Glycoproteomics of a Single Protein: Revealing Tens of Thousands of Myozyme Glycoforms by Hybrid HPLC-MS Approaches.

Characterization of highly glycosylated biopharma-ceuticals by mass spectrometry is challenging because of the huge chemical space of coexistent glycoforms present. Here, we report the use of an array of HPLC-mass spectrometry-based approaches at different structural levels of released glycan, glycopeptide, and hitherto unexplored intact glycoforms to scrutinize the biopharmaceutical Myozyme, containing the highly complex lysosomal enzyme recombinant acid α-glucosidase. The intrinsic heterogeneity of recombinant acid α-glucosidase glycoforms was unraveled using a novel strong anion exchange HPLC-mass spectrometry approach involving a pH-gradient of volatile buffers to facilitate chromatographic separation of glycoforms based on their degree of sialylation, followed by the acquisition of native mass spectra in an Orbitrap mass spectrometer. Upon considering the structures of 60 different glycans attached to seven glycosylation sites in the intact protein, the large set of interdependent data acquired at different structural levels was integrated using a set of bioinformatic tools and allowed the annotation of intact glycoforms unraveling more than 1,000,000 putative intact glycoforms. Detectable isoforms also included several mannose-6-phosphate variants, which are essential for directing the drug toward its target, the lysosomes. Finally, for the first time, we sought to validate the intact glycoform annotations by integrating experimental data on the enzymatically dissected proteoforms, which reduced the number of glycoforms supported by experimental evidence to 42,104. The latter verification clearly revealed the strengths but also intrinsic limitations of this approach for fully characterizing such highly complex glycoproteins by mass spectrometry.

The NMR signature of maltose-based glycation in full-length proteins.

Reducing sugars can spontaneously react with free amines in protein side chains leading to posttranslational modifications (PTMs) called glycation. In contrast to glycosylation, glycation is a non-enzymatic modification with consequences on the overall charge, solubility, aggregation susceptibility and functionality of a protein. Glycation is a critical quality attribute of therapeutic monoclonal antibodies. In addition to glucose, also disaccharides like maltose can form glycation products. We present here a detailed NMR analysis of the Amadori product formed between proteins and maltose. For better comparison, data collection was done under denaturing conditions using 7 M urea-d4 in D2O. The here presented correlation patterns serve as a signature and can be used to identify maltose-based glycation in any protein that can be denatured. In addition to the model protein BSA, which can be readily glycated, we present data of the biotherapeutic abatacept containing maltose in its formulation buffer. With this contribution, we demonstrate that NMR spectroscopy is an independent method for detecting maltose-based glycation, that is suited for cross-validation with other methods.

Selectivity and Resolving Power of Hydrophobic Interaction Chromatography Targeting the Separation of Monoclonal Antibody Variants.

This study presents a comprehensive investigation of the mechanistic understanding of retention and selectivity in hydrophobic interaction chromatography. It provides valuable insights into crucial method-development parameters involved in achieving chromatographic resolution for profiling molecular variants of trastuzumab. Retention characteristics have been assessed for three column chemistries, i.e., butyl, alkylamide, and long-stranded multialkylamide ligands, while distinguishing column hydrophobicity and surface area. Salt type and specifically chloride ions proved to be the key driver for improving chromatographic selectivity, and this was attributed to the spatial distribution of ions at the protein surface, which is ion-specific. The effect was notably more pronounced on the multialkylamide column, as proteins intercalated between the multiamide polymer strands, enabling steric effects. Column coupling proved to be an effective approach for maximizing resolution between molecular variants present in the trastuzumab reference sample and trastuzumab variants induced by forced oxidation. Liquid chromatography-mass spectrometry (LC-MS)/MS peptide mapping experiments after fraction collection indicate that the presence of chloride in the mobile phase enables the selectivity of site-specific deamidation (N30) situated at the heavy chain. Moreover, site-specific oxidation of peptides (M255, W420, and M431) was observed for peptides situated at the Fc region close to the CH2-CH3 interface, previously reported to activate unfolding of trastuzumab, increasing the accessible surface area and hence resulting in an increase in chromatographic retention.

