The proteomic landscape of genome-wide genetic perturbations.

Functional genomic strategies have become fundamental for annotating gene function and regulatory networks. Here, we combined functional genomics with proteomics by quantifying protein abundances in a genome-scale knockout library in Saccharomyces cerevisiae, using data-independent acquisition mass spectrometry. We find that global protein expression is driven by a complex interplay of (1) general biological properties, including translation rate, protein turnover, the formation of protein complexes, growth rate, and genome architecture, followed by (2) functional properties, such as the connectivity of a protein in genetic, metabolic, and physical interaction networks. Moreover, we show that functional proteomics complements current gene annotation strategies through the assessment of proteome profile similarity, protein covariation, and reverse proteome profiling. Thus, our study reveals principles that govern protein expression and provides a genome-spanning resource for functional annotation.

Maturation Kinetics of a Multiprotein Complex Revealed by Metabolic Labeling.

All proteins interact with other cellular components to fulfill their function. While tremendous progress has been made in the identification of protein complexes, their assembly and dynamics remain difficult to characterize. Here, we present a high-throughput strategy to analyze the native assembly kinetics of protein complexes. We apply our approach to characterize the co-assembly for 320 pairs of nucleoporins (NUPs) constituting the ≈50 MDa nuclear pore complex (NPC) in yeast. Some NUPs co-assemble fast via rapid exchange whereas others require lengthy maturation steps. This reveals a hierarchical principle of NPC biogenesis where individual subcomplexes form on a minute timescale and then co-assemble from center to periphery in a ∼1 h-long maturation process. Intriguingly, the NUP Mlp1 stands out as joining very late and associating preferentially with aged NPCs. Our approach is readily applicable beyond the NPC, making it possible to analyze the intracellular dynamics of a variety of multiprotein assemblies.

Assembly and Translocation of a CRISPR-Cas Primed Acquisition Complex.

CRISPR-Cas systems confer an adaptive immunity against viruses. Following viral injection, Cas1-Cas2 integrates segments of the viral genome (spacers) into the CRISPR locus. In type I CRISPR-Cas systems, efficient "primed" spacer acquisition and viral degradation (interference) require both the Cascade complex and the Cas3 helicase/nuclease. Here, we present single-molecule characterization of the Thermobifida fusca (Tfu) primed acquisition complex (PAC). We show that TfuCascade rapidly samples non-specific DNA via facilitated one-dimensional diffusion. Cas3 loads at target-bound Cascade and the Cascade/Cas3 complex translocates via a looped DNA intermediate. Cascade/Cas3 complexes stall at diverse protein roadblocks, resulting in a double strand break at the stall site. In contrast, Cas1-Cas2 samples DNA transiently via 3D collisions. Moreover, Cas1-Cas2 associates with Cascade and translocates with Cascade/Cas3, forming the PAC. PACs can displace different protein roadblocks, suggesting a mechanism for long-range spacer acquisition. This work provides a molecular basis for the coordinated steps in CRISPR-based adaptive immunity.

Cleavage of viral DNA by restriction endonucleases stimulates the type II CRISPR-Cas immune response.

Prokaryotic organisms have developed multiple defense systems against phages; however, little is known about whether and how these interact with each other. Here, we studied the connection between two of the most prominent prokaryotic immune systems: restriction-modification and CRISPR. While both systems employ enzymes that cleave a specific DNA sequence of the invader, CRISPR nucleases are programmed with phage-derived spacer sequences, which are integrated into the CRISPR locus upon infection. We found that restriction endonucleases provide a short-term defense, which is rapidly overcome through methylation of the phage genome. In a small fraction of the cells, however, restriction results in the acquisition of spacer sequences from the cleavage site, which mediates a robust type II-A CRISPR-Cas immune response against the methylated phage. This mechanism is reminiscent of eukaryotic immunity in which the innate response offers a first temporary line of defense and also activates a second and more robust adaptive response.

Efflux-linked accelerated evolution of antibiotic resistance at a population edge.

