Diagnosing and staging epithelial ovarian cancer by serum glycoproteomic profiling.

BACKGROUND: There is a need for diagnostic tests for screening, triaging and staging of epithelial ovarian cancer (EOC). Glycoproteomics of blood samples has shown promise for biomarker discovery. METHODS: We applied glycoproteomics to serum of people with EOC or benign pelvic masses and healthy controls. A total of 653 analytes were quantified and assessed in multivariable models, which were tested in an independent cohort. Additionally, we analyzed glycosylation patterns in serum markers and in tissues. RESULTS: We identified a biomarker panel that distinguished benign lesions from EOC with sensitivity and specificity of 83.5% and 90.1% in the training set, and of 86.7 and 86.7% in the test set, respectively. ROC analysis demonstrated strong performance across a range of cutoffs. Fucosylated multi-antennary glycopeptide markers were higher in late-stage than in early-stage EOC. A comparable pattern was found in late-stage EOC tissues. CONCLUSIONS: Blood glycopeptide biomarkers have the potential to distinguish benign from malignant pelvic masses, and early- from late-stage EOC. Glycosylation of circulating and tumor tissue proteins may be related. This study supports the hypothesis that blood glycoproteomic profiling can be used for EOC diagnosis and staging and it warrants further clinical evaluation.

Decoding the glycoproteome: a new frontier for biomarker discovery in cancer.

Cancer early detection and treatment response prediction continue to pose significant challenges. Cancer liquid biopsies focusing on detecting circulating tumor cells (CTCs) and DNA (ctDNA) have shown enormous potential due to their non-invasive nature and the implications in precision cancer management. Recently, liquid biopsy has been further expanded to profile glycoproteins, which are the products of post-translational modifications of proteins and play key roles in both normal and pathological processes, including cancers. The advancements in chemical and mass spectrometry-based technologies and artificial intelligence-based platforms have enabled extensive studies of cancer and organ-specific changes in glycans and glycoproteins through glycomics and glycoproteomics. Glycoproteomic analysis has emerged as a promising tool for biomarker discovery and development in early detection of cancers and prediction of treatment efficacy including response to immunotherapies. These biomarkers could play a crucial role in aiding in early intervention and personalized therapy decisions. In this review, we summarize the significant advance in cancer glycoproteomic biomarker studies and the promise and challenges in integration into clinical practice to improve cancer patient care.

Mass Spectrometry Analysis of Glycopeptides Enriched by Anion Exchange-Mediated Methods Reveals PolyLacNAc-Extended N-Glycans in Integrins and Tetraspanins in Melanoma Cells.

Glycosylation is a key modulator of the functional state of proteins. Recent developments in large-scale analysis of intact glycopeptides have enabled the identification of numerous glycan structures that are relevant in pathophysiological processes. However, one motif found in N-glycans, poly-N-acetyllactosamine (polyLacNAc), still poses a substantial challenge to mass spectrometry-based glycoproteomic analysis due to its relatively low abundance and large size. In this work, we developed approaches for the systematic mapping of polyLacNAc-elongated N-glycans in melanoma cells. We first evaluated five anion exchange-based matrices for enriching intact glycopeptides and selected two materials that provided better overall enrichment efficiency. We then tested the robustness of the methodology by quantifying polyLacNAc-containing glycopeptides as well as changes in protein fucosylation and sialylation. Finally, we applied the optimal enrichment methods to discover glycopeptides containing polyLacNAc motifs in melanoma cells and found that integrins and tetraspanins are substantially modified with these structures. This study demonstrates the feasibility of glycoproteomic approaches for identification of glycoproteins with polyLacNAc motifs.

Variable PD-1 glycosylation modulates the activity of immune checkpoint inhibitors.

Monoclonal antibodies targeting the immune checkpoint PD-1 have provided significant clinical benefit across a number of solid tumors, with differences in efficacy and toxicity profiles possibly related to their intrinsic molecular properties. Here, we report that camrelizumab and cemiplimab engage PD-1 through interactions with its fucosylated glycan. Using a combination of protein and cell glycoengineering, we demonstrate that the two antibodies bind preferentially to PD-1 with core fucose at the asparagine N58 residue. We then provide evidence that the concentration of fucosylated PD-1 in the blood of non-small-cell lung cancer patients varies across different stages of disease. This study illustrates how glycoprofiling of surface receptors and related circulating forms can inform the development of differentiated antibodies that discriminate glycosylation variants and achieve enhanced selectivity, and paves the way toward the implementation of personalized therapeutic approaches.