Comparing the latent state-trait structure of the PANSS in cariprazine-medicated and placebo-controlled patients with acute schizophrenia.

After over a hundred years of research, the question whether the symptoms of schizophrenia are rather trait-like (being a relatively stable quality of individuals) or state-like (being substance to change) is still unanswered. To assess the trait and the state component in patients with acute schizophrenia, one group receiving antipsychotic treatment, the other not. Data from four phase II/III, 6-week, randomized, double-blind, placebo-controlled trials of similar design that included patients with acute exacerbation of schizophrenia were pooled. In every trial, one treatment group received a third-generation antipsychotic, cariprazine, and the other group placebo. To assess symptoms of schizophrenia, the Positive and Negative Symptom Scale (PANSS) was applied. Further analyses were conducted using the five subscales as proposed by Wallwork and colleagues. A latent state-trait (LST) model was developed to estimate the trait and state components of the total variance of the observed scores. All symptom dimensions behaved more in a trait-like manner. The proportions of all sources of variability changed over the course of the observational period, with a bent around weeks 3 and 4. Visually inspected, no major differences were found between the two treatment groups regarding the LST structure of symptom dimensions. This high proportion of inter-individual stability may represent an inherent part of symptomatology that behaves independently from treatment status.

Overexpression of UDP-sugar pyrophosphorylase leads to higher sensitivity towards galactose, providing new insights into the mechanisms of galactose toxicity in plants.

Galactose toxicity (Gal-Tox) is a widespread phenomenon ranging from Escherichia coli to mammals and plants. In plants, the predominant pathway for the conversion of galactose into UDP-galactose (UDP-Gal) and UDP-glucose is catalyzed by the enzymes galactokinase, UDP-sugar pyrophosphorylase (USP) and UDP-galactose 4-epimerase. Galactose is a major component of cell wall polymers, glycolipids and glycoproteins; therefore, it becomes surprising that exogenous addition of galactose leads to drastic root phenotypes including cessation of primary root growth and induction of lateral root formation. Currently, little is known about galactose-mediated toxicity in plants. In this study, we investigated the role of galactose-containing metabolites like galactose-1-phosphate (Gal-1P) and UDP-Gal in Gal-Tox. Recently published data from mouse models suggest that a reduction of the Gal-1P level via an mRNA-based therapy helps to overcome Gal-Tox. To test this hypothesis in plants, we created Arabidopsis thaliana lines overexpressing USP from Pisum sativum. USP enzyme assays confirmed a threefold higher enzyme activity in the overexpression lines leading to a significant reduction of the Gal-1P level in roots. Interestingly, the overexpression lines are phenotypically more sensitive to the exogenous addition of galactose (0.5 mmol L-1 Gal). Nucleotide sugar analysis via high-performance liquid chromatography-mass spectrometry revealed highly elevated UDP-Gal levels in roots of seedlings grown on 1.5 mmol L-1 galactose versus 1.5 mmol L-1 sucrose. Analysis of plant cell wall glycans by comprehensive microarray polymer profiling showed a high abundance of antibody binding recognizing arabinogalactanproteins and extensins under Gal-feeding conditions, indicating that glycoproteins are a major target for elevated UDP-Gal levels in plants.

Unambiguous Identification of Glucose-Induced Glycation in mAbs and other Proteins by NMR Spectroscopy.

