A Workflow for Improved Analysis of Cross-Linking Mass Spectrometry Data Integrating Parallel Accumulation-Serial Fragmentation with MeroX and Skyline.

Cross-linking mass spectrometry (XL-MS) has evolved into a pivotal technique for probing protein interactions. This study describes the implementation of Parallel Accumulation-Serial Fragmentation (PASEF) on timsTOF instruments, enhancing the detection and analysis of protein interactions by XL-MS. Addressing the challenges in XL-MS, such as the interpretation of complex spectra, low abundant cross-linked peptides, and a data acquisition bias, our current study integrates a peptide-centric approach for the analysis of XL-MS data and presents the foundation for integrating data-independent acquisition (DIA) in XL-MS with a vendor-neutral and open-source platform. A novel workflow is described for processing data-dependent acquisition (DDA) of PASEF-derived information. For this, software by Bruker Daltonics is used, enabling the conversion of these data into a format that is compatible with MeroX and Skyline software tools. Our approach significantly improves the identification of cross-linked products from complex mixtures, allowing the XL-MS community to overcome current analytical limitations.

Structural surfaceomics reveals an AML-specific conformation of integrin ß2 as a CAR T cellular therapy target.

Safely expanding indications for cellular therapies has been challenging given a lack of highly cancer-specific surface markers. Here we explore the hypothesis that tumor cells express cancer-specific surface protein conformations that are invisible to standard target discovery pipelines evaluating gene or protein expression, and these conformations can be identified and immunotherapeutically targeted. We term this strategy integrating cross-linking mass spectrometry with glycoprotein surface capture 'structural surfaceomics'. As a proof of principle, we apply this technology to acute myeloid leukemia (AML), a hematologic malignancy with dismal outcomes and no known optimal immunotherapy target. We identify the activated conformation of integrin ß2 as a structurally defined, widely expressed AML-specific target. We develop and characterize recombinant antibodies to this protein conformation and show that chimeric antigen receptor T cells eliminate AML cells and patient-derived xenografts without notable toxicity toward normal hematopoietic cells. Our findings validate an AML conformation-specific target antigen and demonstrate a tool kit for applying these strategies more broadly.

Structural assessment of the full-length wild-type tumor suppressor protein p53 by mass spectrometry-guided computational modeling.

The tetrameric tumor suppressor p53 represents a great challenge for 3D-structural analysis due to its high degree of intrinsic disorder (ca. 40%). We aim to shed light on the structural and functional roles of p53's C-terminal region in full-length, wild-type human p53 tetramer and their importance for DNA binding. For this, we employed complementary techniques of structural mass spectrometry (MS) in an integrated approach with computational modeling. Our results show no major conformational differences in p53 between DNA-bound and DNA-free states, but reveal a substantial compaction of p53's C-terminal region. This supports the proposed mechanism of unspecific DNA binding to the C-terminal region of p53 prior to transcription initiation by specific DNA binding to the core domain of p53. The synergies between complementary structural MS techniques and computational modeling as pursued in our integrative approach is envisioned to serve as general strategy for studying intrinsically disordered proteins (IDPs) and intrinsically disordered region (IDRs).

Direct Observation of "Elongated" Conformational States in α-Synuclein upon Liquid-Liquid Phase Separation.

α-Synuclein (α-syn) is an intrinsically disordered protein (IDP) that undergoes liquid-liquid phase separation (LLPS), fibrillation, and forms insoluble intracellular Lewy bodies in neurons, which are the hallmark of Parkinson's Disease (PD). Neurotoxicity precedes the formation of aggregates and might be related to α-syn LLPS. The molecular mechanisms underlying the early stages of LLPS are still elusive. To obtain structural insights into α-syn upon LLPS, we take advantage of cross-linking/mass spectrometry (XL-MS) and introduce an innovative approach, termed COMPASS (COMPetitive PAiring StatisticS). In this work, we show that the conformational ensemble of α-syn shifts from a "hairpin-like" structure towards more "elongated" conformational states upon LLPS. We obtain insights into the critical initial stages of LLPS and establish a novel mass spectrometry-based approach that will aid to solve open questions in LLPS structural biology.

Different Oligomeric States of the Tumor Suppressor p53 Show Identical Binding Behavior towards the S100ß Homodimer.