Proteoform-Resolved Profiling of Plasminogen Activation Reveals Novel Abundant Phosphorylation Site and Primary N-Terminal Cleavage Site.

Plasminogen (Plg), the zymogen of plasmin (Plm), is a glycoprotein involved in fibrinolysis and a wide variety of other physiological processes. Plg dysregulation has been implicated in a range of diseases. Classically, human Plg is categorized into two types, supposedly having different functional features, based on the presence (type I) or absence (type II) of a single N-linked glycan. Using high-resolution native mass spectrometry, we uncovered that the proteoform profiles of human Plg (and Plm) are substantially more extensive than this simple binary classification. In samples derived from human plasma, we identified up to 14 distinct proteoforms of Plg, including a novel highly stoichiometric phosphorylation site at Ser339. To elucidate the potential functional effects of these post-translational modifications, we performed proteoform-resolved kinetic analyses of the Plg-to-Plm conversion using several canonical activators. This conversion is thought to involve at least two independent cleavage events: one to remove the N-terminal peptide and another to release the active catalytic site. Our analyses reveal that these processes are not independent but are instead tightly regulated and occur in a step-wise manner. Notably, N-terminal cleavage at the canonical site (Lys77) does not occur directly from intact Plg. Instead, an activation intermediate corresponding to cleavage at Arg68 is initially produced, which only then is further processed to the canonical Lys77 product. Based on our results, we propose a refined categorization for human Plg proteoforms. In addition, we reveal that the proteoform profile of human Plg is more extensive than that of rat Plg, which lacks, for instance, the here-described phosphorylation at Ser339.

Warning Signs From the Crypt: Aberrant Protein Glycosylation Marks Opportunities for Early Colorectal Cancer Detection.

Colorectal cancer (CRC) remains a leading cause of cancer-related deaths despite being the most preventable and treatable forms of cancer when caught early through screening. There is an unmet need for novel screening approaches with improved accuracy, less invasiveness, and reduced costs. In recent years, evidence has accumulated around particular biological events that happen during the adenoma-to-carcinoma transition, especially focusing on precancerous immune responses in the colonic crypt. Protein glycosylation plays a central role in driving those responses, and recently, numerous reports have been published on how aberrant protein glycosylation both in colonic tissue and on circulating glycoproteins reflects these precancerous developments. The complex field of glycosylation, which exceeds complexity of proteins by several orders of magnitude, can now be studied primarily because of the availability of new high-throughput technologies such as mass spectrometry and artificial intelligence-powered data processing. This has now opened new avenues for studying novel biomarkers for CRC screening. This review summarizes the early events taking place from the normal colon mucosa toward adenoma and adenocarcinoma formation and associated critical protein glycosylation phenomena, both on the tissue level and in the circulation. These insights will help establish an understanding in the interpretation of novel CRC detection modalities that involve high-throughput glycomics.

Roles of glycosylation at the cancer cell surface: opportunities for large scale glycoproteomics.

Cell surface glycosylation has a variety of functions, and its dysregulation in cancer contributes to impaired signaling, metastasis and the evasion of the immune responses. Recently, a number of glycosyltransferases that lead to altered glycosylation have been linked to reduced anti-tumor immune responses: B3GNT3, which is implicated in PD-L1 glycosylation in triple negative breast cancer, FUT8, through fucosylation of B7H3, and B3GNT2, which confers cancer resistance to T cell cytotoxicity. Given the increased appreciation of the relevance of protein glycosylation, there is a critical need for the development of methods that allow for an unbiased interrogation of cell surface glycosylation status. Here we provide an overview of the broad changes in glycosylation at the surface of cancer cell and describe selected examples of receptors with aberrant glycosylation leading to functional changes, with emphasis on immune checkpoint inhibitors, growth-promoting and growth-arresting receptors. Finally, we posit that the field of glycoproteomics has matured to an extent where large-scale profiling of intact glycopeptides from the cell surface is feasible and is poised for discovery of new actionable targets against cancer.

A universal GlycoDesign for lysosomal replacement enzymes to improve circulation time and biodistribution.

