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.

Quantitative Proteomics of the Cancer Cell Line Encyclopedia.

Proteins are essential agents of biological processes. To date, large-scale profiling of cell line collections including the Cancer Cell Line Encyclopedia (CCLE) has focused primarily on genetic information whereas deep interrogation of the proteome has remained out of reach. Here, we expand the CCLE through quantitative profiling of thousands of proteins by mass spectrometry across 375 cell lines from diverse lineages to reveal information undiscovered by DNA and RNA methods. We observe unexpected correlations within and between pathways that are largely absent from RNA. An analysis of microsatellite instable (MSI) cell lines reveals the dysregulation of specific protein complexes associated with surveillance of mutation and translation. These and other protein complexes were associated with sensitivity to knockdown of several different genes. These data in conjunction with the wider CCLE are a broad resource to explore cellular behavior and facilitate cancer research.

A Quantitative Proteome Map of the Human Body.

Determining protein levels in each tissue and how they compare with RNA levels is important for understanding human biology and disease as well as regulatory processes that control protein levels. We quantified the relative protein levels from over 12,000 genes across 32 normal human tissues. Tissue-specific or tissue-enriched proteins were identified and compared to transcriptome data. Many ubiquitous transcripts are found to encode tissue-specific proteins. Discordance of RNA and protein enrichment revealed potential sites of synthesis and action of secreted proteins. The tissue-specific distribution of proteins also provides an in-depth view of complex biological events that require the interplay of multiple tissues. Most importantly, our study demonstrated that protein tissue-enrichment information can explain phenotypes of genetic diseases, which cannot be obtained by transcript information alone. Overall, our results demonstrate how understanding protein levels can provide insights into regulation, secretome, metabolism, and human diseases.

Horizontal Cell Biology: Monitoring Global Changes of Protein Interaction States with the Proteome-Wide Cellular Thermal Shift Assay (CETSA).

The cellular thermal shift assay (CETSA) is a biophysical technique allowing direct studies of ligand binding to proteins in cells and tissues. The proteome-wide implementation of CETSA with mass spectrometry detection (MS-CETSA) has now been successfully applied to discover targets for orphan clinical drugs and hits from phenotypic screens, to identify off-targets, and to explain poly-pharmacology and drug toxicity. Highly sensitive multidimensional MS-CETSA implementations can now also access binding of physiological ligands to proteins, such as metabolites, nucleic acids, and other proteins. MS-CETSA can thereby provide comprehensive information on modulations of protein interaction states in cellular processes, including downstream effects of drugs and transitions between different physiological cell states. Such horizontal information on ligandmodulation in cells is largely orthogonal to vertical information on the levels of different proteins and therefore opens novel opportunities to understand operational aspects of cellular proteomes.

Modulation of Protein-Interaction States through the Cell Cycle.

Global profiling of protein expression through the cell cycle has revealed subsets of periodically expressed proteins. However, expression levels alone only give a partial view of the biochemical processes determining cellular events. Using a proteome-wide implementation of the cellular thermal shift assay (CETSA) to study specific cell-cycle phases, we uncover changes of interaction states for more than 750 proteins during the cell cycle. Notably, many protein complexes are modulated in specific cell-cycle phases, reflecting their roles in processes such as DNA replication, chromatin remodeling, transcription, translation, and disintegration of the nuclear envelope. Surprisingly, only small differences in the interaction states were seen between the G1 and the G2 phase, suggesting similar hardwiring of biochemical processes in these two phases. The present work reveals novel molecular details of the cell cycle and establishes proteome-wide CETSA as a new strategy to study modulation of protein-interaction states in intact cells.

K6-linked ubiquitylation marks formaldehyde-induced RNA-protein crosslinks for resolution.

Reactive aldehydes are produced by normal cellular metabolism or after alcohol consumption, and they accumulate in human tissues if aldehyde clearance mechanisms are impaired. Their toxicity has been attributed to the damage they cause to genomic DNA and the subsequent inhibition of transcription and replication. However, whether interference with other cellular processes contributes to aldehyde toxicity has not been investigated. We demonstrate that formaldehyde induces RNA-protein crosslinks (RPCs) that stall the ribosome and inhibit translation in human cells. RPCs in the messenger RNA (mRNA) are recognized by the translating ribosomes, marked by atypical K6-linked ubiquitylation catalyzed by the RING-in-between-RING (RBR) E3 ligase RNF14, and subsequently resolved by the ubiquitin- and ATP-dependent unfoldase VCP. Our findings uncover an evolutionary conserved formaldehyde-induced stress response pathway that protects cells against RPC accumulation in the cytoplasm, and they suggest that RPCs contribute to the cellular and tissue toxicity of reactive aldehydes.

