conformational change may reflect what is happening by binding of antibodies to their cognate antigens, e.g., in the form of pathogen-associated molecular patterns on the surface of microorganisms, where the antibodies have an intrinsic ability to change conformation and expose their effector function sites. Thus, in the absence of their cognate antigen, antibodies circulate as closed, m-shaped, inactive molecules.

Immunoglobulin G (IgG) is fundamental to adaptive immunity and numerous monoclonal IgGs (monoclonal antibodies (MAbs)) have been developed as therapeutics for various diseases, including ocrelizumab (OMAb), a CD20 MAb used for treating multiple sclerosis, and infliximab (IMAb), a tumor necrosis factor MAb used for treating rheumatoid arthritis and other conditions. Understanding structure-function relationships are essential for understanding the mechanisms of action of IgG MAbs and previous results have shown that IgG has a "closed", "m"-shaped conformation in native form, which may switch to an "open", "Y"-shaped conformation upon antigen binding or physico-chemical stress. Supported by immunochemical and biophysical methods and by chemical crosslinking mass spectrometry (XL-MS) we show that both OMAb and IMAb conform to this paradigm. By XL-MS, we identified eighty-five high-confidence cross-links that support the native closed state of OMAb, refining our understanding of IgG architecture. Molecular modeling based on these data further corroborates a compact IgG structure, shielding the Fc domain. This structural insight may increase our understanding of immunoglobulin biology and enhance therapeutic MAb design by optimizing stability and efficacy.

The development of biologics necessitates reliable assays to characterize and control Host Cell Protein (HCP) impurities. Liquid Chromatography-Mass Spectrometry (LC-MS)-based HCP assays have emerged as a powerful orthogonal method to HCP ELISA, providing detailed information on individual HCPs. In response to a growing need, the U.S Pharmacopeia (USP) introduced General Chapter < 1132.1 > , which provides the best practices and outlines three quantitative LC-MS methods. This study explores the practical application and validation readiness of the following strategies: A - Relative to Product Protein, B - Relative to Spiked-in Protein, and C - Relative to Spiked-in Peptide. Two common HCPs-Clusterin and Lipoprotein Lipase-were quantified using LC-MS in a purified mAb drug substance spiked with a CHO cell culture harvest to simulate in-process HCP levels. All three methods were demonstrated in the same samples and dilutions, enabling direct comparison of the three methods from a single dataset. Method performance was assessed according to ICH Q2(R2) guidelines for analytical method validation, focusing on linearity, accuracy, precision, and specificity. Results include a comparative assessment and discussion of the advantages and disadvantages, and application of each aforementioned HCP quantification method. This study provides practical insights into the implementation of USP < 1132.1 > supporting the growing role of LC-MS in HCP analysis for biologics development.

In the quality control of synthetic peptides, mass spectroscopy (MS) serves as an optimal method for evaluating authenticity and integrity. Typically, the sequence of a synthetic peptide is already established, thereby directing the focus of analysis towards validating its identity and purity. This chapter outlines straightforward methodologies for conducting MS analyses specifically tailored for synthetic peptides.

Amino acid analysis is an accurate method for the composition and quantitation of polypeptides and among these synthetic peptides. Combined with mass spectrometry, it yields a reliable control of peptide quality and quantity prior to conjugation and immunization.Initially peptides are hydrolyzed, preferably in the gas phase, with 6-M HCl at 110 °C for 20-24 h and the resulting amino acids analyzed by chromatography, where the most reliable form is ion exchange chromatography with post-column ninhydrin derivatization. Depending on the hydrolysis conditions, tryptophan is destroyed, and likewise cysteine, unless derivatized, and the amides, glutamine, and asparagine are deamidated to glutamic acid and aspartic acid, respectively. Three different ways of calculating results are suggested, and taking the above limitations into account, a quantitation better than 5% can usually be obtained.