Currently available enzyme replacement therapies for lysosomal storage diseases are limited in their effectiveness due in part to short circulation times and suboptimal biodistribution of the therapeutic enzymes. We previously engineered Chinese hamster ovary (CHO) cells to produce α-galactosidase A (GLA) with various N-glycan structures and demonstrated that elimination of mannose-6-phosphate (M6P) and conversion to homogeneous sialylated N-glycans prolonged circulation time and improved biodistribution of the enzyme following a single-dose infusion into Fabry mice. Here, we confirmed these findings using repeated infusions of the glycoengineered GLA into Fabry mice and further tested whether this glycoengineering approach, Long-Acting-GlycoDesign (LAGD), could be implemented on other lysosomal enzymes. LAGD-engineered CHO cells stably expressing a panel of lysosomal enzymes [aspartylglucosamine (AGA), beta-glucuronidase (GUSB), cathepsin D (CTSD), tripeptidyl peptidase (TPP1), alpha-glucosidase (GAA) or iduronate 2-sulfatase (IDS)] successfully converted all M6P-containing N-glycans to complex sialylated N-glycans. The resulting homogenous glycodesigns enabled glycoprotein profiling by native mass spectrometry. Notably, LAGD extended the plasma half-life of all three enzymes tested (GLA, GUSB, AGA) in wildtype mice. LAGD may be widely applicable to lysosomal replacement enzymes to improve their circulatory stability and therapeutic efficacy.

Charting the Proteoform Landscape of Serum Proteins in Individual Donors by High-Resolution Native Mass Spectrometry.

Most proteins in serum are glycosylated, with several annotated as biomarkers and thus diagnostically important and of interest for their role in disease. Most methods for analyzing serum glycoproteins employ either glycan release or glycopeptide centric mass spectrometry-based approaches, which provide excellent tools for analyzing known glycans but neglect previously undefined or unknown glycosylation and/or other co-occurring modifications. High-resolution native mass spectrometry is a relatively new technique for the analysis of intact glycoproteins, providing a "what you see is what you get" mass profile of a protein, allowing the qualitative and quantitative observation of all modifications present. So far, a disadvantage of this approach has been that it centers mostly on just one specific serum glycoprotein at the time. To address this issue, we introduce an ion-exchange chromatography-based fractionation method capable of isolating and analyzing, in parallel, over 20 serum (glyco)proteins, covering a mass range between 30 and 190 kDa, from 150 µL of serum. Although generating data in parallel for all these 20 proteins, we focus the discussion on the very complex proteoform profiles of four selected proteins, i.e., α-1-antitrypsin, ceruloplasmin, hemopexin, and complement protein C3. Our analyses provide an insight into the extensive proteoform landscape of serum proteins in individual donors, caused by the occurrence of various N- and O-glycans, protein cysteinylation, and co-occurring genetic variants. Moreover, native mass intact mass profiling also provided an edge over alternative approaches revealing the presence of apo- and holo-forms of ceruloplasmin and the endogenous proteolytic processing in plasma of among others complement protein C3. We also applied our approach to a small cohort of serum samples from healthy and diseased individuals. In these, we qualitatively and quantitatively monitored the changes in proteoform profiles of ceruloplasmin and revealed a substantial increase in fucosylation and glycan occupancy in patients with late-stage hepatocellular carcinoma and pancreatic cancer as compared to healthy donor samples.

Exploring the glycosylation of mucins by use of O-glycodomain reporters recombinantly expressed in glycoengineered HEK293 cells.

Mucins and glycoproteins with mucin-like regions contain densely O-glycosylated domains often found in tandem repeat (TR) sequences. These O-glycodomains have traditionally been difficult to characterize because of their resistance to proteolytic digestion, and knowledge of the precise positions of O-glycans is particularly limited for these regions. Here, we took advantage of a recently developed glycoengineered cell-based platform for the display and production of mucin TR reporters with custom-designed O-glycosylation to characterize O-glycodomains derived from mucins and mucin-like glycoproteins. We combined intact mass and bottom-up site-specific analysis for mapping O-glycosites in the mucins, MUC2, MUC20, MUC21, protein P-selectin-glycoprotein ligand 1, and proteoglycan syndecan-3. We found that all the potential Ser/Thr positions in these O-glycodomains were O-glycosylated when expressed in human embryonic kidney 293 SimpleCells (Tn-glycoform). Interestingly, we found that all potential Ser/Thr O-glycosites in TRs derived from secreted mucins and most glycosites from transmembrane mucins were almost fully occupied, whereas TRs from a subset of transmembrane mucins were less efficiently processed. We further used the mucin TR reporters to characterize cleavage sites of glycoproteases StcE (secreted protease of C1 esterase inhibitor from EHEC) and BT4244, revealing more restricted substrate specificities than previously reported. Finally, we conducted a bottom-up analysis of isolated ovine submaxillary mucin, which supported our findings that mucin TRs in general are efficiently O-glycosylated at all potential glycosites. This study provides insight into O-glycosylation of mucins and mucin-like domains, and the strategies developed open the field for wider analysis of native mucins.