The tumor suppressor protein p53 is a transcription factor that is referred to as the "guardian of the genome" and plays an important role in cancer development. p53 is active as a homotetramer; the S100ß homodimer binds to the intrinsically disordered C-terminus of p53 affecting its transcriptional activity. The p53/S100ß complex is regarded as highly promising therapeutic target in cancer. It has been suggested that S100ß exerts its oncogenic effects by altering the p53 oligomeric state. Our aim was to study the structures and oligomerization behavior of different p53/S100ß complexes by ESI-MS, XL-MS, and SPR. Wild-type p53 and single amino acid variants, representing different oligomeric states of p53 were individually investigated regarding their binding behavior towards S100ß. The stoichiometry of the different p53/S100ß complexes were determined by ESI-MS showing that tetrameric, dimeric, and monomeric p53 variants all bind to an S100ß dimer. In addition, XL-MS revealed the topologies of the p53/S100ß complexes to be independent of p53's oligomeric state. With SPR, the thermodynamic parameters were determined for S100ß binding to tetrameric, dimeric, or monomeric p53 variants. Our data prove that the S100ß homodimer binds to different oligomeric states of p53 with similar binding affinities. This emphasizes the need for alternative explanations to describe the molecular mechanisms underlying p53/S100ß interaction.

Cross-Linking/Mass Spectrometry Combined with Ion Mobility on a timsTOF Pro Instrument for Structural Proteomics.

The combination of cross-linking/mass spectrometry (XL-MS) and ion mobility is still underexplored for conducting protein conformational and protein-protein interaction studies. We present a method for analyzing cross-linking mixtures on a timsTOF Pro mass spectrometer that allows separating ions based on their gas-phase mobilities. Cross-linking was performed with three urea-based MS-cleavable cross-linkers that deliver distinct fragmentation patterns for cross-linked species upon collisional activation. The discrimination of cross-linked species from non-cross-linked peptides was readily performed based on their collisional cross sections. We demonstrate the general feasibility of our combined XL-MS/ion mobility approach for three protein systems of increasing complexity: (i) bovine serum albumin (BSA), (ii) Escherichia coli ribosome, and (iii) HEK293T cell nuclear lysates. We identified a total of 623 unique cross-linking sites for BSA, 670 for the E. coli ribosome, and 1623 unique cross-links for nuclear lysates, corresponding to 1088 intra- and 535 interprotein interactions and yielding 564 distinct protein-protein interactions. Our results underline the strength of combining XL-MS with ion mobility not only for deriving three-dimensional (3D) structures of single proteins but also for performing system-wide protein interaction studies.

First 3D-Structural Data of Full-Length Guanylyl Cyclase 1 in Rod-Outer-Segment Preparations of Bovine Retina by Cross-Linking/Mass Spectrometry.

The rod-outer-segment guanylyl cyclase 1 (ROS-GC1) is a key transmembrane protein for retinal phototransduction. Mutations of ROS-GC1 correlate with different retinal diseases that often lead to blindness. No structural data are available for ROS-GC1 so far. We performed a 3D-structural analysis of native ROS-GC1 from bovine retina by cross-linking/mass spectrometry (XL-MS) and computational modeling. Absolute quantification and activity measurements of native ROS-GC1 were performed by MS-based assays directly in bovine retina samples. Our data present the first 3D-structural analysis of active, full-length ROS-GC1 derived from bovine retina. We propose a novel domain organization for the intracellular domain ROS-GC1. Our XL-MS data of native ROS-GC1 from rod-outer-segment preparations of bovine retina agree with a dimeric architecture. Our integrated approach can serve as a blueprint for conducting 3D-structural studies of membrane proteins in their native environment.

Structural analysis of 70S ribosomes by cross-linking/mass spectrometry reveals conformational plasticity.

The ribosome is not only a highly complex molecular machine that translates the genetic information into proteins, but also an exceptional specimen for testing and optimizing cross-linking/mass spectrometry (XL-MS) workflows. Due to its high abundance, ribosomal proteins are frequently identified in proteome-wide XL-MS studies of cells or cell extracts. Here, we performed in-depth cross-linking of the E. coli ribosome using the amine-reactive cross-linker disuccinimidyl diacetic urea (DSAU). We analyzed 143 E. coli ribosomal structures, mapping a total of 10,771 intramolecular distances for 126 cross-link-pairs and 3,405 intermolecular distances for 97 protein pairs. Remarkably, 44% of intermolecular cross-links covered regions that have not been resolved in any high-resolution E. coli ribosome structure and point to a plasticity of cross-linked regions. We systematically characterized all cross-links and discovered flexible regions, conformational changes, and stoichiometric variations in bound ribosomal proteins, and ultimately remodeled 2,057 residues (15,794 atoms) in total. Our working model explains more than 95% of all cross-links, resulting in an optimized E. coli ribosome structure based on the cross-linking data obtained. Our study might serve as benchmark for conducting biochemical experiments on newly modeled protein regions, guided by XL-MS. Data are available via ProteomeXchange with identifier PXD018935.

