An automated workflow based on data independent acquisition for practical and high-throughput personalized assay development and minimal residual disease monitoring in multiple myeloma patients.

OBJECTIVES: Minimal residual disease (MRD) status in multiple myeloma (MM) is an important prognostic biomarker. Personalized blood-based targeted mass spectrometry detecting M-proteins (MS-MRD) was shown to provide a sensitive and minimally invasive alternative to MRD-assessment in bone marrow. However, MS-MRD still comprises of manual steps that hamper upscaling of MS-MRD testing. Here, we introduce a proof-of-concept for a novel workflow using data independent acquisition-parallel accumulation and serial fragmentation (dia-PASEF) and automated data processing. METHODS: Using automated data processing of dia-PASEF measurements, we developed a workflow that identified unique targets from MM patient sera and personalized protein sequence databases. We generated patient-specific libraries linked to dia-PASEF methods and subsequently quantitated and reported M-protein concentrations in MM patient follow-up samples. Assay performance of parallel reaction monitoring (prm)-PASEF and dia-PASEF workflows were compared and we tested mixing patient intake sera for multiplexed target selection. RESULTS: No significant differences were observed in lowest detectable concentration, linearity, and slope coefficient when comparing prm-PASEF and dia-PASEF measurements of serial dilutions of patient sera. To improve assay development times, we tested multiplexing patient intake sera for target selection which resulted in the selection of identical clonotypic peptides for both simplex and multiplex dia-PASEF. Furthermore, assay development times improved up to 25× when measuring multiplexed samples for peptide selection compared to simplex. CONCLUSIONS: Dia-PASEF technology combined with automated data processing and multiplexed target selection facilitated the development of a faster MS-MRD workflow which benefits upscaling and is an important step towards the clinical implementation of MS-MRD.

The GlycoPaSER Prototype as a Real-Time N-Glycopeptide Identification Tool Based on the PaSER Parallel Computing Platform.

Real-time database searching allows for simpler and automated proteomics workflows as it eliminates technical bottlenecks in high-throughput experiments. Most importantly, it enables results-dependent acquisition (RDA), where search results can be used to guide data acquisition during acquisition. This is especially beneficial for glycoproteomics since the wide range of physicochemical properties of glycopeptides lead to a wide range of optimal acquisition parameters. We established here the GlycoPaSER prototype by extending the Parallel Search Engine in Real-time (PaSER) functionality for real-time glycopeptide identification from fragmentation spectra. Glycopeptide fragmentation spectra were decomposed into peptide and glycan moiety spectra using common N-glycan fragments. Each moiety was subsequently identified by a specialized algorithm running in real-time. GlycoPaSER can keep up with the rate of data acquisition for real-time analysis with similar performance to other glycoproteomics software and produces results that are in line with the literature reference data. The GlycoPaSER prototype presented here provides the first proof-of-concept for real-time glycopeptide identification that unlocks the future development of RDA technology to transcend data acquisition.

The linear ubiquitin-specific deubiquitinase gumby regulates angiogenesis.

A complex interaction of signalling events, including the Wnt pathway, regulates sprouting of blood vessels from pre-existing vasculature during angiogenesis. Here we show that two distinct mutations in the (uro)chordate-specific gumby (also called Fam105b) gene cause an embryonic angiogenic phenotype in gumby mice. Gumby interacts with disheveled 2 (DVL2), is expressed in canonical Wnt-responsive endothelial cells and encodes an ovarian tumour domain class of deubiquitinase that specifically cleaves linear ubiquitin linkages. A crystal structure of gumby in complex with linear diubiquitin reveals how the identified mutations adversely affect substrate binding and catalytic function in line with the severity of their angiogenic phenotypes. Gumby interacts with HOIP (also called RNF31), a key component of the linear ubiquitin assembly complex, and decreases linear ubiquitination and activation of NF-κB-dependent transcription. This work provides support for the biological importance of linear (de)ubiquitination in angiogenesis, craniofacial and neural development and in modulating Wnt signalling.