Understanding protein-protein interactions is crucial for drug design and investigating biological processes. Various techniques, such as CryoEM, X-ray spectroscopy, linear epitope mapping, and mass spectrometry-based methods, can be employed to map binding regions on proteins. Commonly used mass spectrometry-based techniques are cross-linking and hydrogen‑deuterium exchange (HDX). Another approach, hydroxyl radical protein footprinting (HRPF), identifies binding residues on proteins but faces challenges due to high initial costs and complex setups.This study introduces a generally applicable method using Fenton chemistry for epitope mapping in a standard mass spectrometry laboratory. It emphasizes the importance of controls, particularly the inclusion of a negative antibody control, not widely utilized in HRPF epitope mapping. Quantification by TMT labelling is introduced to reduce false positives, enabling direct comparison between sample conditions and biological triplicates. Additionally, six technical replicates were incorporated to enhance the depth of analysis.Observations on the receptor-binding domain (RBD) of SARS-CoV-2 Spike Protein, Alpha and Delta variants, revealed both binding and opening regions. Significantly changed peptides upon mixing with a negative control antibody suggested structural alterations or nonspecific binding induced by the antibody alone. Integration of negative control antibody experiments and high overlap between biological triplicates led to the exclusion of 40% of significantly changed regions. The final identified binding region correlated with existing literature on neutralizing antibodies against RBD.The presented method offers a straightforward implementation for HRPF analysis in a generic mass spectrometry-based laboratory. Enhanced data reliability was achieved through increased technical and biological replicates alongside negative antibody controls.

Lipopolysaccharide O-antigen is an immunodominant target of protective antibodies. Variation in O-antigen structures limits antibody-mediated cross-protection between closely-related pathogens including Salmonella Typhimurium (STm) and S . Enteritidis (SEn). Bacterial outer membrane vesicles (OMV) are vaccine platforms presenting surface antigens in their natural conformations. To assess how O-antigen lengths impact antibody responses and control of homologous or heterologous infection, mice were immunized with STm-OMV containing wild-type O-antigen unit repeats (wt-OMV), ≤1 O-antigen unit (wzy-OMV), or no O-antigen units (wbaP-OMV) respectively and challenged with either STm or SEn. Unexpectedly, anti-STm LPS IgG and protection to STm were comparable after immunization with either wt-OMV or wzy-OMV. Anti-porin responses were elevated after immunization with wzy-OMV and wbaP-OMV. A single immunization with any OMV induced minimal cross-protection against SEn, except in blood. In contrast, boosting with O-antigen-expressing OMV enhanced control of SEn infections by >10-fold. These results suggest that i) Antibody to single or variable-length O-antigen units are comparably protective against Salmonella ; ii) Antigens other than immunodominant O-antigens may be targets of cross-reactive antibodies that moderate bacterial burdens; iii) Boosting can enhance the level of cross-protection against related Salmonella serovars and iv) High tissue burdens of Salmonella can be present in the absence of detectable bacteraemia. ### Competing Interest Statement The authors have declared no competing interest.

Abstract Fibrolamellar hepatocellular carcinoma (FLC) is a rare liver cancer that is driven by the fusion of DNAJB1 and PRKACA, the catalytic subunit of protein kinase A (PKA). PKA activity is controlled through regulatory proteins that both inhibit catalytic activity and control localization, and an excess of regulatory subunits ensures PRKACA activity is inhibited. Here, we found an increase in the ratio of catalytic to regulatory units in FLC patient tumors driven by DNAJB1::PRKACA using mass spectrometry, biochemistry, and immunofluorescence, with increased nuclear localization of the kinase. Overexpression of DNAJB1::PRKACA, ATP1B1::PRKACA, or PRKACA, but not catalytically inactive kinase, caused similar transcriptomic changes in primary human hepatocytes, recapitulating the changes observed in FLC. Consistently, tumors in patients missing a regulatory subunit or harboring an ATP1B1::PRKACA fusion were indistinguishable from FLC based on the histopathological, transcriptomic, and drug–response profiles. Together, these findings indicate that the DNAJB1 domain of DNAJB1::PRKACA is not required for FLC. Instead, changes in PKA activity and localization determine the FLC phenotype. Significance: Alterations leading to unconstrained protein kinase A signaling, regardless of the presence or absence of PRKACA fusions, drive the phenotypes of fibrolamellar hepatocellular carcinoma, reshaping understanding of the pathogenesis of this rare liver cancer.