Flexibility of intrinsically disordered degrons in AUX/IAA proteins reinforces auxin co-receptor assemblies.

Cullin RING-type E3 ubiquitin ligases SCFTIR1/AFB1-5 and their AUX/IAA targets perceive the phytohormone auxin. The F-box protein TIR1 binds a surface-exposed degron in AUX/IAAs promoting their ubiquitylation and rapid auxin-regulated proteasomal degradation. Here, by adopting biochemical, structural proteomics and in vivo approaches we unveil how flexibility in AUX/IAAs and regions in TIR1 affect their conformational ensemble allowing surface accessibility of degrons. We resolve TIR1·auxin·IAA7 and TIR1·auxin·IAA12 complex topology, and show that flexible intrinsically disordered regions (IDRs) in the degron's vicinity, cooperatively position AUX/IAAs on TIR1. We identify essential residues at the TIR1 N- and C-termini, which provide non-native interaction interfaces with IDRs and the folded PB1 domain of AUX/IAAs. We thereby establish a role for IDRs in modulating auxin receptor assemblies. By securing AUX/IAAs on two opposite surfaces of TIR1, IDR diversity supports locally tailored positioning for targeted ubiquitylation, and might provide conformational flexibility for a multiplicity of functional states.

The Isotope-Labeled, MS-Cleavable Cross-Linker Disuccinimidyl Dibutyric Urea for Improved Cross-Linking/Mass Spectrometry Studies.

Previous studies have shown the benefits of the amine-reactive, CID-MS/MS-cleavable cross-linker disuccinimidyl dibutyric urea (DSBU) for structural proteomics studies via cross-linking/MS (XL-MS). To further facilitate the automation of XL-MS experiments, we synthesized a deuterated (D12) version of the DSBU cross-linker combining the advantages of MS-cleavable linkers and isotope labeling. The rationale of conducting XL-MS with a mixture of unlabeled and stable isotope-labeled DSBU is to obtain characteristic mass differences at the MS level indicating cross-linked species. These cross-linked species can then be selected for fragmentation by collisional activation. At the MS/MS level, the characteristic 26-u doublets arising from cleavage of the central urea group in DSBU confirm the amino acid sequences of cross-linked peptides as well as the exact cross-linking sites. D12-labeled DSBU was tested on three systems with increasing complexity: (i) bovine serum albumin as purified protein, (ii) Escherichia coli ribosome as large, multimeric protein assembly, and (iii) Drosophila embryo extract as complete proteome. We demonstrate the benefits arising from the use of isotope-labeled DSBU for an automated assignment of cross-linked products. Combining isotope labeling and MS cleavability in one cross-linker resulted in higher cross-link identification numbers especially for highly complex protein mixtures.

Delineating the Molecular Basis of the Calmodulin‒bMunc13-2 Interaction by Cross-Linking/Mass Spectrometry-Evidence for a Novel CaM Binding Motif in bMunc13-2.

Exploring the interactions between the Ca2+ binding protein calmodulin (CaM) and its target proteins remains a challenging task. Members of the Munc13 protein family play an essential role in short-term synaptic plasticity, modulated via the interaction with CaM at the presynaptic compartment. In this study, we focus on the bMunc13-2 isoform expressed in the brain, as strong changes in synaptic transmission were observed upon its mutagenesis or deletion. The CaM‒bMunc13-2 interaction was previously characterized at the molecular level using short bMunc13-2-derived peptides only, revealing a classical 1‒5‒10 CaM binding motif. Using larger protein constructs, we have now identified for the first time a novel and unique CaM binding site in bMunc13-2 that contains an N-terminal extension of a classical 1‒5‒10 CaM binding motif. We characterize this motif using a range of biochemical and biophysical methods and highlight its importance for the CaM‒bMunc13-2 interaction.

A Simple Cross-Linking/Mass Spectrometry Workflow for Studying System-wide Protein Interactions.

We present a cross-linking/mass spectrometry workflow for performing proteome-wide cross-linking analyses within 1 week. The workflow is based on the commercially available mass spectrometry-cleavable cross-linker disuccinimidyl dibutyric urea and can be employed by every lab having access to a mass spectrometer with tandem mass spectrometry capabilities. We provide an updated version 2.0 of the freeware software tool MeroX, available at www.StavroX.com , that allows us to conduct fully automated and reliable studies delivering insights into protein-protein interaction networks and protein conformations at the proteome level. We exemplify our optimized workflow for mapping protein-protein interaction networks in Drosophila melanogaster embryos on a system-wide level. From cross-linked Drosophila embryo extracts, we detected 29931 cross-link spectrum matches corresponding to 7436 unique cross-linked residues in biological triplicate experiments at a 1% false discovery rate. Among these, 1611 interprotein cross-linking sites were identified and yielded valuable information about protein-protein interactions. The 5825 remaining intraprotein cross-links yield information about the conformational landscape of proteins in their cellular environment.