Oxygen-dependent Regulation of Erythropoietin Receptor Turnover and Signaling.

von Hippel-Lindau (VHL) disease is a rare familial cancer predisposition syndrome caused by a loss or mutation in a single gene,VHL, but it exhibits a wide phenotypic variability that can be categorized into distinct subtypes. The phenotypic variability has been largely argued to be attributable to the extent of deregulation of the α subunit of hypoxia-inducible factor α, a well established target of VHL E3 ubiquitin ligase, ECV (Elongins/Cul2/VHL). Here, we show that erythropoietin receptor (EPOR) is hydroxylated on proline 419 and 426 via prolyl hydroxylase 3. EPOR hydroxylation is required for binding to the ß domain of VHL and polyubiquitylation via ECV, leading to increased EPOR turnover. In addition, several type-specific VHL disease-causing mutants, including those that have retained proper binding and regulation of hypoxia-inducible factor α, showed a severe defect in binding prolyl hydroxylated EPOR peptides. These results identify EPOR as the secondbona fidehydroxylation-dependent substrate of VHL that potentially influences oxygen homeostasis and contributes to the complex genotype-phenotype correlation in VHL disease.

Global map of SUMO function revealed by protein-protein interaction and genetic networks.

Systematic functional genomics approaches were used to map a network centered on the small ubiquitin-related modifier (SUMO) system. Over 250 physical interactions were identified using the SUMO protein as bait in affinity purification-mass spectrometry and yeast two-hybrid screens. More than 500 genes and 1400 synthetic genetic interactions were mapped by synthetic genetic array (SGA) analysis using eight different SUMO pathway query genes. The resultant global SUMO network highlights its role in 15 major biological processes and better defines functional relationships between the different components of the SUMO pathway. Using this information-rich resource, we have identified roles for the SUMO system in the function of the AAA ATPase Cdc48p, the regulation of lipid metabolism, localization of the ATP-dependent endonuclease Dna2p, and recovery from the DNA-damage checkpoint.

KCMF1 (potassium channel modulatory factor 1) Links RAD6 to UBR4 (ubiquitin N-recognin domain-containing E3 ligase 4) and lysosome-mediated degradation.

RAD6 is a ubiquitin E2 protein with roles in a number of different biological processes. Here, using affinity purification coupled with mass spectrometry, we identify a number of new RAD6 binding partners, including the poorly characterized ubiquitin E3 ligases KCMF1 (potassium channel modulatory factor 1) and UBR4 (ubiquitin N-recognin domain-containing E3 ligase 4), a protein that can bind N-end rule substrates, and which was recently linked to lysosome-mediated degradation and autophagy. NMR, combined with in vivo and in vitro interaction mapping, demonstrate that the KCMF1 C terminus binds directly to RAD6, whereas N-terminal domains interact with UBR4 and other intracellular vesicle- and mitochondria-associated proteins. KCMF1 and RAD6 colocalize at late endosomes and lysosomes, and cells disrupted for KCMF1 or RAD6 function display defects in late endosome vesicle dynamics. Notably, we also find that two different RAD6A point mutants (R7W and R11Q) found in X-linked intellectual disability (XLID) patients specifically lose the interaction with KCMF1 and UBR4, but not with other previously identified RAD6 interactors. We propose that RAD6-KCMF1-UBR4 represents a unique new E2-E3 complex that targets unknown N-end rule substrates for lysosome-mediated degradation, and that disruption of this complex via RAD6A mutations could negatively affect neuronal function in XLID patients.

Src promotes GTPase activity of Ras via tyrosine 32 phosphorylation.

Mutations in Ras GTPase and various other components of the Ras signaling pathways are among the most common genetic alterations in human cancers and also have been identified in several familial developmental syndromes. Over the past few decades it has become clear that the activity or the oncogenic potential of Ras is dependent on the nonreceptor tyrosine kinase Src to promote the Ras/Raf/MAPK pathway essential for proliferation, differentiation, and survival of eukaryotic cells. However, no direct relationship between Ras and Src has been established. We show here that Src binds to and phosphorylates GTP-, but not GDP-, loaded Ras on a conserved Y32 residue within the switch I region in vitro and that in vivo, Ras-Y32 phosphorylation markedly reduces the binding to effector Raf and concomitantly increases binding to GTPase-activating proteins and the rate of GTP hydrolysis. These results suggest that, in the context of predetermined crystallographic structures, Ras-Y32 serves as an Src-dependent keystone regulatory residue that modulates Ras GTPase activity and ensures unidirectionality to the Ras GTPase cycle.