OBJECTIVE: Glycation is a non-enzymatic and spontaneous post-translational modification (PTM) generated by the reaction between reducing sugars and primary amine groups within proteins. Because glycation can alter the properties of proteins, it is a critical quality attribute of therapeutic monoclonal antibodies (mAbs) and should therefore be carefully monitored. The most abundant product of glycation is formed by glucose and lysine side chains resulting in fructoselysine after Amadori rearrangement. In proteomics, which routinely uses a combination of chromatography and mass spectrometry to analyze PTMs, there is no straight-forward way to distinguish between glycation products of a reducing monosaccharide and an additional hexose within a glycan, since both lead to a mass difference of 162 Da. METHODS: To verify that the observed mass change is indeed a glycation product, we developed an approach based on 2D NMR spectroscopy spectroscopy and full-length protein samples denatured using high concentrations of deuterated urea. RESULTS: The dominating ß-pyranose form of the Amadori product shows a characteristic chemical shift correlation pattern in 1H-13C HSQC spectra suited to identify glucose-induced glycation. The same pattern was observed in spectra of a variety of artificially glycated proteins, including two mAbs, as well as natural proteins. CONCLUSION: Based on this unique correlation pattern, 2D NMR spectroscopy can be used to unambiguously identify glucose-induced glycation in any protein of interest. We provide a robust method that is orthogonal to MS-based methods and can also be used for cross-validation.

Legumain Functions as a Transient TrkB Sheddase.

While primarily found in endo-lysosomal compartments, the cysteine protease legumain can also translocate to the cell surface if stabilized by the interaction with the RGD-dependent integrin receptor αVß3. Previously, it has been shown that legumain expression is inversely related to BDNF-TrkB activity. Here we show that legumain can conversely act on TrkB-BDNF by processing the C-terminal linker region of the TrkB ectodomain in vitro. Importantly, when in complex with BDNF, TrkB was not cleaved by legumain. Legumain-processed TrkB was still able to bind BDNF, suggesting a potential scavenger function of soluble TrkB towards BDNF. The work thus presents another mechanistic link explaining the reciprocal TrkB signaling and δ-secretase activity of legumain, with relevance for neurodegeneration.

[The Attitude of the Public Concerning Coercive Measures in Psychiatric Patients]. / Die Haltung der Öffentlichkeit zu Zwangsmassnahmen bei psychiatrischen PatientInnen.

OBJECTIVE: To examine the attitude of the general public in Basel concerning the use of coercive measures while dealing with psychiatric patients. The common population indirectly governs the use of coercive measures in psychiatry by its stigmatization of people with psychiatric illnesses, and its attitude towards treatment in psychiatry and by local opinion leaders and reactions of social networks. METHODS: The answers of 1,112 persons from a representative population survey were evaluated. Participants were mailed case vignettes and questionnaires, and asked if they considered involuntary admission, coercive medication, and/or seclusion as acceptable measures in dealing with psychiatric patients. RESULTS: When symptoms of a psychotic disorder were present, 31.5% approved of at least one coercive measure, with 22% approval in the case of a borderline personality disorder, and 20.7% in the case of alcohol dependency. However, the overall rejection of coercive measures by the general public in Basel was high. The differential approval of the examined coercive measures depending on psychiatric symptoms was in line with professional medical and ethical guidelines. CONCLUSION: Public attitudes have an indirect influence on the local use of coercive measures and should be included in the specialist psychiatric discourse.

Glycosphingolipid-Glycan Signatures of Acute Myeloid Leukemia Cell Lines Reflect Hematopoietic Differentiation.

Aberrant expression of certain glycosphingolipids (GSLs) is associated with the differentiation of acute myeloid leukemia (AML) cells. However, the expression patterns of GSLs in AML are still poorly explored because of their complexity, the presence of multiple isomeric structures, and tedious analytical procedures. In this study, we performed an in-depth GSL glycan analysis of 19 AML cell lines using porous graphitized carbon liquid chromatography-mass spectrometry revealing strikingly different GSL glycan profiles between the various AML cell lines. The cell lines of the M6 subtype showed a high expression of gangliosides with α2,3-sialylation and Neu5Gc, while the M2 and M5 subtypes were characterized by high expression of (neo)lacto-series glycans and Lewis A/X antigens. Integrated analysis of glycomics and available transcriptomics data revealed the association of GSL glycan abundances with the transcriptomics expression of certain glycosyltransferases (GTs) and transcription factors (TFs). In addition, correlations were found between specific GTs and TFs. Our data reveal TFs GATA2, GATA1, and RUNX1 as candidate inducers of the expression of gangliosides and sialylation via regulation of the GTs ST3GAL2 and ST8SIA1. In conclusion, we show that GSL glycan expression levels are associated with hematopoietic AML classifications and TF and GT gene expression. Further research is needed to dissect the regulation of GSL expression and its role in hematopoiesis and associated malignancies.