The First MS-Cleavable, Photo-Thiol-Reactive Cross-Linker for Protein Structural Studies.

Cleavable cross-linkers are gaining increasing importance for chemical cross-linking/mass spectrometry (MS) as they permit a reliable and automated data analysis in structural studies of proteins and protein assemblies. Here, we introduce 1,3-diallylurea (DAU) as the first CID-MS/MS-cleavable, photo-thiol-reactive cross-linker. DAU is a commercially available, inexpensive reagent that efficiently undergoes an anti-Markovnikov hydrothiolation with cysteine residues in the presence of a radical initiator upon UV-A irradiation. Radical cysteine cross-linking proceeds via an orthogonal "click reaction" and yields stable alkyl sulfide products. DAU reacts at physiological pH and cross-linking reactions with peptides, and proteins can be performed at temperatures as low as 4 °C. The central urea bond is efficiently cleaved upon collisional activation during tandem MS experiments generating characteristic product ions. This improves the reliability of automated cross-link identification. Different radical initiators have been screened for the cross-linking reaction of DAU using the thiol-containing compounds cysteine and glutathione. Our concept has also been exemplified for the biologically relevant proteins bMunc13-2 and retinal guanylyl cyclase-activating protein-2. Graphical abstract ᅟ.

A cross-linking/mass spectrometry workflow based on MS-cleavable cross-linkers and the MeroX software for studying protein structures and protein-protein interactions.

Chemical cross-linking in combination with mass spectrometric analysis of the created cross-linked products is an emerging technology aimed at deriving valuable structural information from proteins and protein complexes. The goal of our protocol is to obtain distance constraints for structure determination of proteins and to investigate protein-protein interactions. We present an integrated workflow for cross-linking/mass spectrometry (MS) based on protein cross-linking with MS-cleavable reagents, followed by enzymatic digestion, enrichment of cross-linked peptides by strong cation-exchange chromatography (SCX), and LC/MS/MS analysis. To exploit the full potential of MS-cleavable cross-linkers, we developed an updated version of the freely available MeroX software for automated data analysis. The commercially available, MS-cleavable cross-linkers (DSBU and CDI) used herein possess different lengths and react with amine as well as hydroxy groups. Owing to the formation of two characteristic 26-u doublets in their MS/MS spectra, many fewer false positives are found than when using classic, non-cleavable cross-linkers. The protocol, exemplified herein for BSA and the whole Escherichia coli ribosome, is robust and widely applicable, and it allows facile identification of cross-links for deriving spatial constraints from purified proteins and protein complexes. The cross-linking/MS procedure takes 2-3 days to complete.

Molecular Details of Retinal Guanylyl Cyclase 1/GCAP-2 Interaction.

The rod outer segment guanylyl cyclase 1 (ROS-GC1) is an essential component of photo-transduction in the retina. In the light-induced signal cascade, membrane-bound ROS-GC1 restores cGMP levels in the dark in a calcium-dependent manner. With decreasing calcium concentration in the intracellular compartment, ROS-GC1 is activated via the intracellular site by guanylyl cyclase-activating proteins (GCAP-1/-2). Presently, the exact activation mechanism is elusive. To obtain structural insights into the ROS-GC1 regulation by GCAP-2, chemical cross-linking/mass spectrometry studies using GCAP-2 and three ROS-GC1 peptides were performed in the presence and absence of calcium. The majority of cross-links were identified with the C-terminal lobe of GCAP-2 and a peptide comprising parts of ROS-GC1's catalytic domain and C-terminal extension. Consistently with the cross-linking results, surface plasmon resonance and fluorescence measurements confirmed specific binding of this ROS-GC peptide to GCAP-2 with a dissociation constant in the low micromolar range. These results imply that a region of the catalytic domain of ROS-GC1 can participate in the interaction with GCAP-2. Additional binding surfaces upstream of the catalytic domain, in particular the juxtamembrane domain, can currently not be excluded.

How Honeybees Defy Gravity with Royal Jelly to Raise Queens.