Discrepancies between High-Resolution Native and Glycopeptide-Centric Mass Spectrometric Approaches: A Case Study into the Glycosylation of Erythropoietin Variants.

Glycosylation represents a critical quality attribute modulating a myriad of physiochemical properties and effector functions of biotherapeutics. Furthermore, a rising landscape of glycosylated biotherapeutics including biosimilars, biobetters, and fusion proteins harboring complicated and dynamic glycosylation profiles requires tailored analytical approaches capable of characterizing their heterogeneous nature. In this work, we perform in-depth evaluation of the glycosylation profiles of three glycoengineered variants of the widely used biotherapeutic erythropoietin. We analyzed these samples in parallel using a glycopeptide-centric liquid chromatography/mass spectrometry approach and high-resolution native mass spectrometry. Although for all of the studied variants the glycopeptide and native mass spectrometry data were in good qualitative agreement, we observed substantial quantitative differences arising from ionization deficiencies and unwanted neutral losses, in particular, for sialylated glycopeptides in the glycoproteomics approach. However, the latter provides direct information about glycosite localization. We conclude that the combined parallel use of native mass spectrometry and bottom-up glycoproteomics offers superior characterization of glycosylated biotherapeutics and thus provides a valuable attribute in the characterization of glycoengineered proteins and other complex biotherapeutics.

The lysosomal endopeptidases Cathepsin D and L are selective and effective proteases for the middle-down characterization of antibodies.

Mass spectrometry is gaining momentum as a method of choice to de novo sequence antibodies (Abs). Adequate sequence coverage of the hypervariable regions remains one of the toughest identification challenges by either bottom-up or top-down workflows. Methods that efficiently generate mid-size Ab fragments would further facilitate top-down MS and decrease data complexity. Here, we explore the proteases Cathepsins L and D for forming protein fragments from three IgG1s, one IgG2, and one bispecific, knob-and-hole IgG1. We demonstrate that high-resolution native MS provides a sensitive method for the detection of clipping sites. Both Cathepsins produced multiple, albeit specific cleavages. The Abs were cleaved immediately after the CDR3 region, yielding ~ 12 kDa fragments, that is, ideal sequencing-sized. Cathepsin D, but not Cathepsin L, also cleaved directly below the Ab hinge, releasing the F(ab')2. When constrained by the different disulfide bonds found in the IgG2 subtype or by the tertiary structure of the hole-containing bispecific IgG1, the hinge region digest product was not produced. The Cathepsin L and Cathepsin D clipping motifs were related to sequences of neutral amino acids and the tertiary structure of the Ab. A single pot (L + D) digestion protocol was optimized to achieve 100% efficiency. Nine protein fragments, corresponding to the VL, VH, CL, CH1, CH2, CH3, CL + CH1, and F(ab')2, constituted ~ 70% of the summed intensities of all deconvolved proteolytic products. Cleavage sites were confirmed by the Edman degradation and validated with top-down sequencing. The described work offers a complementary method for middle-down analysis that may be applied to top-down Ab sequencing. ENZYMES: Cathepsin L-EC 3.4.22.15, Cathepsin D-EC 3.4.23.5.

Meta-heterogeneity: Evaluating and Describing the Diversity in Glycosylation Between Sites on the Same Glycoprotein.

Mass spectrometry-based glycoproteomics has gone through some incredible developments over the last few years. Technological advances in glycopeptide enrichment, fragmentation methods, and data analysis workflows have enabled the transition of glycoproteomics from a niche application, mainly focused on the characterization of isolated glycoproteins, to a mature technology capable of profiling thousands of intact glycopeptides at once. In addition to numerous biological discoveries catalyzed by the technology, we are also observing an increase in studies focusing on global protein glycosylation and the relationship between multiple glycosylation sites on the same protein. It has become apparent that just describing protein glycosylation in terms of micro- and macro-heterogeneity, respectively, the variation and occupancy of glycans at a given site, is not sufficient to describe the observed interactions between sites. In this perspective we propose a new term, meta-heterogeneity, to describe a higher level of glycan regulation: the variation in glycosylation across multiple sites of a given protein. We provide literature examples of extensive meta-heterogeneity on relevant proteins such as antibodies, erythropoietin, myeloperoxidase, and a number of serum and plasma proteins. Furthermore, we postulate on the possible biological reasons and causes behind the intriguing meta-heterogeneity observed in glycoproteins.