Purpose of the study: Calreticulin is an endoplasmic reticulum chaperone protein, which is involved in protein folding and in peptide loading of major histocompatibility complex class I molecules together with its homo-log calnexin. Mutated calreticulin is associated with a group of hemopoietic disorders, especially myeloprolif-erative neoplasms. Currently only the cellular immune response to mutated calreticulin has been described, although preliminary findings have indicated that antibodies to mutated calreticulin are not specific for mye-loproliferative disorders. These findings have prompted us to characterize the humoral immune response to mutated calreticulin and its chaperone homologue calnexin.Patients and methods: We analyzed sera from myeloproliferative neoplasm patients, healthy donors and relapsing-remitting multiple sclerosis patients for the occurrence of autoantibodies to wild type and mutated calreticulin forms and to calnexin by enzyme-linked immunosorbent assay.Results: Antibodies to mutated calreticulin and calnexin were present at similar levels in serum samples of myeloproliferative neoplasm and multiple sclerosis patients as well as healthy donors. Moreover, a high cor-relation between antibodies to mutated calreticulin and calnexin was seen for all patient and control groups. Epitope binding studies indicated that cross-reactive antibodies bound to a three-dimensional epitope encompassing a short linear sequence in the C-terminal of mutated calreticulin and calnexin.Conclusion: Collectively, these findings indicate that calreticulin mutations may be common and not neces-sarily lead to onset of myeloproliferative neoplasm, possibly due to elimination of cells with mutations. This, in turn, may suggest that additional molecular changes may be required for development of myeloprolifera-tive neoplasm.(c) 2023 The Author(s). Published by Elsevier Masson SAS. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)

Proteinase 3 (PR3) is a neutrophil granulocyte enzyme and an autoantigen found in several forms of vasculitis. Due to the diagnostic and clinical importance of antibodies (Abs) to PR3, it is important to characterize the protein and the nature of its epitopes. Here, we have characterized PR3 monoclonal antibodies (MAbs) and disease-associated Abs and their dependency on the PR3 structure and modifications, especially interactions with α-defensins. Three MAbs (HYB 172-01, 172-04, 172-05), which bind to PR3 in its native and denatured forms and provide the disulphide bridges, were intact. α-1-antitrypsin (AT) binds to purified human neutrophil granulocyte PR3 and inhibits its proteolytic activity, towards a small synthetic peptide substrate and a large protein substrate (casein). AT also inhibited the binding of the three MAbs to PR3, indicating that they bind in a region affected by AT binding. However, the MAbs did not inhibit PR3 proteolytic activity with a small substrate, showing that they bound at the active site without restricting access to the substrate cleft. Patient-derived Abs showed essentially the same characteristics as the MAbs, with important implications for vasculitis diagnostics and pathophysiology. Current findings illustrate that PR3 epitopes depend on the three-dimensional structure of the PR3/defensin complex, and that the epitopes depend to a smaller or larger degree on PR3/defensin associations.

Understanding protein-protein interaction is essential when designing drugs or investigating biological processes. A variety of techniques can be employed in order to map the regions on proteins that are involved in binding eg., CryoEM, X-ray spectroscopy, linear epitope mapping, or mass spectrometry-based methods. The most commonly utilized mass spectrometry-based techniques are cross-linking and hydrogen-deuterium exchange (HDX). An alternative technique for identifying residues on the three-dimensional structure of proteins, that are involved in binding, can be hydroxyl radical protein footprinting (HRPF). However, this method is currently hampered by high initial cost and complex experimental setup. Here we set out to present a generally applicable method using Fenton chemistry for mapping of epitopes in a standard mass spectrometry laboratory. Furthermore, the described method illustrates the importance of controls on several levels when performing mass spectrometry-based epitope mapping. In particular, the inclusion of a negative antibody control has not previously been widely utilized in epitope mapping by HRPF analysis. In order to limit the number of false positives, we further introduced quantification by TMT labelling, thereby allowing for direct comparison between sample conditions and biological triplicates. Lastly, up to six technical replicates were incorporated in the experimental setup in order to achieve increased depth of the final analysis. Both binding and opening of regions on receptor-binding domain (RBD) from SARS-CoV-2 Spike Protein, Alpha, and Delta variants, were observed. The negative control antibody experiment combined with the high overlap between biological triplicates resulted in the exclusion of 40% of the significantly changed regions, including both binding and opening regions. The final identified binding region was mapped to a three-dimensional structure and agrees with the literature for neutralizing antibodies towards SARS-CoV-2 Spike Protein. The presented method is straightforward to implement for the analysis of HRPF in a generic MS-based laboratory. The high reliability of the data was achieved by increasing the number of technical and biological replicates combined with negative antibody controls.

