RESUMO
Ribosomes are produced in large quantities during oogenesis and are stored in the egg. However, the egg and early embryo are translationally repressed1-4. Here, using mass spectrometry and cryo-electron microscopy analyses of ribosomes isolated from zebrafish (Danio rerio) and Xenopus laevis eggs and embryos, we provide molecular evidence that ribosomes transition from a dormant state to an active state during the first hours of embryogenesis. Dormant ribosomes are associated with four conserved factors that form two modules, consisting of Habp4-eEF2 and death associated protein 1b (Dap1b) or Dap in complex with eIF5a. Both modules occupy functionally important sites and act together to stabilize ribosomes and repress translation. Dap1b (also known as Dapl1 in mammals) is a newly discovered translational inhibitor that stably inserts into the polypeptide exit tunnel. Addition of recombinant zebrafish Dap1b protein is sufficient to block translation and reconstitute the dormant egg ribosome state in a mammalian translation extract in vitro. Thus, a developmentally programmed, conserved ribosome state has a key role in ribosome storage and translational repression in the egg.
Assuntos
Sequência Conservada , Evolução Molecular , Óvulo , Biossíntese de Proteínas , Ribossomos , Proteínas de Xenopus , Proteínas de Peixe-Zebra , Animais , Microscopia Crioeletrônica/métodos , Peptídeos/metabolismo , Ribossomos/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Espectrometria de Massas , Xenopus laevis/embriologia , Óvulo/metabolismo , Estruturas Embrionárias , Desenvolvimento Embrionário , Feminino , Fator de Iniciação de Tradução Eucariótico 5ARESUMO
Nuclear Argonaute proteins, guided by their bound small RNAs to nascent target transcripts, mediate cotranscriptional silencing of transposons and repetitive genomic loci through heterochromatin formation. The molecular mechanisms involved in this process are incompletely understood. Here, we show that the SFiNX complex, a silencing mediator downstream from nuclear Piwi-piRNA complexes in Drosophila, facilitates cotranscriptional silencing as a homodimer. The dynein light chain protein Cut up/LC8 mediates SFiNX dimerization, and its function can be bypassed by a heterologous dimerization domain, arguing for a constitutive SFiNX dimer. Dimeric, but not monomeric SFiNX, is capable of forming molecular condensates in a nucleic acid-stimulated manner. Mutations that prevent SFiNX dimerization result in loss of condensate formation in vitro and the inability of Piwi to initiate heterochromatin formation and silence transposons in vivo. We propose that multivalent SFiNX-nucleic acid interactions are critical for heterochromatin establishment at piRNA target loci in a cotranscriptional manner.
Assuntos
Proteínas Argonautas/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Inativação Gênica/fisiologia , Complexos Multiproteicos/metabolismo , Animais , Dimerização , Proteínas de Drosophila/química , Drosophila melanogaster/metabolismo , Dineínas/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático/química , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
All multicellular organisms rely on differential gene transcription regulated by genomic enhancers, which function through cofactors that are recruited by transcription factors1,2. Emerging evidence suggests that not all cofactors are required at all enhancers3-5, yet whether these observations reflect more general principles or distinct types of enhancers remained unknown. Here we categorized human enhancers by their cofactor dependencies and show that these categories provide a framework to understand the sequence and chromatin diversity of enhancers and their roles in different gene-regulatory programmes. We quantified enhancer activities along the entire human genome using STARR-seq6 in HCT116 cells, following the rapid degradation of eight cofactors. This analysis identified different types of enhancers with distinct cofactor requirements, sequences and chromatin properties. Some enhancers were insensitive to the depletion of the core Mediator subunit MED14 or the bromodomain protein BRD4 and regulated distinct transcriptional programmes. In particular, canonical Mediator7 seemed dispensable for P53-responsive enhancers, and MED14-depleted cells induced endogenous P53 target genes. Similarly, BRD4 was not required for the transcription of genes that bear CCAAT boxes and a TATA box (including histone genes and LTR12 retrotransposons) or for the induction of heat-shock genes. This categorization of enhancers through cofactor dependencies reveals distinct enhancer types that can bypass broadly utilized cofactors, which illustrates how alternative ways to activate transcription separate gene expression programmes and provide a conceptual framework to understand enhancer function and regulatory specificity.