RNF168 ubiquitylates 53BP1 and controls its response to DNA double-strand breaks.

Defective signaling or repair of DNA double-strand breaks has been associated with developmental defects and human diseases. The E3 ligase RING finger 168 (RNF168), mutated in the human radiosensitivity, immunodeficiency, dysmorphic features, and learning difficulties syndrome, was shown to ubiquitylate H2A-type histones, and this ubiquitylation was proposed to facilitate the recruitment of p53-binding protein 1 (53BP1) to the sites of DNA double-strand breaks. In contrast to more upstream proteins signaling DNA double-strand breaks (e.g., RNF8), deficiency of RNF168 fully prevents both the initial recruitment to and retention of 53BP1 at sites of DNA damage; however, the mechanism for this difference has remained unclear. Here, we identify mechanisms that regulate 53BP1 recruitment to the sites of DNA double-strand breaks and provide evidence that RNF168 plays a central role in the regulation of 53BP1 functions. RNF168 mediates K63-linked ubiquitylation of 53BP1 which is required for the initial recruitment of 53BP1 to sites of DNA double-strand breaks and for its function in DNA damage repair, checkpoint activation, and genomic integrity. Our findings highlight the multistep roles of RNF168 in signaling DNA damage.

The use of ubiquitin lysine mutants to characterize E2-E3 linkage specificity: Mass spectrometry offers a cautionary "tail".

Oligomeric ubiquitin structures (i.e. ubiquitin "chains") may be formed through any of seven different lysine residues in the polypeptide, or via the amine group of Met 1. Different types of ubiquitin chains can confer very different biological outcomes to a protein substrate, yet the structural characteristics of E2s and E3s that determine ubiquitin linkage specificity remain poorly understood. In vitro autoubiquitylation assays combined with ubiquitin protein variants bearing individually mutated lysine residues ("K-to-R" mutants) have thus been widely used to characterize E2-E3 linkage specificity. However, how this type of assay compares to direct identification of ubiquitin linkage types using mass spectrometry (MS) has not been rigorously tested. Here, we characterize the linkage specificity of 12 different E2-E3 combinations using both approaches. The simple MS-based method described here is more robust, requires less material and is less prone to bias introduced by, e.g. the use of mutant proteins with unknown effects on E1, E2 or E3 recognition, antibodies with uncharacterized epitopes, the low dynamic range of X-ray film, and additional sources of experimental error. Indeed, our results suggest that the K-to-R assay be approached with some caution.

Identification of SUMO-2/3-modified proteins associated with mitotic chromosomes.

Sumoylation is essential for progression through mitosis, but the specific protein targets and functions remain poorly understood. In this study, we used chromosome spreads to more precisely define the localization of SUMO-2/3 (small ubiquitin-related modifier) to the inner centromere and protein scaffold of mitotic chromosomes. We also developed methods to immunopurify proteins modified by endogenous, untagged SUMO-2/3 from mitotic chromosomes. Using these methods, we identified 149 chromosome-associated SUMO-2/3 substrates by nLC-ESI-MS/MS. Approximately one-third of the identified proteins have reported functions in mitosis. Consistent with SUMO-2/3 immunolocalization, we identified known centromere- and kinetochore-associated proteins, as well as chromosome scaffold associated proteins. Notably, >30 proteins involved in chromatin modification or remodeling were identified. Our results provide insights into the roles of sumoylation as a regulator of chromatin structure and other diverse processes in mitosis. Furthermore, our purification and fractionation methodologies represent an important compliment to existing approaches to identify sumoylated proteins using exogenously expressed and tagged SUMOs.

A pathogen type III effector with a novel E3 ubiquitin ligase architecture.