Dissecting the Structural Dynamics of the Nuclear Pore Complex.

Cellular processes are largely carried out by macromolecular assemblies, most of which are dynamic, having components that are in constant flux. One such assembly is the nuclear pore complex (NPC), an ∼50 MDa assembly comprised of ∼30 different proteins called Nups that mediates selective macromolecular transport between the nucleus and cytoplasm. We developed a proteomics method to provide a comprehensive picture of the yeast NPC component dynamics. We discovered that, although all Nups display uniformly slow turnover, their exchange rates vary considerably. Surprisingly, this exchange rate was relatively unrelated to each Nup's position, accessibility, or role in transport but correlated with its structural role; scaffold-forming Nups exchange slowly, whereas flexible connector Nups threading throughout the NPC architecture exchange more rapidly. Targeted perturbations in the NPC structure revealed a dynamic resilience to damage. Our approach opens a new window into macromolecular assembly dynamics.

Proteome-wide mapping of short-lived proteins in human cells.

Rapid protein degradation enables cells to quickly modulate protein abundance. Dysregulation of short-lived proteins plays essential roles in disease pathogenesis. A focused map of short-lived proteins remains understudied. Cycloheximide, a translational inhibitor, is widely used in targeted studies to measure degradation kinetics for short-lived proteins. Here, we combined cycloheximide chase assays with advanced quantitative proteomics to map short-lived proteins under translational inhibition in four human cell lines. Among 11,747 quantified proteins, we identified 1,017 short-lived proteins (half-lives ≤ 8 h). These short-lived proteins are less abundant, evolutionarily younger, and less thermally stable than other proteins. We quantified 103 proteins with different stabilities among cell lines. We showed that U2OS and HCT116 cells express truncated forms of ATRX and GMDS, respectively, which have lower stability than their full-length counterparts. This study provides a large-scale resource of human short-lived proteins under translational arrest, leading to untapped avenues of protein regulation for therapeutic interventions.

Global Landscape and Dynamics of Parkin and USP30-Dependent Ubiquitylomes in iNeurons during Mitophagic Signaling.

The ubiquitin ligase Parkin, protein kinase PINK1, USP30 deubiquitylase, and p97 segregase function together to regulate turnover of damaged mitochondria via mitophagy, but our mechanistic understanding in neurons is limited. Here, we combine induced neurons (iNeurons) derived from embryonic stem cells with quantitative proteomics to reveal the dynamics and specificity of Parkin-dependent ubiquitylation under endogenous expression conditions. Targets showing elevated ubiquitylation in USP30-/- iNeurons are concentrated in components of the mitochondrial translocon, and the ubiquitylation kinetics of the vast majority of Parkin targets are unaffected, correlating with a modest kinetic acceleration in accumulation of pS65-Ub and mitophagic flux upon mitochondrial depolarization without USP30. Basally, ubiquitylated translocon import substrates accumulate, suggesting a quality control function for USP30. p97 was dispensable for Parkin ligase activity in iNeurons. This work provides an unprecedented quantitative landscape of the Parkin-modified ubiquitylome in iNeurons and reveals the underlying specificity of central regulatory elements in the pathway.

PIKES Analysis Reveals Response to Degraders and Key Regulatory Mechanisms of the CRL4 Network.

Co-opting Cullin4 RING ubiquitin ligases (CRL4s) to inducibly degrade pathogenic proteins is emerging as a promising therapeutic strategy. Despite intense efforts to rationally design degrader molecules that co-opt CRL4s, much about the organization and regulation of these ligases remains elusive. Here, we establish protein interaction kinetics and estimation of stoichiometries (PIKES) analysis, a systematic proteomic profiling platform that integrates cellular engineering, affinity purification, chemical stabilization, and quantitative mass spectrometry to investigate the dynamics of interchangeable multiprotein complexes. Using PIKES, we show that ligase assemblies of Cullin4 with individual substrate receptors differ in abundance by up to 200-fold and that Cand1/2 act as substrate receptor exchange factors. Furthermore, degrader molecules can induce the assembly of their cognate CRL4, and higher expression of the associated substrate receptor enhances degrader potency. Beyond the CRL4 network, we show how PIKES can reveal systems level biochemistry for cellular protein networks important to drug development.

Light Changes Promote Distinct Responses of Plastid Protein Acetylation Marks.