Assuntos
Elementos Facilitadores Genéticos , Fatores de Transcrição , Proteínas de Ciclo Celular/metabolismo , Cromatina/genética , Elementos Facilitadores Genéticos/genética , Humanos , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
Recruitment of RNA polymerase II (Pol II) to promoters is essential for transcription. Despite conflicting evidence, the Pol II preinitiation complex (PIC) is often thought to have a uniform composition and to assemble at all promoters via an identical mechanism. Here, using Drosophila melanogaster S2 cells as a model, we demonstrate that different promoter classes function via distinct PICs. Promoter DNA of developmentally regulated genes readily associates with the canonical Pol II PIC, whereas housekeeping promoters do not, and instead recruit other factors such as DREF. Consistently, TBP and DREF are differentially required by distinct promoter types. TBP and its paralog TRF2 also function at different promoter types in a partially redundant manner. In contrast, TFIIA is required at all promoters, and we identify factors that can recruit and/or stabilize TFIIA at housekeeping promoters and activate transcription. Promoter activation by tethering these factors is sufficient to induce the dispersed transcription initiation patterns characteristic of housekeeping promoters. Thus, different promoter classes utilize distinct mechanisms of transcription initiation, which translate into different focused versus dispersed initiation patterns.
Assuntos
Proteínas de Drosophila , Fatores de Transcrição , Animais , Fatores de Transcrição/genética , Fator de Transcrição TFIIA/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteína de Ligação a TATA-Box/genética , Proteína de Ligação a TATA-Box/metabolismo , Regiões Promotoras Genéticas , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Transcrição Gênica , Proteínas de Drosophila/genéticaRESUMO
Protein expression and turnover are controlled through a complex interplay of transcriptional, post-transcriptional and post-translational mechanisms to enable spatial and temporal regulation of cellular processes. To systematically elucidate such gene regulatory networks, we developed a CRISPR screening assay based on time-controlled Cas9 mutagenesis, intracellular immunostaining and fluorescence-activated cell sorting that enables the identification of regulatory factors independent of their effects on cellular fitness. We pioneered this approach by systematically probing the regulation of the transcription factor MYC, a master regulator of cell growth1-3. Our screens uncover a highly conserved protein, AKIRIN2, that is essentially required for nuclear protein degradation. We found that AKIRIN2 forms homodimers that directly bind to fully assembled 20S proteasomes to mediate their nuclear import. During mitosis, proteasomes are excluded from condensing chromatin and re-imported into newly formed daughter nuclei in a highly dynamic, AKIRIN2-dependent process. Cells undergoing mitosis in the absence of AKIRIN2 become devoid of nuclear proteasomes, rapidly causing accumulation of MYC and other nuclear proteins. Collectively, our study reveals a dedicated pathway controlling the nuclear import of proteasomes in vertebrates and establishes a scalable approach to decipher regulators in essential cellular processes.
Assuntos
Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Fatores de Transcrição/metabolismo , Transporte Ativo do Núcleo Celular , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Feminino , Genes myc , Humanos , Masculino , Mitose , Complexo de Endopeptidases do Proteassoma/química , Ligação Proteica , ProteóliseRESUMO
Multiplex proteomics using isobaric labeling tags has emerged as a powerful tool for the simultaneous relative quantification of peptides and proteins across multiple experimental conditions. However, the quantitative accuracy of the approach is largely compromised by ion interference, a phenomenon that causes fold changes to appear compressed. The degree of compression is generally unknown, and the contributing factors are poorly understood. In this study, we thoroughly characterized ion interference at the MS2 level using a defined two-proteome experimental system with known ground-truth. We discovered remarkably poor agreement between the apparent precursor purity in the isolation window and the actual level of observed reporter ion interference in MS2 scans-a discrepancy that we found resolved by considering cofragmentation of peptide ions hidden within the spectral "noise" of the MS1 isolation window. To address this issue, we developed a regression modeling strategy to accurately predict reporter ion interference in any dataset. Finally, we demonstrate the utility of our procedure for improved fold change estimation and unbiased PTM site-to-protein normalization. All computational tools and code required to apply this method to any MS2 TMT dataset are documented and freely available.