The Fluorescent Enzyme Cascade Detects Low Abundance Protein Modifications Suitable for the Assembly of Functionally Annotated Modificatome Databases.

Pathophysiological functions of proteins critically depend on both their chemical composition, including post-translational modifications, and their three-dimensional structure, commonly referred to as structure-activity relationship. Current analytical methods, like capillary electrophoresis or mass spectrometry, suffer from limitations, such as the detection of unexpected modifications at low abundance and their insensitivity to conformational changes. Building on previous enzyme-based analytical methods, we here introduce a fluorescence-based enzyme cascade (fEC), which can detect diverse chemical and conformational variations in protein samples and assemble them into digital databases. Together with complementary analytical methods an automated fEC analysis established unique modification-function relationships, which can be expanded to a proteome-wide scale, i. e. a functionally annotated modificatome. The fEC offers diverse applications, including hypersensitive biomarker detection in complex samples.

Hydrogen/deuterium exchange memory NMR reveals structural epitopes involved in IgE cross-reactivity of allergenic lipid transfer proteins.

Identification of antibody-binding epitopes is crucial to understand immunological mechanisms. It is of particular interest for allergenic proteins with high cross-reactivity as observed in the lipid transfer protein (LTP) syndrome, which is characterized by severe allergic reactions. Art v 3, a pollen LTP from mugwort, is frequently involved in this cross-reactivity, but no antibody-binding epitopes have been determined so far. To reveal human IgE-binding regions of Art v 3, we produced three murine high-affinity mAbs, which showed 70-90% coverage of the allergenic epitopes from mugwort pollen-allergic patients. As reliable methods to determine structural epitopes with tightly interacting intact antibodies under native conditions are lacking, we developed a straightforward NMR approach termed hydrogen/deuterium exchange memory (HDXMEM). It relies on the slow exchange between the invisible antigen-mAb complex and the free 15N-labeled antigen whose 1H-15N correlations are detected. Due to a memory effect, changes of NH protection during antibody binding are measured. Differences in H/D exchange rates and analyses of mAb reactivity to homologous LTPs revealed three structural epitopes: two partially cross-reactive regions around α-helices 2 and 4 as well as a novel Art v 3-specific epitope at the C terminus. Protein variants with exchanged epitope residues confirmed the antibody-binding sites and revealed strongly reduced IgE reactivity. Using the novel HDXMEM for NMR epitope mapping allowed identification of the first structural epitopes of an allergenic pollen LTP. This knowledge enables improved cross-reactivity prediction for patients suffering from LTP allergy and facilitates design of therapeutics.

Detecting aspartate isomerization and backbone cleavage after aspartate in intact proteins by NMR spectroscopy.