Protein acetylation is a key co- and post-translational modification. However, how different types of acetylation respond to environmental stress is still unknown. To address this, we investigated the role of a member of the newly discovered family of plastid acetyltransferases (GNAT2), which features both lysine- and N-terminal acetyltransferase activities. Our study aimed to provide a holistic multi-omics acetylation-dependent view of plant acclimation to short-term light changes. We found that both the yield and coverage of the N-terminal acetylome remained unchanged in WT and gnat2-KO backgrounds after 2 h of exposure to high light or darkness. Similarly, no differences in transcriptome or adenylate energy charge were observed between the genotypes under the tested light conditions. In contrast, the lysine acetylome proved to be sensitive to the changes in light conditions, especially in the gnat2 background. This suggests unique strategies of plant acclimation for quick responses to environmental changes involving lysine, but not N-terminal, GNAT2-mediated acetylation activity.

Quantitative Proteomic Analysis Reveals Functional Alterations of the Peripheral Immune System in Colorectal Cancer.

Colorectal cancer (CRC) is characterized by high morbidity, high mortality, and limited response to immunotherapies. The peripheral immune system is an important component of tumor immunity, and enhancements of peripheral immunity help to suppress tumor progression. However, the functional alterations of the peripheral immune system in CRC are unclear. Here, we used mass spectrometry-based quantitative proteomics to establish a protein expression atlas for the peripheral immune system in CRC, including plasma and five types of immune cells (CD4+ T cells, CD8+ T cells, monocytes, natural killer cells, and B cells). Synthesizing the results of the multidimensional analysis, we observed an enhanced inflammatory phenotype in CRC, including elevated expression of plasma inflammatory proteins, activation of the inflammatory pathway in monocytes, and increased inflammation-related ligand-receptor interactions. Notably, we observed tumor effects on peripheral T cells, including altered cell subpopulation ratios and suppression of cell function. Suppression of CD4+ T cell function is mainly mediated by high expression levels of protein tyrosine phosphatases. Among them, the expression of protein tyrosine phosphatase receptor type J (PTPRJ) gradually increased with CRC progression; knockdown of PTPRJ in vitro could promote T cell activation, thereby enhancing peripheral immunity. We also found that the combination of leucine-rich α-2 glycoprotein 1 (LRG1) and apolipoprotein A4 (APOA4) had the best predictive ability for colorectal cancer and has the potential to be a biomarker. Overall, this study provides a comprehensive understanding of the peripheral immune system in CRC. It also offers insights regarding the potential clinical utilities of these peripheral immune characteristics as diagnostic indicators and therapeutic targets.

Derailed protein turnover in the aging mammalian brain.

Efficient protein turnover is essential for cellular homeostasis and organ function. Loss of proteostasis is a hallmark of aging culminating in severe dysfunction of protein turnover. To investigate protein turnover dynamics as a function of age, we performed continuous in vivo metabolic stable isotope labeling in mice along the aging continuum. First, we discovered that the brain proteome uniquely undergoes dynamic turnover fluctuations during aging compared to heart and liver tissue. Second, trends in protein turnover in the brain proteome during aging showed sex-specific differences that were tightly tied to cellular compartments. Next, parallel analyses of the insoluble proteome revealed that several cellular compartments experience hampered turnover, in part due to misfolding. Finally, we found that age-associated fluctuations in proteasome activity were associated with the turnover of core proteolytic subunits, which was recapitulated by pharmacological suppression of proteasome activity. Taken together, our study provides a proteome-wide atlas of protein turnover across the aging continuum and reveals a link between the turnover of individual proteasome subunits and the age-associated decline in proteasome activity.

Proteomic dissection of vanishing white matter pathogenesis.

Vanishing white matter (VWM) is a leukodystrophy caused by biallelic pathogenic variants in eukaryotic translation initiation factor 2B. To date, it remains unclear which factors contribute to VWM pathogenesis. Here, we investigated the basis of VWM pathogenesis using the 2b5ho mouse model. We first mapped the temporal proteome in the cerebellum, corpus callosum, cortex, and brainstem of 2b5ho and wild-type (WT) mice. Protein changes observed in 2b5ho mice were then cross-referenced with published proteomic datasets from VWM patient brain tissue to define alterations relevant to the human disease. By comparing 2b5ho mice with their region- and age-matched WT counterparts, we showed that the proteome in the cerebellum and cortex of 2b5ho mice was already dysregulated prior to pathology development, whereas proteome changes in the corpus callosum only occurred after pathology onset. Remarkably, protein changes in the brainstem were transient, indicating that a compensatory mechanism might occur in this region. Importantly, 2b5ho mouse brain proteome changes reflect features well-known in VWM. Comparison of the 2b5ho mouse and VWM patient brain proteomes revealed shared changes. These could represent changes that contribute to the disease or even drive its progression in patients. Taken together, we show that the 2b5ho mouse brain proteome is affected in a region- and time-dependent manner. We found that the 2b5ho mouse model partly replicates the human disease at the protein level, providing a resource to study aspects of VWM pathogenesis by highlighting alterations from early to late disease stages, and those that possibly drive disease progression.