Assuntos
Peptídeos , Proteômica , Proteômica/métodos , Proteoma/metabolismo , ÍonsRESUMO
Sister chromatid cohesion is essential for chromosome segregation and is mediated by cohesin bound to DNA. Cohesin-DNA interactions can be reversed by the cohesion-associated protein Wapl, whereas a stably DNA-bound form of cohesin is thought to mediate cohesion. In vertebrates, Sororin is essential for cohesion and stable cohesin-DNA interactions, but how Sororin performs these functions is unknown. We show that DNA replication and cohesin acetylation promote binding of Sororin to cohesin, and that Sororin displaces Wapl from its binding partner Pds5. In the absence of Wapl, Sororin becomes dispensable for cohesion. We propose that Sororin maintains cohesion by inhibiting Wapl's ability to dissociate cohesin from DNA. Sororin has only been identified in vertebrates, but we show that many invertebrate species contain Sororin-related proteins, and that one of these, Dalmatian, is essential for cohesion in Drosophila. The mechanism we describe here may therefore be widely conserved among different species.
Assuntos
Cromátides/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Replicação do DNA , Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Animais , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/antagonistas & inibidores , Proteínas Cromossômicas não Histona/química , Proteínas de Drosophila/antagonistas & inibidores , Proteínas de Drosophila/química , Humanos , Fase S , Xenopus/metabolismo , CoesinasRESUMO
Multiplexed and label-free mass spectrometry-based approaches with single-cell resolution have attributed surprising heterogeneity to presumed homogenous cell populations. Even though specialized experimental designs and instrumentation have demonstrated remarkable advances, the efficient sample preparation of single cells still lags. Here, we introduce the proteoCHIP, a universal option for single-cell proteomics sample preparation including multiplexed labeling up to 16-plex with high sensitivity and throughput. The automated processing using a commercial system combining single-cell isolation and picoliter dispensing, the cellenONE, reduces final sample volumes to low nanoliters submerged in a hexadecane layer simultaneously eliminating error-prone manual sample handling and overcoming evaporation. The specialized proteoCHIP design allows direct injection of single cells via a standard autosampler resulting in around 1500 protein groups per TMT10-plex with reduced or eliminated need for a carrier proteome. We evaluated the effect of wider precursor isolation windows at single-cell input levels and found that using 2 Da isolation windows increased overall sensitivity without significantly impacting interference. Using the dedicated mass spectrometry acquisition strategies detailed here, we identified on average close to 2000 proteins per TMT10-plex across 170 multiplexed single cells that readily distinguished human cell types. Overall, our workflow combines highly efficient sample preparation, chromatographic and ion mobility-based filtering, rapid wide-window data-dependent acquisition analysis, and intelligent data analysis for optimal multiplexed single-cell proteomics. This versatile and automated proteoCHIP-based sample preparation approach is sufficiently sensitive to drive biological applications of single-cell proteomics and can be readily adopted by proteomics laboratories.
Assuntos
Proteoma , Proteômica , Humanos , Proteômica/métodos , Fluxo de Trabalho , Espectrometria de Massas/métodos , Proteoma/metabolismoRESUMO
HOIP, the catalytic component of the linear ubiquitin chain assembly complex (LUBAC), is a critical regulator of inflammation. However, how HOIP itself is regulated to control inflammatory responses is unclear. Here, we discover that site-specific ubiquitination of K784 within human HOIP promotes tumor necrosis factor (TNF)-induced inflammatory signaling. A HOIP K784R mutant is catalytically active but shows reduced induction of an NF-κB reporter relative to wild-type HOIP. HOIP K784 is evolutionarily conserved, equivalent to HOIP K778 in mice. We generated HoipK778R/K778R knock-in mice, which show no overt developmental phenotypes; however, in response to TNF, HoipK778R/K778R mouse embryonic fibroblasts display mildly suppressed NF-κB activation and increased apoptotic markers. On the other hand, HOIP K778R enhances the TNF-induced formation of TNFR complex II and an interaction between TNFR complex II and LUBAC. Loss of the LUBAC component SHARPIN leads to embryonic lethality in HoipK778R/K778R mice, which is rescued by knockout of TNFR1. We propose that site-specific ubiquitination of HOIP regulates a LUBAC-dependent switch between survival and apoptosis in TNF signaling.