The monitoring of non-enzymatic post-translational modifications (PTMs) in therapeutic proteins is important to ensure drug safety and efficacy. Together with methionine and asparagine, aspartic acid (Asp) is very sensitive to spontaneous alterations. In particular, Asp residues can undergo isomerization and peptide-bond hydrolysis, especially when embedded in sequence motifs that are prone to succinimide formation or when followed by proline (Pro). As Asp and isoAsp have the same mass, and the Asp-Pro peptide-bond cleavage may lead to an unspecific mass difference of + 18 Da under native conditions or in the case of disulfide-bridged cleavage products, it is challenging to directly detect and characterize such modifications by mass spectrometry (MS). Here we propose a 2D NMR-based approach for the unambiguous identification of isoAsp and the products of Asp-Pro peptide-bond cleavage, namely N-terminal Pro and C-terminal Asp, and demonstrate its applicability to proteins including a therapeutic monoclonal antibody (mAb). To choose the ideal pH conditions under which the NMR signals of isoAsp and C-terminal Asp are distinct from other random coil signals, we determined the pKa values of isoAsp and C-terminal Asp in short peptides. The characteristic 1H-13C chemical shift correlations of isoAsp, N-terminal Pro and C-terminal Asp under standardized conditions were used to identify these PTMs in lysozyme and in the therapeutic mAb rituximab (MabThera) upon prolonged storage under acidic conditions (pH 4-5) and 40 °C. The results show that the application of our 2D NMR-based protocol is straightforward and allows detecting chemical changes of proteins that may be otherwise unnoticed with other analytical methods.

Exploring the Chemical Space of Protein Glycosylation in Noncovalent Protein Complexes: An Expedition along Different Structural Levels of Human Chorionic Gonadotropin by Employing Mass Spectrometry.

Modern analytical approaches employing high-resolution mass spectrometry (MS) facilitate the generation of a vast amount of structural data of highly complex glycoproteins. Nevertheless, systematic interpretation of this data at different structural levels remains an analytical challenge. The glycoprotein utilized as a model system in this study, human chorionic gonadotropin (hCG), exists as a heterodimer composed of two heavily glycosylated subunits. In order to unravel the multitude of glycoforms of recombinant hCG (drug product Ovitrelle), we combine established techniques, such as released glycan and glycopeptide analysis, with novel approaches employing high-performance liquid chromatography-mass spectrometry (HPLC-MS) to characterize protein subunits and native MS to analyze the noncovalent hCG complex. Starting from the deconvoluted mass spectrum of dimeric hCG comprising about 50 signals, it was possible to explore the chemical space of hCG glycoforms and elucidate the complexity that hides behind just 50 signals. Systematic, stepwise integration of data obtained at the levels of released glycans, glycopeptides, and subunits using a computational annotation tool allowed us to reveal 1031 underlying glycoforms. Additionally, critical quality attributes such as sialylation and core fucosylation were compared for two batches of Ovitrelle to assess the potential product variability.

Crystal Structure of Plant Legumain Reveals a Unique Two-Chain State with pH-Dependent Activity Regulation.

The vacuolar cysteine protease legumain can cleave and selectively rebuild peptide bonds, thereby vastly expanding the sequential repertoire of biomolecules. In this context, plant legumains have recently attracted particular interest. Furthermore, legumains have important roles in many physiological processes, including programmed cell death. Their efficient peptide bond ligase activity has gained tremendous interest in the design of cyclic peptides for drug design. However, the mechanistic understanding of these dual activities is incomplete and partly conflicting. Here, we present the crystal structure of a plant legumain, Arabidopsis thaliana isoform-γ (AtLEGγ). Employing a conserved legumain fold, the plant legumain AtLEGγ revealed unique mechanisms of autoactivation, including a plant-specific two-chain activation state, which remains conformationally stable at neutral pH, which is a prerequisite for full ligase activity and survival in different cell compartments. The charge distribution around the α6-helix mediates the pH-dependent dimerization and serves as a gatekeeper for the active site, thus regulating its protease and ligase activity.

Simultaneous Monitoring of Monoclonal Antibody Variants by Strong Cation-Exchange Chromatography Hyphenated to Mass Spectrometry to Assess Quality Attributes of Rituximab-Based Biotherapeutics.