Efflux is a common mechanism of resistance to antibiotics. We show that efflux itself promotes accumulation of antibiotic-resistance mutations (ARMs). This phenomenon was initially discovered in a bacterial swarm where the linked phenotypes of high efflux and high mutation frequencies spatially segregated to the edge, driven there by motility. We have uncovered and validated a global regulatory network connecting high efflux to downregulation of specific DNA-repair pathways even in non-swarming states. The efflux-DNA repair link was corroborated in a clinical "resistome" database: genomes with mutations that increase efflux exhibit a significant increase in ARMs. Accordingly, efflux inhibitors decreased evolvability to antibiotic resistance. Swarms also revealed how bacterial populations serve as a reservoir of ARMs even in the absence of antibiotic selection pressure. High efflux at the edge births mutants that, despite compromised fitness, survive there because of reduced competition. This finding is relevant to biofilms where efflux activity is high.

PAM binding ensures orientational integration during Cas4-Cas1-Cas2-mediated CRISPR adaptation.

Adaptation in CRISPR-Cas systems immunizes bacteria and archaea against mobile genetic elements. In many DNA-targeting systems, the Cas4-Cas1-Cas2 complex is required for selection and processing of DNA segments containing PAM sequences prior to integration of these "prespacer" substrates as spacers in the CRISPR array. We determined cryo-EM structures of the Cas4-Cas1-Cas2 adaptation complex from the type I-C system that encodes standalone Cas1 and Cas4 proteins. The structures reveal how Cas4 specifically reads out bases within the PAM sequence and how interactions with both Cas1 and Cas2 activate Cas4 endonuclease activity. The Cas4-PAM interaction ensures tight binding between the adaptation complex and the prespacer, significantly enhancing integration of the non-PAM end into the CRISPR array and ensuring correct spacer orientation. Corroborated with our biochemical results, Cas4-Cas1-Cas2 structures with substrates representing various stages of CRISPR adaptation reveal a temporally resolved mechanism for maturation and integration of functional spacers into the CRISPR array.

Molecular Mechanisms of CRISPR-Cas Immunity in Bacteria.

Prokaryotes have developed numerous defense strategies to combat the constant threat posed by the diverse genetic parasites that endanger them. Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas loci guard their hosts with an adaptive immune system against foreign nucleic acids. Protection starts with an immunization phase, in which short pieces of the invader's genome, known as spacers, are captured and integrated into the CRISPR locus after infection. Next, during the targeting phase, spacers are transcribed into CRISPR RNAs (crRNAs) that guide CRISPR-associated (Cas) nucleases to destroy the invader's DNA or RNA. Here we describe the many different molecular mechanisms of CRISPR targeting and how they are interconnected with the immunization phase through a third phase of the CRISPR-Cas immune response: primed spacer acquisition. In this phase, Cas proteins direct the crRNA-guided acquisition of additional spacers to achieve a more rapid and robust immunization of the population.

Molecular Connectivity of Mitochondrial Gene Expression and OXPHOS Biogenesis.

Mitochondria contain their own gene expression systems, including membrane-bound ribosomes dedicated to synthesizing a few hydrophobic subunits of the oxidative phosphorylation (OXPHOS) complexes. We used a proximity-dependent biotinylation technique, BioID, coupled with mass spectrometry to delineate in baker's yeast a comprehensive network of factors involved in biogenesis of mitochondrial encoded proteins. This mitochondrial gene expression network (MiGENet) encompasses proteins involved in transcription, RNA processing, translation, or protein biogenesis. Our analyses indicate the spatial organization of these processes, thereby revealing basic mechanistic principles and the proteins populating strategically important sites. For example, newly synthesized proteins are directly handed over to ribosomal tunnel exit-bound factors that mediate membrane insertion, co-factor acquisition, or their mounting into OXPHOS complexes in a special early assembly hub. Collectively, the data reveal the connectivity of mitochondrial gene expression, reflecting a unique tailoring of the mitochondrial gene expression system.

A Functional Mini-Integrase in a Two-Protein-type V-C CRISPR System.