Assuntos
Apoptose/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/efeitos dos fármacos , Animais , Feminino , Técnicas de Introdução de Genes , Células HEK293 , Humanos , Masculino , Camundongos , NF-kappa B/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Receptores Tipo II do Fator de Necrose Tumoral , Transcriptoma , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/farmacologiaRESUMO
Somatic cells acclimate to changes in the environment by temporary reprogramming. Much has been learned about transcription factors that induce these cell-state switches in both plants and animals, but how cells rapidly modulate their proteome remains elusive. Here, we show rapid induction of autophagy during temporary reprogramming in plants triggered by phytohormones, immune, and danger signals. Quantitative proteomics following sequential reprogramming revealed that autophagy is required for timely decay of previous cellular states and for tweaking the proteome to acclimate to the new conditions. Signatures of previous cellular programs thus persist in autophagy-deficient cells, affecting cellular decision-making. Concordantly, autophagy-deficient cells fail to acclimatize to dynamic climate changes. Similarly, they have defects in dedifferentiating into pluripotent stem cells, and redifferentiation during organogenesis. These observations indicate that autophagy mediates cell-state switches that underlie somatic cell reprogramming in plants and possibly other organisms, and thereby promotes phenotypic plasticity.
Assuntos
Arabidopsis/fisiologia , Autofagia , Reprogramação Celular , Proteoma , Transdução de Sinais , Aclimatação , Arabidopsis/citologia , Arabidopsis/imunologia , Fenótipo , Reguladores de Crescimento de Plantas/metabolismo , ProteômicaRESUMO
Single-cell transcriptomics has revolutionized our understanding of basic biology and disease. Since transcript levels often do not correlate with protein expression, it is crucial to complement transcriptomics approaches with proteome analyses at single-cell resolution. Despite continuous technological improvements in sensitivity, mass-spectrometry-based single-cell proteomics ultimately faces the challenge of reproducibly comparing the protein expression profiles of thousands of individual cells. Here, we combine two hitherto opposing analytical strategies, DIA and Tandem-Mass-Tag (TMT)-multiplexing, to generate highly reproducible, quantitative proteome signatures from ultralow input samples. We developed a novel, identification-independent proteomics data-analysis pipeline that allows to quantitatively compare DIA-TMT proteome signatures across hundreds of samples independent of their biological origin to identify cell types and single protein knockouts. These proteome signatures overcome the need to impute quantitative data due to accumulating detrimental amounts of missing data in standard multibatch TMT experiments. We validate our approach using integrative data analysis of different human cell lines and standard database searches for knockouts of defined proteins. Our data establish a novel and reproducible approach to markedly expand the numbers of proteins one detects from ultralow input samples.
Assuntos
Proteoma , Espectrometria de Massas em Tandem , Linhagem Celular , Humanos , Processamento de Proteína Pós-Traducional , Proteoma/metabolismo , ProteômicaRESUMO
Post-transcriptional regulation of gene expression is crucial during the oocyte-to-embryo transition, a highly dynamic process characterized by the absence of nuclear transcription. Thus, changes to the RNA content are solely dependent on RNA degradation. Although several mechanisms that promote RNA decay during embryogenesis have been identified, it remains unclear which machineries contribute to remodeling the maternal transcriptome. Here, we focused on the degradation factor Ski7 in zebrafish. Homozygous ski7 mutant fish had higher proportions of both poor quality eggs and eggs that were unable to develop beyond the one-cell stage. Consistent with the idea that Ski7 participates in remodeling the maternal RNA content, transcriptome profiling identified hundreds of misregulated mRNAs in the absence of Ski7. Furthermore, upregulated genes were generally lowly expressed in wild type, suggesting that Ski7 maintains low transcript levels for this subset of genes. Finally, GO enrichment and proteomic analyses of misregulated factors implicated Ski7 in the regulation of redox processes. This was confirmed experimentally by an increased resistance of ski7 mutant embryos to reductive stress. Our results provide first insights into the physiological role of vertebrate Ski7 as a post-transcriptional regulator during the oocyte-to-embryo transition.
Assuntos
Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Oócitos/metabolismo , RNA/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Animais , Embrião não Mamífero/embriologia , Desenvolvimento Embrionário/genética , Exossomos/genética , Exossomos/metabolismo , Perfilação da Expressão Gênica/métodos , Mutação , Oócitos/citologia , Ligação Proteica , RNA/metabolismo , Estabilidade de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcriptoma/genética , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/metabolismoRESUMO
Multiple placental pathologies are associated with failures in trophoblast differentiation, yet the underlying transcriptional regulation is poorly understood. Here, we discovered msh homeobox 2 (MSX2) as a key transcriptional regulator of trophoblast identity using the human trophoblast stem cell model. Depletion of MSX2 resulted in activation of the syncytiotrophoblast transcriptional program, while forced expression of MSX2 blocked it. We demonstrated that a large proportion of the affected genes were directly bound and regulated by MSX2 and identified components of the SWItch/Sucrose nonfermentable (SWI/SNF) complex as strong MSX2 interactors and target gene cobinders. MSX2 cooperated specifically with the SWI/SNF canonical BAF (cBAF) subcomplex and cooccupied, together with H3K27ac, a number of differentiation genes. Increased H3K27ac and cBAF occupancy upon MSX2 depletion imply that MSX2 prevents premature syncytiotrophoblast differentiation. Our findings established MSX2 as a repressor of the syncytiotrophoblast lineage and demonstrated its pivotal role in cell fate decisions that govern human placental development and disease.