Different manufacturing processes and storage conditions of biotherapeutics can lead to a significant variability in drug products arising from chemical and enzymatic post-translational modifications (PTMs), resulting in the co-existence of a plethora of proteoforms with different physicochemical properties. To unravel the heterogeneity of these proteoforms, novel approaches employing strong cation-exchange (SCX) high-performance liquid chromatography (HPLC) hyphenated to mass spectrometry (MS) using a pH gradient of volatile salts have been developed in recent years. Here, we apply an established SCX-HPLC-MS method to characterize and compare two rituximab-based biotherapeutics, the originator MabThera® and its Indian copy product Reditux™. The study assessed molecular differences between the two drug products in terms of C-terminal lysine variants, glycosylation patterns, and other basic and acidic variants. Overall, MabThera® and Reditux™ displayed differences at the molecular level. MabThera® showed a higher degree of galactosylated and sialylated glycoforms, while Reditux™ showed increased levels of oligomannose and afucosylated glycoforms. Moreover, the two drug products showed differences in terms of basic variants such as C-terminal lysine and N-terminal truncation, present in Reditux™ but not in MabThera®. This study demonstrates the capability of this fast SCX-HPLC-MS approach to compare different drug products and simultaneously assess some of their quality attributes.

Proteolytic Profiling of Streptococcal Pyrogenic Exotoxin B (SpeB) by Complementary HPLC-MS Approaches.

Streptococcal pyrogenic exotoxin B (SpeB) is a cysteine protease expressed during group A streptococcal infection that represents a major virulence factor. Although subject to several studies, its role during infection is still under debate, and its proteolytic properties remain insufficiently characterized. Here, we revisited this protease through a set of complementary approaches relying on state of-the-art HPLC-MS methods. After conceiving an efficient protocol to recombinantly express SpeB, the zymogen of the protease and its activation were characterized. Employing proteome-derived peptide libraries, a strong preference for hydrophobic and aromatic residues at P2 alongside negatively charged amino acids at P3' to P6' was revealed. To identify relevant in vivo substrates, native proteins were obtained from monocytic secretome and plasma to assess their cleavage under physiological conditions. Besides corroborating our findings concerning specificity, more than 200 cleaved proteins were identified, including proteins of the extracellular matrix, proteins of the immune system, and proteins involved in inflammation. Finally, the cleavage of IgG subclasses was studied in detail. This study precisely depicts the proteolytic properties of SpeB and provides a library of potential host substrates, including their exact cleavage positions, as a valuable source for further research to unravel the role of SpeB during streptococcal infection.

Identification and Quantification of Oxidation Products in Full-Length Biotherapeutic Antibodies by NMR Spectroscopy.

Therapeutic proteins are an indispensable class of drugs and often therapeutics of last resort. They are sensitive to oxidation, which is of critical concern, because it can affect drug safety and efficacy. Protein oxidation, with methionine and tryptophan as the most susceptible moieties, is mainly monitored by HPLC-MS techniques. However, since several oxidation products display the same mass difference, their identification by MS is often ambiguous. Therefore, an alternative analytical method able to unambiguously identify and, ideally, also quantify oxidation species in proteins is highly desired. Here, we present an NMR-based approach to monitor oxidation in full-length proteins under denaturing conditions, as demonstrated on two biotherapeutic monoclonal antibodies (mAbs). We show that methionine sulfoxide, methionine sulfone, N-formylkynurenine, kynurenine, oxindolylalanine, hydroxypyrroloindole, and 5-hydroxytryptophan result in characteristic chemical shift correlations suited for their identification and quantification. We identified the five most abundant oxidation products in forced degradation studies of two full-length therapeutic mAbs and can also unambiguously distinguish oxindolylalanine from 5-hydroxytryptophan, which are undistinguishable by MS due to the same mass shift. Quantification of the abundant methionine sulfoxide by NMR and MS gave highly comparable values. These results underline the suitability of NMR spectroscopy for the identification and quantification of critical quality attributes of biotherapeutics.

An overlapping pattern of cerebral cortical thinning is associated with both positive symptoms and aggression in schizophrenia via the ENIGMA consortium.