Quantitative characterization of O-GalNAc glycosylation.

O-GalNAc type glycosylation is an abundant and complex protein modification. Recent developments in mass spectrometry resulted in significant success in quantitative analysis of O-GalNAc glycosylation. The analysis of released O-GalNAc type glycans expanded our horizons of understanding the glycome of various biological models. The site-specific analysis of glycosylation micro-heterogeneity of purified proteins opened perspectives for the improved design of glycoprotein therapeutics. Advanced gene editing and chemical technologies applied to O-glycoproteomics enabled to identify O-GalNAc glycosylation at unprecedented depth. Progress in the analysis of intact glycoproteins under native and reduced conditions enabled the monitoring of glycosylation proteoform variants. Despite of the astonishing results in quantitative O-GalNAc glycoproteomics, site-specific mapping of the full O-GalNAc structural repertoire in complex samples is yet a long way off. Here, we summarize the most common quantitative strategies in O-GalNAc glycoproteomics, review recent progress and discuss benefits and limitations of the various approaches in the field.

Glycoproteoform Profiles of Individual Patients' Plasma Alpha-1-Antichymotrypsin are Unique and Extensively Remodeled Following a Septic Episode.

Sepsis and septic shock remain the leading causes of death in intensive care units (ICUs), yet the pathogenesis originating from the inflammatory response during sepsis remains ambiguous. Acute-phase proteins are typically highly glycosylated, and the nature of the glycans have been linked to the incidence and severity of such inflammatory responses. To further build upon these findings we here monitored, the longitudinal changes in the plasma proteome and, in molecular detail, glycoproteoform profiles of alpha-1-antichymotrypsin (AACT) extracted from plasma of ten individual septic patients. For each patient we included four different time-points, including post-operative (before sepsis) and following discharge from the ICU. We isolated AACT from plasma depleted for albumin, IgG and serotransferrin and used high-resolution native mass spectrometry to qualitatively and quantitatively monitor the multifaceted glycan microheterogeneity of desialylated AACT, which allowed us to monitor how changes in the glycoproteoform profiles reflected the patient's physiological state. Although we observed a general trend in the remodeling of the AACT glycoproteoform profiles, e.g. increased fucosylation and branching/LacNAc elongation, each patient exhibited unique features and responses, providing a resilient proof-of-concept for the importance of personalized longitudinal glycoproteoform profiling. Importantly, we observed that the AACT glycoproteoform changes induced by sepsis did not readily subside after discharge from ICU.

Functional diversification of hybridoma-produced antibodies by CRISPR/HDR genomic engineering.

Hybridoma technology is instrumental for the development of novel antibody therapeutics and diagnostics. Recent preclinical and clinical studies highlight the importance of antibody isotype for therapeutic efficacy. However, since the sequence encoding the constant domains is fixed, tuning antibody function in hybridomas has been restricted. Here, we demonstrate a versatile CRISPR/HDR platform to rapidly engineer the constant immunoglobulin domains to obtain recombinant hybridomas, which secrete antibodies in the preferred format, species, and isotype. Using this platform, we obtained recombinant hybridomas secreting Fab' fragments, isotype-switched chimeric antibodies, and Fc-silent mutants. These antibody products are stable, retain their antigen specificity, and display their intrinsic Fc-effector functions in vitro and in vivo. Furthermore, we can site-specifically attach cargo to these antibody products via chemoenzymatic modification. We believe that this versatile platform facilitates antibody engineering for the entire scientific community, empowering preclinical antibody research.

Simply Extending the Mass Range in Electron Transfer Higher Energy Collisional Dissociation Increases Confidence in N-Glycopeptide Identification.