Type III effectors are virulence factors of Gram-negative bacterial pathogens delivered directly into host cells by the type III secretion nanomachine where they manipulate host cell processes such as the innate immunity and gene expression. Here, we show that the novel type III effector XopL from the model plant pathogen Xanthomonas campestris pv. vesicatoria exhibits E3 ubiquitin ligase activity in vitro and in planta, induces plant cell death and subverts plant immunity. E3 ligase activity is associated with the C-terminal region of XopL, which specifically interacts with plant E2 ubiquitin conjugating enzymes and mediates formation of predominantly K11-linked polyubiquitin chains. The crystal structure of the XopL C-terminal domain revealed a single domain with a novel fold, termed XL-box, not present in any previously characterized E3 ligase. Mutation of amino acids in the central cavity of the XL-box disrupts E3 ligase activity and prevents XopL-induced plant cell death. The lack of cysteine residues in the XL-box suggests the absence of thioester-linked ubiquitin-E3 ligase intermediates and a non-catalytic mechanism for XopL-mediated ubiquitination. The crystal structure of the N-terminal region of XopL confirmed the presence of a leucine-rich repeat (LRR) domain, which may serve as a protein-protein interaction module for ubiquitination target recognition. While the E3 ligase activity is required to provoke plant cell death, suppression of PAMP responses solely depends on the N-terminal LRR domain. Taken together, the unique structural fold of the E3 ubiquitin ligase domain within the Xanthomonas XopL is unprecedented and highlights the variation in bacterial pathogen effectors mimicking this eukaryote-specific activity.

A global S. cerevisiae small ubiquitin-related modifier (SUMO) system interactome.

The small ubiquitin-related modifier (SUMO) system has been implicated in a number of biological functions, yet the individual components of the SUMO machinery involved in each of these activities were largely unknown. Here we report the first global SUMO system interactome. Using affinity purification coupled with mass spectrometry, we identify >450 protein-protein interactions surrounding the SUMO E2, Siz type E3s and SUMO-specific proteases in budding yeast. Exploiting this information-rich resource, we validate several Siz1- and Siz2-specific substrates, identify a nucleoporin required for proper Ulp1 localization, and uncover important new roles for Ubc9 and Ulp2 in the maintenance of ribosomal DNA.

A human ubiquitin conjugating enzyme (E2)-HECT E3 ligase structure-function screen.

Here we describe a systematic structure-function analysis of the human ubiquitin (Ub) E2 conjugating proteins, consisting of the determination of 15 new high-resolution three-dimensional structures of E2 catalytic domains, and autoubiquitylation assays for 26 Ub-loading E2s screened against a panel of nine different HECT (homologous to E6-AP carboxyl terminus) E3 ligase domains. Integration of our structural and biochemical data revealed several E2 surface properties associated with Ub chain building activity; (1) net positive or neutral E2 charge, (2) an "acidic trough" located near the catalytic Cys, surrounded by an extensive basic region, and (3) similarity to the previously described HECT binding signature in UBE2L3 (UbcH7). Mass spectrometry was used to characterize the autoubiquitylation products of a number of functional E2-HECT pairs, and demonstrated that HECT domains from different subfamilies catalyze the formation of very different types of Ub chains, largely independent of the E2 in the reaction. Our data set represents the first comprehensive analysis of E2-HECT E3 interactions, and thus provides a framework for better understanding the molecular mechanisms of ubiquitylation.

The dynamics and mechanism of SUMO chain deconjugation by SUMO-specific proteases.

SUMOylation of proteins is a cyclic process that requires both conjugation and deconjugation of SUMO moieties. Besides modification by a single SUMO, SUMO chains have also been observed, yet the dynamics of SUMO conjugation/deconjugation remain poorly understood. Using a non-deconjugatable form of SUMO we demonstrate the underappreciated existence of SUMO chains in vivo, we highlight the importance of SUMO deconjugation, and we demonstrate the highly dynamic nature of the SUMO system. We show that SUMO-specific proteases (SENPs) play a crucial role in the dynamics of SUMO chains in vivo by constant deconjugation. Preventing deSUMOylation in Schizosaccharomyces pombe results in slow growth and a sensitivity to replication stress, highlighting the biological requirement for deSUMOylation dynamics. Furthermore, we present the mechanism of SUMO chain deconjugation by SENPs, which occurs via a stochastic mechanism, resulting in cleavage anywhere within a chain. Our results offer mechanistic insights into the workings of deSUMOylating proteases and highlight their importance in the homeostasis of (poly)SUMO-modified substrates.