BACKGROUND: Positive symptoms are a useful predictor of aggression in schizophrenia. Although a similar pattern of abnormal brain structures related to both positive symptoms and aggression has been reported, this observation has not yet been confirmed in a single sample. METHOD: To study the association between positive symptoms and aggression in schizophrenia on a neurobiological level, a prospective meta-analytic approach was employed to analyze harmonized structural neuroimaging data from 10 research centers worldwide. We analyzed brain MRI scans from 902 individuals with a primary diagnosis of schizophrenia and 952 healthy controls. RESULTS: The result identified a widespread cortical thickness reduction in schizophrenia compared to their controls. Two separate meta-regression analyses revealed that a common pattern of reduced cortical gray matter thickness within the left lateral temporal lobe and right midcingulate cortex was significantly associated with both positive symptoms and aggression. CONCLUSION: These findings suggested that positive symptoms such as formal thought disorder and auditory misperception, combined with cognitive impairments reflecting difficulties in deploying an adaptive control toward perceived threats, could escalate the likelihood of aggression in schizophrenia.

How many human proteoforms are there?

Despite decades of accumulated knowledge about proteins and their post-translational modifications (PTMs), numerous questions remain regarding their molecular composition and biological function. One of the most fundamental queries is the extent to which the combinations of DNA-, RNA- and PTM-level variations explode the complexity of the human proteome. Here, we outline what we know from current databases and measurement strategies including mass spectrometry-based proteomics. In doing so, we examine prevailing notions about the number of modifications displayed on human proteins and how they combine to generate the protein diversity underlying health and disease. We frame central issues regarding determination of protein-level variation and PTMs, including some paradoxes present in the field today. We use this framework to assess existing data and to ask the question, "How many distinct primary structures of proteins (proteoforms) are created from the 20,300 human genes?" We also explore prospects for improving measurements to better regularize protein-level biology and efficiently associate PTMs to function and phenotype.

Phosphoproteomics of short-term hedgehog signaling in human medulloblastoma cells.

BACKGROUND: Aberrant hedgehog (HH) signaling is implicated in the development of various cancer entities such as medulloblastoma. Activation of GLI transcription factors was revealed as the driving force upon pathway activation. Increased phosphorylation of essential effectors such as Smoothened (SMO) and GLI proteins by kinases including Protein Kinase A, Casein Kinase 1, and Glycogen Synthase Kinase 3 ß controls effector activity, stability and processing. However, a deeper and more comprehensive understanding of phosphorylation in the signal transduction remains unclear, particularly during early response processes involved in SMO activation and preceding GLI target gene regulation. METHODS: We applied temporal quantitative phosphoproteomics to reveal phosphorylation dynamics underlying the short-term chemical activation and inhibition of early hedgehog signaling in HH responsive human medulloblastoma cells. Medulloblastoma cells were treated for 5.0 and 15 min with Smoothened Agonist (SAG) to induce and with vismodegib to inhibit the HH pathway. RESULTS: Our phosphoproteomic profiling resulted in the quantification of 7700 and 10,000 phosphosites after 5.0 and 15 min treatment, respectively. The data suggest a central role of phosphorylation in the regulation of ciliary assembly, trafficking, and signal transduction already after 5.0 min treatment. ERK/MAPK signaling, besides Protein Kinase A signaling and mTOR signaling, were differentially regulated after short-term treatment. Activation of Polo-like Kinase 1 and inhibition of Casein Kinase 2A1 were characteristic for vismodegib treatment, while SAG treatment induced Aurora Kinase A activity. Distinctive phosphorylation of central players of HH signaling such as SMO, SUFU, GLI2 and GLI3 was observed only after 15 min treatment. CONCLUSIONS: This study provides evidence that phosphorylation triggered in response to SMO modulation dictates the localization of hedgehog pathway components within the primary cilium and affects the regulation of the SMO-SUFU-GLI axis. The data are relevant for the development of targeted therapies of HH-associated cancers including sonic HH-type medulloblastoma. A deeper understanding of the mechanisms of action of SMO inhibitors such as vismodegib may lead to the development of compounds causing fewer adverse effects and lower frequencies of drug resistance. Video Abstract.