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Hybrid mass spectrometry (MS) is an emerging technique for characterizing glycoproteins, which typically display pronounced microheterogeneity. Since hybrid MS combines information from different experimental levels, it crucially depends on computational methods. Here, we describe a novel software tool, MoFi, which integrates hybrid MS data to assign glycans and other post-translational modifications (PTMs) in deconvoluted mass spectra of intact proteins. Its two-stage search algorithm first assigns monosaccharide/PTM compositions to each peak and then compiles a hierarchical list of glycan combinations compatible with these compositions. Importantly, the program only includes those combinations which are supported by a glycan library as derived from glycopeptide or released glycan analysis. By applying MoFi to mass spectra of rituximab, ado-trastuzumab emtansine, and recombinant human erythropoietin, we demonstrate how integration of bottom-up data may be used to refine information collected at the intact protein level. Accordingly, our software reveals that a single mass frequently can be explained by a considerable number of glycoforms. Yet, it simultaneously ranks proteoforms according to their probability, based on a score which is calculated from relative glycan abundances. Notably, glycoforms that comprise identical glycans may nevertheless differ in score if those glycans occupy different sites. Hence, MoFi exposes different layers of complexity that are present in the annotation of a glycoprotein mass spectrum.

Robust manufacturing processes resulting in consistent glycosylation are critical for the efficacy and safety of biopharmaceuticals. Information on glycosylation can be obtained by conventional bottom–up methods but is often limited to the glycan or glycopeptide level. Here, we apply high-resolution native mass spectrometry (MS) for the characterization of the therapeutic fusion protein Etanercept to unravel glycoform heterogeneity in conditions of hitherto unmatched mass spectral complexity. Higher spatial resolution at lower charge states, an inherent characteristic of native MS, represents a key component for the successful revelation of glycan heterogeneity. Combined with enzymatic dissection using a set of proteases and glycosidases, assignment of specific glycoforms is achieved by transferring information from subunit to whole protein level. The application of native mass spectrometric analysis of intact Etanercept as a fingerprinting tool for the assessment of batch-to-batch variability is exemplified and may be extended to demonstrate comparability after changes in the biologic manufacturing process., The specific glycosylation patterns of biological drugs often impact the efficacy and safety of the therapeutic product. Here the authors describe a native mass spectrometry approach that allows the resolution of highly complex glycosylation patterns on large proteins, which they apply to the therapeutic Fc-fusion protein Etanercept.

Human kallikrein-related peptidase 2 (KLK2) is a key serine protease in semen liquefaction and prostate cancer together with KLK3/prostate-specific antigen. In order to decipher the function of its potential N-glycosylation site, we produced pro-KLK2 in Leishmania tarentolae cells and compared it with its non-glycosylated counterpart from Escherichia coli expression. Mass spectrometry revealed that Asn-95 carries a core glycan, consisting of two GlcNAc and three hexoses. Autocatalytic activation was retarded in glyco-pro-KLK2, whereas the activated glyco-form exhibited an increased proteolytic resistance. The specificity patterns obtained by the PICS (proteomic identification of protease cleavage sites) method are similar for both KLK2 variants, with a major preference for P1-Arg. However, glycosylation changes the enzymatic activity of KLK2 in a drastically substrate-dependent manner. Although glyco-KLK2 has a considerably lower catalytic efficiency than glycan-free KLK2 toward peptidic substrates with P2-Phe, the situation was reverted toward protein substrates, such as glyco-pro-KLK2 itself. These findings can be rationalized by the glycan-carrying 99-loop that prefers to cover the active site like a lid. By contrast, the non-glycosylated 99-loop seems to favor a wide open conformation, which mostly increases the apparent affinity for the substrates (i.e. by a reduction of Km). Also, the cleavage pattern and kinetics in autolytic inactivation of both KLK2 variants can be explained by a shift of the target sites due to the presence of the glycan. These striking effects of glycosylation pave the way to a deeper understanding of kallikrein-related peptidase biology and pathology.

Background: Serine proteases KLK2 and KLK3 clear the way for spermatozoa before impregnation. Results: Enzymatic assays and structures of KLK2 elucidate its catalytic action, especially when compared with conformations of similar proteases. Conclusion: Flexible loops around the active site of serine proteases open concertedly upon substrate binding. Significance: This mechanistic model will stimulate the design of pharmaceutical inhibitors., Human kallikrein-related peptidase 2 (KLK2) is a tryptic serine protease predominantly expressed in prostatic tissue and secreted into prostatic fluid, a major component of seminal fluid. Most likely it activates and complements chymotryptic KLK3 (prostate-specific antigen) in cleaving seminal clotting proteins, resulting in sperm liquefaction. KLK2 belongs to the “classical” KLKs 1–3, which share an extended 99- or kallikrein loop near their non-primed substrate binding site. Here, we report the 1.9 Å crystal structures of two KLK2-small molecule inhibitor complexes. In both structures discontinuous electron density for the 99-loop indicates that this loop is largely disordered. We provide evidence that the 99-loop is responsible for two biochemical peculiarities of KLK2, i.e. reversible inhibition by micromolar Zn2+ concentrations and permanent inactivation by autocatalytic cleavage. Indeed, several 99-loop mutants of KLK2 displayed an altered susceptibility to Zn2+, which located the Zn2+ binding site at the 99-loop/active site interface. In addition, we identified an autolysis site between residues 95e and 95f in the 99-loop, whose elimination prevented the mature enzyme from limited autolysis and irreversible inactivation. An exhaustive comparison of KLK2 with related structures revealed that in the KLK family the 99-, 148-, and 220-loop exist in open and closed conformations, allowing or preventing substrate access, which extends the concept of conformational selection in trypsin-related proteases. Taken together, our novel biochemical and structural data on KLK2 identify its 99-loop as a key player in activity regulation.

Most kallikrein-related peptidases (KLKs) are N-glycosylated with N-acetylglucosamine2-mannose9 units at Asn-Xaa-Ser/Thr sequons during protein synthesis and translocation into the endoplasmic reticulum. These N-glycans are modified in the Golgi machinery, where additional O-glycosylation at Ser and Thr takes place, before exocytotic release of the KLKs into the extracellular space. Sequons are present in all 15 members of the KLKs and comparative studies for KLKs from natural and recombinant sources elucidated some aspects of glycosylation. Although glycosylation of mammalian KLKs 1, 3, 4, 6, and 8 has been analyzed in great detail, e.g., by crystal structures, the respective function remains largely unclear. In some cases, altered enzymatic activity was observed for KLKs upon glycosylation. Remarkably, for KLK3/PSA, changes in the glycosylation pattern were observed in samples of benign prostatic hyperplasia and prostate cancer with respect to healthy individuals. Potential functions of KLK glycosylation in structural stabilization, protection against degradation, and activity modulation of substrate specificity can be deduced from a comparison with other glycosylated proteins and their regulation. According to the new concept of protein sectors, glycosylation distant from the active site might significantly influence the activity of proteases. Novel pharmacological approaches can exploit engineered glycans in the therapeutical context.