FAM81A is a postsynaptic protein that regulates the condensation of postsynaptic proteins via liquid-liquid phase separation.

Proteome analyses of the postsynaptic density (PSD), a proteinaceous specialization beneath the postsynaptic membrane of excitatory synapses, have identified several thousands of proteins. While proteins with predictable functions have been well studied, functionally uncharacterized proteins are mostly overlooked. In this study, we conducted a comprehensive meta-analysis of 35 PSD proteome datasets, encompassing a total of 5,869 proteins. Employing a ranking methodology, we identified 97 proteins that remain inadequately characterized. From this selection, we focused our detailed analysis on the highest-ranked protein, FAM81A. FAM81A interacts with PSD proteins, including PSD-95, SynGAP, and NMDA receptors, and promotes liquid-liquid phase separation of those proteins in cultured cells or in vitro. Down-regulation of FAM81A in cultured neurons causes a decrease in the size of PSD-95 puncta and the frequency of neuronal firing. Our findings suggest that FAM81A plays a crucial role in facilitating the interaction and assembly of proteins within the PSD, and its presence is important for maintaining normal synaptic function. Additionally, our methodology underscores the necessity for further characterization of numerous synaptic proteins that still lack comprehensive understanding.

The extracellular matrix supports breast cancer cell growth under amino acid starvation by promoting tyrosine catabolism.

Breast tumours are embedded in a collagen I-rich extracellular matrix (ECM) network, where nutrients are scarce due to limited blood flow and elevated tumour growth. Metabolic adaptation is required for cancer cells to endure these conditions. Here, we demonstrated that the presence of ECM supported the growth of invasive breast cancer cells, but not non-transformed mammary epithelial cells, under amino acid starvation, through a mechanism that required macropinocytosis-dependent ECM uptake. Importantly, we showed that this behaviour was acquired during carcinoma progression. ECM internalisation, followed by lysosomal degradation, contributed to the up-regulation of the intracellular levels of several amino acids, most notably tyrosine and phenylalanine. This resulted in elevated tyrosine catabolism on ECM under starvation, leading to increased fumarate levels, potentially feeding into the tricarboxylic acid (TCA) cycle. Interestingly, this pathway was required for ECM-dependent cell growth and invasive cell migration under amino acid starvation, as the knockdown of p-hydroxyphenylpyruvate hydroxylase-like protein (HPDL), the third enzyme of the pathway, opposed cell growth and motility on ECM in both 2D and 3D systems, without affecting cell proliferation on plastic. Finally, high HPDL expression correlated with poor prognosis in breast cancer patients. Collectively, our results highlight that the ECM in the tumour microenvironment (TME) represents an alternative source of nutrients to support cancer cell growth by regulating phenylalanine and tyrosine metabolism.

Typhoid toxin hijacks Wnt5a to establish host senescence and Salmonella infection.

Damage to our genome causes acute senescence in mammalian cells, which undergo growth arrest and release a senescence-associated secretory phenotype (SASP) that propagates the stress response to bystander cells. Thus, acute senescence is a powerful tumor suppressor. Salmonella enterica hijacks senescence through its typhoid toxin, which usurps unidentified factors in the stress secretome of senescent cells to mediate intracellular infections. Here, transcriptomics of toxin-induced senescent cells (TxSCs) and proteomics of their secretome identify the factors as Wnt5a, INHBA, and GDF15. Wnt5a establishes a positive feedback loop, driving INHBA and GDF15 expression. In fibroblasts, Wnt5a and INHBA mediate autocrine senescence in TxSCs and paracrine senescence in naive cells. Wnt5a synergizes with GDF15 to increase Salmonella invasion. Intestinal TxSCs undergo apoptosis without Wnt5a, which is required for establishing intestinal TxSCs. The study reveals how an innate defense against cancer is co-opted by a bacterial pathogen to cause widespread damage and mediate infections.

A potential histone-chaperone activity for the MIER1 histone deacetylase complex.

Histone deacetylases 1 and 2 (HDAC1/2) serve as the catalytic subunit of six distinct families of nuclear complexes. These complexes repress gene transcription through removing acetyl groups from lysine residues in histone tails. In addition to the deacetylase subunit, these complexes typically contain transcription factor and/or chromatin binding activities. The MIER:HDAC complex has hitherto been poorly characterized. Here, we show that MIER1 unexpectedly co-purifies with an H2A:H2B histone dimer. We show that MIER1 is also able to bind a complete histone octamer. Intriguingly, we found that a larger MIER1:HDAC1:BAHD1:C1QBP complex additionally co-purifies with an intact nucleosome on which H3K27 is either di- or tri-methylated. Together this suggests that the MIER1 complex acts downstream of PRC2 to expand regions of repressed chromatin and could potentially deposit histone octamer onto nucleosome-depleted regions of DNA.