Systemic lupus erythematosus (SLE) is an autoimmune disease, which has been associated with Epstein–Barr virus (EBV) and Cytomegalovirus (CMV) infection. Drug-induced lupus (DIL) is a lupus-like disease caused by the intake of therapeutic drugs, which has been estimated to cause approximately 10–15% of lupus-like cases. Although SLE and DIL share common clinical symptoms, there are some fundamental differences between DIL and SLE onset. Moreover, it remains to be examined whether environmental factors, such as EBV and CMV infections, may contribute to the development of DIL. This study focused on examining the possible association between DIL and EBV and CMV infections, by examining IgG titers to EBV and CMV antigens in serum samples by enzyme-linked immunosorbent assays. Antibody titers to EBV early antigen–diffuse and CMV pp52 were found to be significantly elevated in both SLE and DIL patients compared to healthy controls, although no correlation was found for antibodies to the two virus antigens in the respective disease groups. Moreover, total IgG titers were reduced in SLE and DIL serum samples, which may reflect a general lymphocytopenia, which commonly is associated with SLE. The current findings support that EBV and CMV infections may contribute to the development of DIL and that onset of both diseases are related.

The post-translational modification citrullination has been proposed to play a role in the pathogenesis of multiple sclerosis (MS). Myelin basic protein (MBP) is a candidate autoantigen which is citrullinated to a minor extent under physiological conditions and hypercitrullinated in MS. We examined immune cell responses elicited by hypercitrullinated MBP (citMBP) in cultures of mononuclear cells from 18 patients with MS and 42 healthy donors (HDs). The immunodominant peptide of MBP, MBP85-99, containing citrulline in position 99, outcompeted the binding of native MBP85-99 to HLA-DR15, which is strongly linked to MS. Moreover, using the monoclonal antibody MK16 as probe, we observed that B cells and monocytes from HLA-DR15+ patients with MS presented MBP85-99 more efficiently after challenge with citMBP than with native MBP. Both citMBP and native MBP induced proliferation of CD4+ T cells from patients with MS as well as TNF-α production by their B cells and CD4+ T cells, and citrullination of MBP tended to enhance TNF-α secretion by CD4+ T cells from HLA-DR15+ patients. Unlike native MBP, citMBP induced differentiation into Th17 cells in cultures from HDs, while neither form of MBP induced Th17-cell differentiation in cultures from patients with MS. These data suggest a role for citrullination in the breach of tolerance to MBP in healthy individuals and in maintenance of the autoimmune response to MBP in patients with MS.

Type 1 Ser/Thr protein phosphatases are represented in all fungi by two enzymes, the ubiquitous PP1, with a conserved catalytic polypeptide (PP1c) and numerous regulatory subunits, and PPZ, with a C-terminal catalytic domain related to PP1c and a variable N-terminal extension. Current evidence indicates that, although PP1 and PPZ enzymes might share some cellular targets and regulatory subunits, their functions are quite separated, and they have individual regulation. We explored the structures of PP1c and PPZ across 57 fungal species to identify those features that (1) are distinctive among these enzymes and (2) have been preserved through evolution. PP1c enzymes are more conserved than PPZs. Still, we identified 26 residues in the PP1 and PPZ catalytic moieties that are specific for each kind of phosphatase. In some cases, these differences likely affect the distribution of charges in the surface of the protein. In many fungi, Hal3 is a specific inhibitor of the PPZ phosphatases, although the basis for the interaction of these proteins is still obscure. By in vivo co-purification of the catalytic domain of ScPpz1 and ScHal3, followed by chemical cross-linking and MS analysis, we identified a likely Hal3-interacting region in ScPpz1 characterized by two major and conserved differences, D566 and D615 in ScPpz1, which correspond to K210 and K259 in ScPP1c (Glc7). Functional analysis showed that changing D615 to K renders Ppz1 refractory to Hal3 inhibition. Since ScHal3 does not regulate Glc7 but it inhibits all fungal PPZ tested so far, this conserved D residue could be pivotal for the differential regulation of both enzymes in fungi.