A ubiquitin and ubiquitin-like protein spectral library.

We have developed and validated a ubiquitin (Ub) and ubiquitin-like protein (Ubl) spectral library, consisting of 467 consensus spectra (320 unique peptides derived from autophagy-related protein 8, F-adjacent transcript 10, interferon-stimulated gene 15 kDa protein, neural precursor cell expressed developmentally down-regulated protein 8, small ubiquitin-related modifiers 1-3 and Ub, and nine of the most commonly observed Ub/Ubl chain linkages). The use of the Ub/Ubl library with a spectral matching tool (SpectraST) yields improved performance over database search engines, and can successfully identify many types of Ub/Ubl chain-derived peptides that cannot be identified by standard database search algorithms.

An improved SUMmOn-based methodology for the identification of ubiquitin and ubiquitin-like protein conjugation sites identifies novel ubiquitin-like protein chain linkages.

Ubiquitin (Ub) and the ubiquitin-like proteins (Ubls) comprise a remarkable assortment of polypeptides that are covalently conjugated to target proteins (or other biomolecules) to modulate their intracellular localization, half-life, and/or activity. Identification of Ub/Ubl conjugation sites on a protein of interest can thus be extremely important for understanding how it is regulated. While MS has become a powerful tool for the study of many classes of PTMs, the identification of Ub/Ubl conjugation sites presents a number of unique challenges. Here, we present an improved Ub/Ubl conjugation site identification strategy, utilizing SUMmOn analysis and an additional protease (lysyl endopeptidase C), as a complement to standard approaches. As compared with standard trypsin proteolysis-database search protocols alone, the addition of SUMmOn analysis can (i) identify Ubl conjugation sites that are not detected by standard database searching methods, (ii) better preserve Ub/Ubl conjugate identity, and (iii) increase the number of identifications of Ub/Ubl modifications in lysine-rich protein regions. Using this methodology, we characterize for the first time a number of novel Ubl linkages and conjugation sites, including alternative yeast (K54) and mammalian small ubiquitin-related modifier (SUMO) chain (SUMO-2 K42, SUMO-3 K41) assemblies, as well as previously unreported NEDD8 chain (K27, K33, and K54) topologies.

Using mass spectrometry to identify ubiquitin and ubiquitin-like protein conjugation sites.

Ubiquitin (Ub) and the ubiquitin-like proteins (Ubls) are polypeptides that are covalently conjugated to proteins and other biomolecules to modulate their turnover rate, localization, and/or function. The full range of Ubl functions is only beginning to be understood, and the wide variety of Ubl conjugates is only beginning to be identified. Moreover, how Ubl conjugation is regulated, and how Ubl conjugate populations change, e.g., throughout the cell cycle, in response to hormones, nutrients, or stress, or in various disease states, remains largely enigmatic. MS represents a powerful tool for the characterization of PTMs. However, standard sample preparation and data search methods are not amenable to the identification of many types of Ubl conjugates. Here, we describe the challenges of identifying Ub/Ubl conjugates, and propose an improved workflow for identification of Ub/Ubl conjugation sites. Considering the importance of Ubls in normal cellular physiology, and their roles in disease etiology and progression, it will be critical to develop improved high-throughput MS methods capable of efficiently identifying proteins and other biomolecules modified by these very interesting and important PTMs.

A genome-wide algal mutant library and functional screen identifies genes required for eukaryotic photosynthesis.

Photosynthetic organisms provide food and energy for nearly all life on Earth, yet half of their protein-coding genes remain uncharacterized1,2. Characterization of these genes could be greatly accelerated by new genetic resources for unicellular organisms. Here we generated a genome-wide, indexed library of mapped insertion mutants for the unicellular alga Chlamydomonas reinhardtii. The 62,389 mutants in the library, covering 83% of nuclear protein-coding genes, are available to the community. Each mutant contains unique DNA barcodes, allowing the collection to be screened as a pool. We performed a genome-wide survey of genes required for photosynthesis, which identified 303 candidate genes. Characterization of one of these genes, the conserved predicted phosphatase-encoding gene CPL3, showed that it is important for accumulation of multiple photosynthetic protein complexes. Notably, 21 of the 43 higher-confidence genes are novel, opening new opportunities for advances in understanding of this biogeochemically fundamental process. This library will accelerate the characterization of thousands of genes in algae, plants, and animals.