Evolutionary Quantitative Proteomics of Reproductive Protein Divergence in Drosophila.

Reproductive traits often evolve rapidly between species. Understanding the causes and consequences of this rapid divergence requires characterization of female and male reproductive proteins and their effect on fertilization success. Species in the Drosophila virilis clade exhibit rampant interspecific reproductive incompatibilities, making them ideal for studies on diversification of reproductive proteins and their role in speciation. Importantly, the role of intraejaculate protein abundance and allocation in interspecific divergence is poorly understood. Here, we identify and quantify the transferred male ejaculate proteome using multiplexed isobaric labeling of the lower female reproductive tract before and immediately after mating using three species of the virilis group. We identified over 200 putative male ejaculate proteins, many of which show differential abundance between species, suggesting that males transfer a species-specific allocation of seminal fluid proteins during copulation. We also identified over 2000 female reproductive proteins, which contain female-specific serine-type endopeptidases that showed differential abundance between species and elevated rates of molecular evolution, similar to that of some male seminal fluid proteins. Our findings suggest that reproductive protein divergence can also manifest in terms of species-specific protein abundance patterns.

Proteomic Analysis of Huntington's Disease Medium Spiny Neurons Identifies Alterations in Lipid Droplets.

Huntington's disease (HD) is a neurodegenerative disease caused by a CAG repeat expansion in the Huntingtin (HTT) gene. The resulting polyglutamine (polyQ) tract alters the function of the HTT protein. Although HTT is expressed in different tissues, the medium-spiny projection neurons (MSNs) in the striatum are particularly vulnerable in HD. Thus, we sought to define the proteome of human HD patient-derived MSNs. We differentiated HD72-induced pluripotent stem cells and isogenic controls into MSNs and carried out quantitative proteomic analysis. Using data-dependent acquisitions with FAIMS for label-free quantification on the Orbitrap Lumos mass spectrometer, we identified 6323 proteins with at least two unique peptides. Of these, 901 proteins were altered significantly more in the HD72-MSNs than in isogenic controls. Functional enrichment analysis of upregulated proteins demonstrated extracellular matrix and DNA signaling (DNA replication pathway, double-strand break repair, G1/S transition) with the highest significance. Conversely, processes associated with the downregulated proteins included neurogenesis-axogenesis, the brain-derived neurotrophic factor-signaling pathway, Ephrin-A:EphA pathway, regulation of synaptic plasticity, triglyceride homeostasis cholesterol, plasmid lipoprotein particle immune response, interferon-γ signaling, immune system major histocompatibility complex, lipid metabolism, and cellular response to stimulus. Moreover, proteins involved in the formation and maintenance of axons, dendrites, and synapses (e.g., septin protein members) were dysregulated in HD72-MSNs. Importantly, lipid metabolism pathways were altered, and using quantitative image analysis, we found that lipid droplets accumulated in the HD72-MSN, suggesting a deficit in the turnover of lipids possibly through lipophagy. Our proteomics analysis of HD72-MSNs identified relevant pathways that are altered in MSNs and confirm current and new therapeutic targets for HD.

Effects of pejus and pessimum zone salinity stress on gill proteome networks and energy homeostasis in Oreochromis mossambicus.

Salinity tolerance in fish involves a suite of physiological changes, but a cohesive theory leading to a mechanistic understanding at the organismal level is lacking. To examine the potential of adapting energy homeostasis theory in the context of salinity stress in teleost fish, Oreochromis mossambicus were acclimated to hypersalinity at multiple rates and durations to determine salinity ranges of tolerance and resistance. Over 3000 proteins were quantified simultaneously to analyze molecular phenotypes associated with hypersalinity. A species- and tissue-specific data-independent acquisition (DIA) assay library of MSMS spectra was created. Protein networks representing complex molecular phenotypes associated with salinity acclimation were generated. O. mossambicus has a wide "zone of resistance" from 75 g/kg salinity to 120 g/kg. Crossing into the zone of resistance resulted in marked phenotypic changes including blood osmolality over 400 mOsm/kg, reduced body condition, and cessation of feeding. Protein networks impacted by hypersalinity consist of electron transport chain (ETC) proteins and specific osmoregulatory proteins. Cytoskeletal, cell adhesion, and extracellular matrix proteins are enriched in networks that are sensitive to the critical salinity threshold. These network analyses identify specific proteome changes that are associated with distinct zones described by energy homeostasis theory and distinguish them from general hypersalinity-induced proteome changes.