CRISPR-Cas immunity requires integration of short, foreign DNA fragments into the host genome at the CRISPR locus, a site consisting of alternating repeat sequences and foreign-derived spacers. In most CRISPR systems, the proteins Cas1 and Cas2 form the integration complex and are both essential for DNA acquisition. Most type V-C and V-D systems lack the cas2 gene and have unusually short CRISPR repeats and spacers. Here, we show that a mini-integrase comprising the type V-C Cas1 protein alone catalyzes DNA integration with a preference for short (17- to 19-base-pair) DNA fragments. The mini-integrase has weak specificity for the CRISPR array. We present evidence that the Cas1 proteins form a tetramer for integration. Our findings support a model of a minimal integrase with an internal ruler mechanism that favors shorter repeats and spacers. This minimal integrase may represent the function of the ancestral Cas1 prior to Cas2 adoption.

Babbling opens the sensory phase for imitative vocal learning.

Zebra finches, a species of songbirds, learn to sing by creating an auditory template through the memorization of model songs (sensory learning phase) and subsequently translating these perceptual memories into motor skills (sensorimotor learning phase). It has been traditionally believed that babbling in juvenile birds initiates the sensorimotor phase while the sensory phase of song learning precedes the onset of babbling. However, our findings challenge this notion by demonstrating that testosterone-induced premature babbling actually triggers the onset of the sensory learning phase instead. We reveal that juvenile birds must engage in babbling and self-listening to acquire the tutor song as the template. Notably, the sensory learning of the template in songbirds requires motor vocal activity, reflecting the observation that prelinguistic babbling in humans plays a crucial role in auditory learning for language acquisition.

Leader-follower dynamics during early social interactions matter for infant word learning.

We know little about the mechanisms through which leader-follower dynamics during dyadic play shape infants' language acquisition. We hypothesized that infants' decisions to visually explore a specific object signal focal increases in endogenous attention, and that when caregivers respond to these proactive behaviors by naming the object it boosts infants' word learning. To examine this, we invited caregivers and their 14-mo-old infants to play with novel objects, before testing infants' retention of the novel object-label mappings. Meanwhile, their electroencephalograms were recorded. Results showed that infants' proactive looks toward an object during play associated with greater neural signatures of endogenous attention. Furthermore, when caregivers named objects during these episodes, infants showed greater word learning, but only when caregivers also joined their focus of attention. Our findings support the idea that infants' proactive visual explorations guide their acquisition of a lexicon.

Fragment correlation mass spectrometry: Determining the structures of biopolymers in a complex mixture without isolating individual components.

Fragment correlation mass spectrometry correlates ion pairs generated from the same fragmentation pathway, achieved by covariance mapping of tandem mass spectra generated with an unmodified linear ion trap without preseparation. We enable the identification of different precursors at different charge states in a complex mixture from a large isolation window, empowering an analytical approach for data-independent acquisition. The method resolves and matches isobaric fragments, internal ions, and disulfide bond fragments. We suggest that this method represents a major advance for analyzing structures of biopolymers in mixtures.

How cognitive selection affects language change.

Like biological species, words in language must compete to survive. Previously, it has been shown that language changes in response to cognitive constraints and over time becomes more learnable. Here, we use two complementary research paradigms to demonstrate how the survival of existing word forms can be predicted by psycholinguistic properties that impact language production. In the first study, we analyzed the survival of words in the context of interpersonal communication. We analyzed data from a large-scale serial-reproduction experiment in which stories were passed down along a transmission chain over multiple participants. The results show that words that are acquired earlier in life, more concrete, more arousing, and more emotional are more likely to survive retellings. We reason that the same trend might scale up to language evolution over multiple generations of natural language users. If that is the case, the same set of psycholinguistic properties should also account for the change of word frequency in natural language corpora over historical time. That is what we found in two large historical-language corpora (Study 2): Early acquisition, concreteness, and high arousal all predict increasing word frequency over the past 200 y. However, the two studies diverge with respect to the impact of word valence and word length, which we take up in the discussion. By bridging micro-level behavioral preferences and macro-level language patterns, our investigation sheds light on the cognitive mechanisms underlying word competition.

Acquisition of the spindle assembly checkpoint and its modulation by cell fate and cell size in a chordate embryo.