Assuntos
Diferenciação Celular , Proteínas Cromossômicas não Histona/metabolismo , Histonas/metabolismo , Proteínas de Homeodomínio/metabolismo , Placenta/citologia , Células-Tronco Pluripotentes/citologia , Fatores de Transcrição/metabolismo , Trofoblastos/citologia , Proteínas Cromossômicas não Histona/genética , Feminino , Histonas/genética , Proteínas de Homeodomínio/genética , Humanos , Placenta/metabolismo , Placentação , Células-Tronco Pluripotentes/metabolismo , Gravidez , Fatores de Transcrição/genética , Trofoblastos/metabolismoRESUMO
Selection of C-terminal motifs participated in evolution of distinct histone H2A variants. Hybrid types of variants combining motifs from distinct H2A classes are extremely rare. This suggests that the proximity between the motif cases interferes with their function. We studied this question in flowering plants that evolved sporadically a hybrid H2A variant combining the SQ motif of H2A.X that participates in the DNA damage response with the KSPK motif of H2A.W that stabilizes heterochromatin. Our inventory of PTMs of H2A.W variants showed that in vivo the cell cycle-dependent kinase CDKA phosphorylates the KSPK motif of H2A.W but only in absence of an SQ motif. Phosphomimicry of KSPK prevented DNA damage response by the SQ motif of the hybrid H2A.W/X variant. In a synthetic yeast expressing the hybrid H2A.W/X variant, phosphorylation of KSPK prevented binding of the BRCT-domain protein Mdb1 to phosphorylated SQ and impaired response to DNA damage. Our findings illustrate that PTMs mediate interference between the function of H2A variant specific C-terminal motifs. Such interference could explain the mutual exclusion of motifs that led to evolution of H2A variants.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Histonas/genética , Processamento de Proteína Pós-Traducional , Schizosaccharomyces/genética , Sequência de Aminoácidos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Técnicas de Cultura de Células , Ciclo Celular/genética , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Dano ao DNA , Evolução Molecular , Heterocromatina/química , Heterocromatina/metabolismo , Histonas/metabolismo , Fosforilação , Células Vegetais/metabolismo , Plantas Geneticamente Modificadas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , TransgenesRESUMO
The ability to map a proteomic fingerprint to transcriptomic data would master the understanding of how gene expression translates into actual phenotype. In contrast to nucleic acid sequencing, in vitro protein amplification is impossible and no single cell proteomic workflow has been established as gold standard yet. Advances in microfluidic sample preparation, multi-dimensional sample separation, sophisticated data acquisition strategies, and intelligent data analysis algorithms have resulted in major improvements to successfully analyze such tiny sample amounts with steadily boosted performance. However, among the broad variation of published approaches, it is commonly accepted that highest possible sensitivity, robustness, and throughput are still the most urgent needs for the field. While many labs have focused on multiplexing to achieve these goals, label-free SCP is a highly promising strategy as well whenever high dynamic range and unbiased accurate quantification are needed. We here focus on recent advances in label-free single-cell mass spectrometry workflows and try to guide our readers to choose the best method or combinations of methods for their specific applications. We further highlight which techniques are most propitious in the future and which applications but also limitations we foresee for the field.
Assuntos
Algoritmos , Proteômica , Proteômica/métodos , Espectrometria de Massas/métodos , Proteoma/análiseRESUMO
Spectral library search can enable more sensitive peptide identification in tandem mass spectrometry experiments. However, its drawbacks are the limited availability of high-quality libraries and the added difficulty of creating decoy spectra for result validation. We describe MS Ana, a new spectral library search engine that enables high sensitivity peptide identification using either curated or predicted spectral libraries as well as robust false discovery control through its own decoy library generation algorithm. MS Ana identifies on average 36% more spectrum matches and 4% more proteins than database search in a benchmark test on single-shot human cell-line data. Further, we demonstrate the quality of the result validation with tests on synthetic peptide pools and show the importance of library selection through a comparison of library search performance with different configurations of publicly available human spectral libraries.