Evaluation of a Proteomics-Guided Protein Signature for Breast Cancer Detection in Breast Tissue.

The distinction between noncancerous and cancerous breast tissues is challenging in clinical settings, and discovering new proteomics-based biomarkers remains underexplored. Through a pilot proteomic study (discovery cohort), we aimed to identify a protein signature indicative of breast cancer for subsequent validation using six published proteomics/transcriptomics data sets (validation cohorts). Sequential window acquisition of all theoretical (SWATH)-based mass spectrometry revealed 370 differentially abundant proteins between noncancerous tissue and breast cancer. Protein-protein interaction-based networks and enrichment analyses revealed dysregulation in pathways associated with extracellular matrix organization, platelet degranulation, the innate immune system, and RNA metabolism in breast cancer. Through multivariate unsupervised analysis, we identified a four-protein signature (OGN, LUM, DCN, and COL14A1) capable of distinguishing breast cancer. This dysregulation pattern was consistently verified across diverse proteomics and transcriptomics data sets. Dysregulation magnitude was notably higher in poor-prognosis breast cancer subtypes like Basal-Like and HER2 compared to Luminal A. Diagnostic evaluation (receiver operating characteristic (ROC) curves) of the signature in distinguishing breast cancer from noncancerous tissue revealed area under the curve (AUC) ranging from 0.87 to 0.9 with predictive accuracy of 80% to 82%. Upon stratifying, to solely include the Basal-Like/Triple-Negative subtype, the ROC AUC increased to 0.922-0.959 with predictive accuracy of 84.2%-89%. These findings suggest a potential role for the identified signature in distinguishing cancerous from noncancerous breast tissue, offering insights into enhancing diagnostic accuracy.

LFQRatio: A Normalization Method to Decipher Quantitative Proteome Changes in Microbial Coculture Systems.

The value of synthetic microbial communities in biotechnology is gaining traction due to their ability to undertake more complex metabolic tasks than monocultures. However, a thorough understanding of strain interactions, productivity, and stability is often required to optimize growth and scale up cultivation. Quantitative proteomics can provide valuable insights into how microbial strains adapt to changing conditions in biomanufacturing. However, current workflows and methodologies are not suitable for simple artificial coculture systems where strain ratios are dynamic. Here, we established a workflow for coculture proteomics using an exemplar system containing two members, Azotobacter vinelandii and Synechococcus elongatus. Factors affecting the quantitative accuracy of coculture proteomics were investigated, including peptide physicochemical characteristics such as molecular weight, isoelectric point, hydrophobicity, and dynamic range as well as factors relating to protein identification such as varying proteome size and shared peptides between species. Different quantification methods based on spectral counts and intensity were evaluated at the protein and cell level. We propose a new normalization method, named "LFQRatio", to reflect the relative contributions of two distinct cell types emerging from cell ratio changes during cocultivation. LFQRatio can be applied to real coculture proteomics experiments, providing accurate insights into quantitative proteome changes in each strain.

Proteomic Evolution from Acute to Post-COVID-19 Conditions.

Many COVID-19 survivors have post-COVID-19 conditions, and females are at a higher risk. We sought to determine (1) how protein levels change from acute to post-COVID-19 conditions, (2) whether females have a plasma protein signature different from that of males, and (3) which biological pathways are associated with COVID-19 when compared to restrictive lung disease. We measured protein levels in 74 patients on the day of admission and at 3 and 6 months after diagnosis. We determined protein concentrations by multiple reaction monitoring (MRM) using a panel of 269 heavy-labeled peptides. The predicted forced vital capacity (FVC) and diffusing capacity of the lungs for carbon monoxide (DLCO) were measured by routine pulmonary function testing. Proteins associated with six key lipid-related pathways increased from admission to 3 and 6 months; conversely, proteins related to innate immune responses and vasoconstriction-related proteins decreased. Multiple biological functions were regulated differentially between females and males. Concentrations of eight proteins were associated with FVC, %, and they together had c-statistics of 0.751 (CI:0.732-0.779); similarly, concentrations of five proteins had c-statistics of 0.707 (CI:0.676-0.737) for DLCO, %. Lipid biology may drive evolution from acute to post-COVID-19 conditions, while activation of innate immunity and vascular regulation pathways decreased over that period. (ProteomeXchange identifiers: PXD041762, PXD029437).