The spindle assembly checkpoint (SAC) is a surveillance system that preserves genome integrity by delaying anaphase onset until all chromosomes are correctly attached to spindle microtubules. Recruitment of SAC proteins to unattached kinetochores generates an inhibitory signal that prolongs mitotic duration. Chordate embryos are atypical in that spindle defects do not delay mitotic progression during early development, implying that either the SAC is inactive or the cell-cycle target machinery is unresponsive. Here, we show that in embryos of the chordate Phallusia mammillata, the SAC delays mitotic progression from the 8th cleavage divisions. Unattached kinetochores are not recognized by the SAC machinery until the 7th cell cycle, when the SAC is acquired. After acquisition, SAC strength, which manifests as the degree of mitotic lengthening induced by spindle perturbations, is specific to different cell types and is modulated by cell size, showing similarity to SAC control in early Caenorhabditis elegans embryos. We conclude that SAC acquisition is a process that is likely specific to chordate embryos, while modulation of SAC efficiency in SAC proficient stages depends on cell fate and cell size, which is similar to non-chordate embryos.

Evidence implicating sequential commitment of the founder lineages in the human blastocyst by order of hypoblast gene activation.

Successful human pregnancy depends upon rapid establishment of three founder lineages: the trophectoderm, epiblast and hypoblast, which together form the blastocyst. Each plays an essential role in preparing the embryo for implantation and subsequent development. Several models have been proposed to define the lineage segregation. One suggests that all lineages specify simultaneously; another favours the differentiation of the trophectoderm before separation of the epiblast and hypoblast, either via differentiation of the hypoblast from the established epiblast, or production of both tissues from the inner cell mass precursor. To begin to resolve this discrepancy and thereby understand the sequential process for production of viable human embryos, we investigated the expression order of genes associated with emergence of hypoblast. Based upon published data and immunofluorescence analysis for candidate genes, we present a basic blueprint for human hypoblast differentiation, lending support to the proposed model of sequential segregation of the founder lineages of the human blastocyst. The first characterised marker, specific initially to the early inner cell mass, and subsequently identifying presumptive hypoblast, is PDGFRA, followed by SOX17, FOXA2 and GATA4 in sequence as the hypoblast becomes committed.

Integrative open workflow for confident annotation and molecular networking of metabolomics MSE/DIA data.

Liquid chromatography coupled with high-resolution mass spectrometry data-independent acquisition (LC-HRMS/DIA), including MSE, enable comprehensive metabolomics analyses though they pose challenges for data processing with automatic annotation and molecular networking (MN) implementation. This motivated the present proposal, in which we introduce DIA-IntOpenStream, a new integrated workflow combining open-source software to streamline MSE data handling. It provides 'in-house' custom database construction, allows the conversion of raw MSE data to a universal format (.mzML) and leverages open software (MZmine 3 and MS-DIAL) all advantages for confident annotation and effective MN data interpretation. This pipeline significantly enhances the accessibility, reliability and reproducibility of complex MSE/DIA studies, overcoming previous limitations of proprietary software and non-universal MS data formats that restricted integrative analysis. We demonstrate the utility of DIA-IntOpenStream with two independent datasets: dataset 1 consists of new data from 60 plant extracts from the Ocotea genus; dataset 2 is a publicly available actinobacterial extract spiked with authentic standard for detailed comparative analysis with existing methods. This user-friendly pipeline enables broader adoption of cutting-edge MS tools and provides value to the scientific community. Overall, it holds promise for speeding up metabolite discoveries toward a more collaborative and open environment for research.

Lactobacillus-Deficient Cervicovaginal Bacterial Communities Are Associated with Increased HIV Acquisition in Young South African Women.

Elevated inflammation in the female genital tract is associated with increased HIV risk. Cervicovaginal bacteria modulate genital inflammation; however, their role in HIV susceptibility has not been elucidated. In a prospective cohort of young, healthy South African women, we found that individuals with diverse genital bacterial communities dominated by anaerobes other than Gardnerella were at over 4-fold higher risk of acquiring HIV and had increased numbers of activated mucosal CD4+ T cells compared to those with Lactobacillus crispatus-dominant communities. We identified specific bacterial taxa linked with reduced (L. crispatus) or elevated (Prevotella, Sneathia, and other anaerobes) inflammation and HIV infection and found that high-risk bacteria increased numbers of activated genital CD4+ T cells in a murine model. Our results suggest that highly prevalent genital bacteria increase HIV risk by inducing mucosal HIV target cells. These findings might be leveraged to reduce HIV acquisition in women living in sub-Saharan Africa.