Glycopeptide-centric mass spectrometry has become a popular approach for studying protein glycosylation. However, current approaches still utilize fragmentation schemes and ranges originally optimized and intended for the analysis of typically much smaller unmodified tryptic peptides. Here, we show that by merely increasing the tandem mass spectrometry m/z range from 2000 to 4000 during electron transfer higher energy collisional dissociation (EThcD) fragmentation, a wealth of highly informative c and z ion fragment ions are additionally detected, facilitating improved identification of glycopeptides. We demonstrate the benefit of this extended mass range on various classes of glycopeptides containing phosphorylated, fucosylated, and/or sialylated N-glycans. We conclude that the current software solutions for glycopeptide identification also require further improvements to realize the full potential of extended mass range glycoproteomics. To stimulate further developments, we provide data sets containing all classes of glycopeptides (high mannose, hybrid, and complex) measured with standard (2000) and extended (4000) m/z range that can be used as test cases for future development of software solutions enhancing automated glycopeptide analysis.

Targeted Analysis of Lysosomal Directed Proteins and Their Sites of Mannose-6-phosphate Modification.

Mannose-6-phosphate (M6P) is a distinctive post-translational modification critical for trafficking of lysosomal acid hydrolases into the lysosome. Improper trafficking into the lysosome, and/or lack of certain hydrolases, results in a toxic accumulation of their substrates within the lysosomes. To gain insight into the enzymes destined to the lysosome these glycoproteins can be distinctively enriched and studied using their unique M6P tag. Here we demonstrate, by adapting a protocol optimized for the enrichment of phosphopeptides using Fe3+-IMAC chromatography, that proteome-wide M6P glycopeptides can be selectively enriched and subsequently analyzed by mass spectrometry, taking advantage of exclusive phosphomannose oxonium fragment marker ions. As proof-of-concept of this protocol, applying it to HeLa cells, we identified hundreds of M6P-modified glycopeptides on 35 M6P-modified glycoproteins. We next targeted CHO cells, either wild-type or cells deficient in Acp2 and Acp5, which are acid phosphatases targeting M6P. In the KO CHO cells we observed a 20-fold increase of the abundance of the M6P-modification on endogenous CHO glycoproteins but also on the recombinantly over-expressed lysosomal human alpha-galactosidase. We conclude that our approach could thus be of general interest for characterization of M6P glycoproteomes as well as characterization of lysosomal enzymes used as treatment in enzyme replacement therapies targeting lysosomal storage diseases.

Direct quality control of glycoengineered erythropoietin variants.

Recombinant production of glycoprotein therapeutics like erythropoietin (EPO) in mammalian CHO cells rely on the heterogeneous N-glycosylation capacity of the cell. Recently, approaches for engineering the glycosylation capacities of mammalian cells for custom designed glycoforms have been developed. With these opportunities there is an increasing need for fast, sensitive, and global analysis of the glycoproteoform landscape produced to evaluate homogeneity and consistency. Here we use high-resolution native mass spectrometry to measure the glycoproteoform profile of 24 glycoengineered variants of EPO. Based on the unique mass and intensity profiles of each variant, we classify them according to similarities in glycosylation profiles. The classification distinguishes EPO variants with varying levels of glycan branchingand sialylation, which are crucial parameters in biotherapeutic efficacy. We propose that our methods could be of great benefit in the characterization of other glycosylated biopharmaceuticals, ranging from the initial clonal selection to batch-to-batch controls, and the assessment of similarity between biosimilar/biobetter products.

Parsimonious Charge Deconvolution for Native Mass Spectrometry.

Charge deconvolution infers the mass from mass over charge (m/z) measurements in electrospray ionization mass spectra. When applied over a wide input m/z or broad target mass range, charge-deconvolution algorithms can produce artifacts, such as false masses at one-half or one-third of the correct mass. Indeed, a maximum entropy term in the objective function of MaxEnt, the most commonly used charge deconvolution algorithm, favors a deconvolved spectrum with many peaks over one with fewer peaks. Here we describe a new "parsimonious" charge deconvolution algorithm that produces fewer artifacts. The algorithm is especially well-suited to high-resolution native mass spectrometry of intact glycoproteins and protein complexes. Deconvolution of native mass spectra poses special challenges due to salt and small molecule adducts, multimers, wide mass ranges, and fewer and lower charge states. We demonstrate the performance of the new deconvolution algorithm on a range of samples. On the heavily glycosylated plasma properdin glycoprotein, the new algorithm could deconvolve monomer and dimer simultaneously and, when focused on the m/z range of the monomer, gave accurate and interpretable masses for glycoforms that had previously been analyzed manually using m/z peaks rather than deconvolved masses. On therapeutic antibodies, the new algorithm facilitated the analysis of extensions, truncations, and Fab glycosylation. The algorithm facilitates the use of native mass spectrometry for the qualitative and quantitative analysis of protein and protein assemblies.