A Photocrosslinking-Based RNA Chemical Proteomics Approach to Profile m6 A-Regulated Protein-RNA Interactions.

Post-transcriptional modifications play an important role in RNA biology. In particular, the addition of small chemical groups to the nucleobases of mRNA can affect how modified transcripts are processed in the cell, thereby impacting gene expression programs. In order to study the molecular mechanisms underlying these modifications, it is necessary to characterize their 'readers', that is, proteins that directly bind to these modifications to mediate their functional consequences; this is a major challenge because we lack approaches to precisely manipulate RNA chemistry in the cell and because protein-modified RNA interactions can be low affinity. In this unit, we describe in detail a photocrosslinking-based RNA chemical proteomics approach to profile the protein-modified RNA interactome modulated by N6 -methyladenosine (m6 A), the most abundant internal modification in eukaryotic mRNA. First, we present protocols for the synthesis and characterization of short, diazirine-containing synthetic RNA probes, followed by a description of their use in mass spectrometry-based proteomics with HeLa cell lysate and a short commentary on data analysis and result interpretation. © 2018 by John Wiley & Sons, Inc.

Cyclic Enterobacterial Common Antigen Maintains the Outer Membrane Permeability Barrier of Escherichia coli in a Manner Controlled by YhdP.

Gram-negative bacteria have an outer membrane (OM) impermeable to many toxic compounds that can be further strengthened during stress. In Enterobacteriaceae, the envelope contains enterobacterial common antigen (ECA), a carbohydrate-derived moiety conserved throughout Enterobacteriaceae, the function of which is poorly understood. Previously, we identified several genes in Escherichia coli K-12 responsible for an RpoS-dependent decrease in envelope permeability during carbon-limited stationary phase. For one of these, yhdP, a gene of unknown function, deletion causes high levels of both vancomycin and detergent sensitivity, independent of growth phase. We isolated spontaneous suppressor mutants of yhdP with loss-of-function mutations in the ECA biosynthesis operon. ECA biosynthesis gene deletions suppressed envelope permeability from yhdP deletion independently of envelope stress responses and interactions with other biosynthesis pathways, demonstrating suppression is caused directly by removing ECA. Furthermore, yhdP deletion changed cellular ECA levels and yhdP was found to co-occur phylogenetically with the ECA biosynthesis operon. Cells make three forms of ECA: ECA lipopolysaccharide (LPS), an ECA chain linked to LPS core; ECA phosphatidylglycerol, a surface-exposed ECA chain linked to phosphatidylglycerol; and cyclic ECA, a cyclized soluble ECA molecule found in the periplasm. We determined that the suppression of envelope permeability with yhdP deletion is caused specifically by the loss of cyclic ECA, despite lowered levels of this molecule found with yhdP deletion. Furthermore, removing cyclic ECA from wild-type cells also caused changes to OM permeability. Our data demonstrate cyclic ECA acts to maintain the OM permeability barrier in a manner controlled by YhdP.IMPORTANCE Enterobacterial common antigen (ECA) is a surface antigen made by all members of Enterobacteriaceae, including many clinically relevant genera (e.g., Escherichia, Klebsiella, Yersinia). Although this surface-exposed molecule is conserved throughout Enterobacteriaceae, very few functions have been ascribed to it. Here, we have determined that the periplasmic form of ECA, cyclic ECA, plays a role in maintaining the outer membrane permeability barrier. This activity is controlled by a protein of unknown function, YhdP, and deletion of yhdP damages the OM permeability barrier in a cyclic ECA-dependent manner, allowing harmful molecules such as antibiotics into the cell. This role in maintenance of the envelope permeability barrier is the first time a phenotype has been described for cyclic ECA. As the Gram-negative envelope is generally impermeable to antibiotics, understanding the mechanisms through which the barrier is maintained and antibiotics are excluded may lead to improved antibiotic delivery.