The female sex in honeybees (Apis spp.) comprises a reproductive queen and a sterile worker caste. Nurse bees feed all larvae progressively with a caste-specific food jelly until the prepupal stage. Only those larvae that are exclusively fed a large amount of royal jelly (RJ) develop into queens [1]. RJ is a composite secretion of two specialized head glands: the mandibular glands, which produce mainly fatty acids [2], and the hypopharyngeal glands, which contribute proteins, primarily belonging to the major royal jelly protein (MRJP) family [3]. Past research on RJ has focused on its nutritional function and overlooked its central role with regard to the orientation of the larva in the royal brood cell. Whereas workers are reared in the regular horizontal cells of the comb, the queen cells are specifically built outside of the normal comb area to accommodate for the larger queen [4, 5]. These cells hang freely along the bottom of the comb and are vertically oriented, opening downward [6]. Queen larvae are attached by their RJ diet to the cell ceiling. Thus, the physical properties of RJ are central to successful retention of larvae in the cell. Here, we show that the main protein of RJ (MRJP1) polymerizes in complex with another protein, apisimin, into long fibrous structures that build the basis for the high viscosity of RJ to hold queen larvae on the RJ surface.

Widely Used Commercial ELISA Does Not Detect Precursor of Haptoglobin2, but Recognizes Properdin as a Potential Second Member of the Zonulin Family.

BACKGROUND: There is increasing evidence for the role of impaired intestinal permeability in obesity and associated metabolic diseases. Zonulin is an established serum marker for intestinal permeability and identical to pre-haptoglobin2. Here, we aimed to investigate the relationship between circulating zonulin and metabolic traits related to obesity. METHODS: Serum zonulin was measured by using a widely used commercial ELISA kit in 376 subjects from the metabolically well-characterized cohort of Sorbs from Germany. In addition, haptoglobin genotype was determined in DNA samples from all study subjects. RESULTS: As zonulin concentrations did not correlate to the haptoglobin genotypes, we investigated the specificity of the zonulin ELISA assay using antibody capture experiments, mass spectrometry, and Western blot analysis. Using serum samples that gave the highest or lowest ELISA signals, we detected several proteins that are likely to be captured by the antibody in the present kit. However, none of these proteins corresponds to pre-haptoglobin2. We used increasing concentrations of recombinant pre-haptoglobin2 and complement C3 as one of the representative captured proteins and the ELISA kit did not detect either. Western blot analysis using both the polyclonal antibodies used in this kit and monoclonal antibodies rose against zonulin showed a similar protein recognition pattern but with different intensity of detection. The protein(s) measured using the ELISA kit was (were) significantly increased in patients with diabetes and obesity and correlated strongly with markers of the lipid and glucose metabolism. Combining mass spectrometry and Western blot analysis using the polyclonal antibodies used in the ELISA kit, we identified properdin as another member of the zonulin family. CONCLUSION: Our study suggests that the zonulin ELISA does not recognize pre-haptoglobin2, rather structural (and possibly functional) analog proteins belonging to the mannose-associated serine protease family, with properdin being the most likely possible candidate.

Combining affinity enrichment, cross-linking with photo amino acids, and mass spectrometry for probing protein kinase D2 interactions.

We present a novel approach that relies on the affinity capture of protein interaction partners from a complex mixture, followed by their covalent fixation via UV-induced activation of incorporated diazirine photoreactive amino acids (photo-methionine and photo-leucine). The captured protein complexes are enzymatically digested and interacting proteins are identified and quantified by label-free LC/MS analysis. Using HeLa cell lysates with photo-methionine and photo-leucine-labeled proteins, we were able to capture and preserve protein interactions that are otherwise elusive in conventional pull-down experiments. Our approach is exemplified for mapping the protein interaction network of protein kinase D2, but has the potential to be applied to any protein system. Data are available via ProteomeXchange with identifiers PXD005346 (photo amino acid incorporation) and PXD005349 (enrichment experiments).

An Integrated Mass Spectrometry Based Approach to Probe the Structure of the Full-Length Wild-Type Tetrameric p53 Tumor Suppressor.

We present an integrated approach for investigating the topology of proteins through native mass spectrometry (MS) and cross-linking/MS, which we applied to the full-length wild-type p53 tetramer. For the first time, the two techniques were combined in one workflow to obtain not only structural insight in the p53 tetramer, but also information on the cross-linking efficiency and the impact of cross-linker modification on the conformation of an intrinsically disordered protein (IDP). P53 cross-linking was monitored by native MS and as such, our strategy serves as a quality control for different cross-linking reagents. Our approach can be applied to the structural investigation of various protein systems, including IDPs and large protein assemblies, which are challenging to study by the conventional methods used for protein structure characterization.