Assuntos
Biblioteca de Peptídeos , Software , Humanos , Peptídeos/análise , Proteínas/química , Algoritmos , Bases de Dados de ProteínasRESUMO
Cross-linking mass spectrometry has become a powerful tool for the identification of protein-protein interactions and for gaining insight into the structures of proteins. We previously published MS Annika, a cross-linking search engine which can accurately identify cross-linked peptides in MS2 spectra from a variety of different MS-cleavable cross-linkers. In this publication, we present MS Annika 2.0, an updated version implementing a new search algorithm that, in addition to MS2 level, only supports the processing of data from MS2-MS3-based approaches for the identification of peptides from MS3 spectra, and introduces a novel scoring function for peptides identified across multiple MS stages. Detected cross-links are validated by estimating the false discovery rate (FDR) using a target-decoy approach. We evaluated the MS3-search-capabilities of MS Annika 2.0 on five different datasets covering a variety of experimental approaches and compared it to XlinkX and MaXLinker, two other cross-linking search engines. We show that MS Annika detects up to 4 times more true unique cross-links while simultaneously yielding less false positive hits and therefore a more accurate FDR estimation than the other two search engines. All mass spectrometry proteomics data along with result files have been deposited to the ProteomeXchange consortium via the PRIDE partner repository with the dataset identifier PXD041955.
Assuntos
Peptídeos , Ferramenta de Busca , Fluxo de Trabalho , Peptídeos/análise , Espectrometria de Massas/métodos , Ferramenta de Busca/métodos , Algoritmos , Reagentes de Ligações Cruzadas/químicaRESUMO
The analysis of ultralow input samples or even individual cells is essential to answering a multitude of biomedical questions, but current proteomic workflows are limited in their sensitivity and reproducibility. Here, we report a comprehensive workflow that includes improved strategies for all steps, from cell lysis to data analysis. Thanks to convenient-to-handle 1 µL sample volume and standardized 384-well plates, the workflow is easy for even novice users to implement. At the same time, it can be performed semi-automatized using CellenONE, which allows for the highest reproducibility. To achieve high throughput, ultrashort gradient lengths down to 5 min were tested using advanced µ-pillar columns. Data-dependent acquisition (DDA), wide-window acquisition (WWA), data-independent acquisition (DIA), and commonly used advanced data analysis algorithms were benchmarked. Using DDA, 1790 proteins covering a dynamic range of four orders of magnitude were identified in a single cell. Using DIA, proteome coverage increased to more than 2200 proteins identified from single-cell level input in a 20 min active gradient. The workflow enabled differentiation of two cell lines, demonstrating its suitability to cellular heterogeneity determination.
Assuntos
Proteoma , Proteômica , Fluxo de Trabalho , Reprodutibilidade dos Testes , Proteoma/análise , Linhagem CelularRESUMO
This work demonstrates the utility of high-throughput nanoLC-MS and label-free quantification (LFQ) for sample-limited bottom-up proteomics analysis, including single-cell proteomics (SCP). Conditions were optimized on a 50 µm internal diameter (I.D.) column operated at 100 nL/min in the direct injection workflow to balance method sensitivity and sample throughput from 24 to 72 samples/day. Multiple data acquisition strategies were also evaluated for proteome coverage, including data-dependent acquisition (DDA), wide-window acquisition (WWA), and wide-window data-independent acquisition (WW-DIA). Analyzing 250 pg HeLa digest with a 10-min LC gradient (72 samples/day) provided >900, >1,800, and >3,000 protein group identifications for DDA, WWA, and WW-DIA, respectively. Total method cycle time was further reduced from 20 to 14.4 min (100 samples/day) by employing a trap-and-elute workflow, enabling 70% mass spectrometer utilization. The method was applied to library-free DIA analysis of single-cell samples, yielding >1,700 protein groups identified. In conclusion, this study provides a high-sensitivity, high-throughput nanoLC-MS configuration for sample-limited proteomics.
Assuntos
Espectrometria de Massa com Cromatografia Líquida , Proteômica , Humanos , Proteômica/métodos , Cromatografia Líquida , Espectrometria de Massas em Tandem/métodos , Proteoma/análiseRESUMO
Proteomics research infrastructures and core facilities within the Core for Life alliance advocate for community policies for quality control to ensure high standards in proteomics services.