Detection of Pancreatic Ductal Adenocarcinoma-Associated Proteins in Serum.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancer types, partly because it is frequently identified at an advanced stage, when surgery is no longer feasible. Therefore, early detection using minimally invasive methods such as blood tests may improve outcomes. However, studies to discover molecular signatures for the early detection of PDAC using blood tests have only been marginally successful. In the current study, a quantitative glycoproteomic approach via data-independent acquisition mass spectrometry was utilized to detect glycoproteins in 29 patient-matched PDAC tissues and sera. A total of 892 N-linked glycopeptides originating from 141 glycoproteins had PDAC-associated changes beyond normal variation. We further evaluated the specificity of these serum-detectable glycoproteins by comparing their abundance in 53 independent PDAC patient sera and 65 cancer-free controls. The PDAC tissue-associated glycoproteins we have identified represent an inventory of serum-detectable PDAC-associated glycoproteins as candidate biomarkers that can be potentially used for the detection of PDAC using blood tests.

Full Mass Range ΦSDM Orbitrap Mass Spectrometry for DIA Proteome Analysis.

Optimizing data-independent acquisition methods for proteomics applications often requires balancing spectral resolution and acquisition speed. Here, we describe a real-time full mass range implementation of the phase-constrained spectrum deconvolution method (ΦSDM) for Orbitrap mass spectrometry that increases mass resolving power without increasing scan time. Comparing its performance to the standard enhanced Fourier transformation signal processing revealed that the increased resolving power of ΦSDM is beneficial in areas of high peptide density and comes with a greater ability to resolve low-abundance signals. In a standard 2 h analysis of a 200 ng HeLa digest, this resulted in an increase of 16% in the number of quantified peptides. As the acquisition speed becomes even more important when using fast chromatographic gradients, we further applied ΦSDM methods to a range of shorter gradient lengths (21, 12, and 5 min). While ΦSDM improved identification rates and spectral quality in all tested gradients, it proved particularly advantageous for the 5 min gradient. Here, the number of identified protein groups and peptides increased by >15% in comparison to enhanced Fourier transformation processing. In conclusion, ΦSDM is an alternative signal processing algorithm for processing Orbitrap data that can improve spectral quality and benefit quantitative accuracy in typical proteomics experiments, especially when using short gradients.

Data-Independent Acquisition: A Milestone and Prospect in Clinical Mass Spectrometry-Based Proteomics.

Data-independent acquisition (DIA) has revolutionized the field of mass spectrometry (MS)-based proteomics over the past few years. DIA stands out for its ability to systematically sample all peptides in a given m/z range, allowing an unbiased acquisition of proteomics data. This greatly mitigates the issue of missing values and significantly enhances quantitative accuracy, precision, and reproducibility compared to many traditional methods. This review focuses on the critical role of DIA analysis software tools, primarily focusing on their capabilities and the challenges they address in proteomic research. Advances in MS technology, such as trapped ion mobility spectrometry, or high field asymmetric waveform ion mobility spectrometry require sophisticated analysis software capable of handling the increased data complexity and exploiting the full potential of DIA. We identify and critically evaluate leading software tools in the DIA landscape, discussing their unique features, and the reliability of their quantitative and qualitative outputs. We present the biological and clinical relevance of DIA-MS and discuss crucial publications that paved the way for in-depth proteomic characterization in patient-derived specimens. Furthermore, we provide a perspective on emerging trends in clinical applications and present upcoming challenges including standardization and certification of MS-based acquisition strategies in molecular diagnostics. While we emphasize the need for continuous development of software tools to keep pace with evolving technologies, we advise researchers against uncritically accepting the results from DIA software tools. Each tool may have its own biases, and some may not be as sensitive or reliable as others. Our overarching recommendation for both researchers and clinicians is to employ multiple DIA analysis tools, utilizing orthogonal analysis approaches to enhance the robustness and reliability of their findings.