Sec22b is a critical and nonredundant regulator of plasma cell maintenance.

Despite the essential role of plasma cells in health and disease, the cellular mechanisms controlling their survival and secretory capacity are still poorly understood. Here, we identified the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) Sec22b as a unique and critical regulator of plasma cell maintenance and function. In the absence of Sec22b, plasma cells were hardly detectable and serum antibody titers were dramatically reduced. Accordingly, Sec22b-deficient mice fail to mount a protective immune response. At the mechanistic level, we demonstrated that Sec22b contributes to efficient antibody secretion and is a central regulator of plasma cell maintenance through the regulation of their transcriptional identity and of the morphology of the endoplasmic reticulum and mitochondria. Altogether, our results unveil an essential and nonredundant role for Sec22b as a regulator of plasma cell fitness and of the humoral immune response.

Low expression of EXOSC2 protects against clinical COVID-19 and impedes SARS-CoV-2 replication.

New therapeutic targets are a valuable resource for treatment of SARS-CoV-2 viral infection. Genome-wide association studies have identified risk loci associated with COVID-19, but many loci are associated with comorbidities and are not specific to host-virus interactions. Here, we identify and experimentally validate a link between reduced expression of EXOSC2 and reduced SARS-CoV-2 replication. EXOSC2 was one of the 332 host proteins examined, all of which interact directly with SARS-CoV-2 proteins. Aggregating COVID-19 genome-wide association studies statistics for gene-specific eQTLs revealed an association between increased expression of EXOSC2 and higher risk of clinical COVID-19. EXOSC2 interacts with Nsp8 which forms part of the viral RNA polymerase. EXOSC2 is a component of the RNA exosome, and here, LC-MS/MS analysis of protein pulldowns demonstrated interaction between the SARS-CoV-2 RNA polymerase and most of the human RNA exosome components. CRISPR/Cas9 introduction of nonsense mutations within EXOSC2 in Calu-3 cells reduced EXOSC2 protein expression and impeded SARS-CoV-2 replication without impacting cellular viability. Targeted depletion of EXOSC2 may be a safe and effective strategy to protect against clinical COVID-19.

The EGFR/ErbB inhibitor neratinib modifies the neutrophil phosphoproteome and promotes apoptosis and clearance by airway macrophages.

Dysregulated neutrophilic inflammation can be highly destructive in chronic inflammatory diseases due to prolonged neutrophil lifespan and continual release of histotoxic mediators in inflamed tissues. Therapeutic induction of neutrophil apoptosis, an immunologically silent form of cell death, may be beneficial in these diseases, provided that the apoptotic neutrophils are efficiently cleared from the tissue. Previous research in our group identified ErbB inhibitors as able to induce neutrophil apoptosis and reduce neutrophilic inflammation both in vitro and in vivo. Here, we extend that work using a clinical ErbB inhibitor, neratinib, which has the potential to be repurposed in inflammatory diseases. We show that neratinib reduces neutrophilic migration o an inflammatory site in zebrafish larvae. Neratinib upregulates efferocytosis and reduces the number of persisting neutrophil corpses in mouse models of acute, but not chronic, lung injury, suggesting that the drug may have therapeutic benefits in acute inflammatory settings. Phosphoproteomic analysis of human neutrophils shows that neratinib modifies the phosphorylation of proteins regulating apoptosis, migration, and efferocytosis. This work identifies a potential mechanism for neratinib in treating acute lung inflammation by upregulating the clearance of dead neutrophils and, through examination of the neutrophil phosphoproteome, provides important insights into the mechanisms by which this may be occurring.

Altered subgenomic RNA abundance provides unique insight into SARS-CoV-2 B.1.1.7/Alpha variant infections.

B.1.1.7 lineage SARS-CoV-2 is more transmissible, leads to greater clinical severity, and results in modest reductions in antibody neutralization. Subgenomic RNA (sgRNA) is produced by discontinuous transcription of the SARS-CoV-2 genome. Applying our tool (periscope) to ARTIC Network Oxford Nanopore Technologies genomic sequencing data from 4400 SARS-CoV-2 positive clinical samples, we show that normalised sgRNA is significantly increased in B.1.1.7 (alpha) infections (n = 879). This increase is seen over the previous dominant lineage in the UK, B.1.177 (n = 943), which is independent of genomic reads, E cycle threshold and days since symptom onset at sampling. A noncanonical sgRNA which could represent ORF9b is found in 98.4% of B.1.1.7 SARS-CoV-2 infections compared with only 13.8% of other lineages, with a 16-fold increase in median sgRNA abundance. We demonstrate that ORF9b protein levels are increased 6-fold in B.1.1.7 compared to a B lineage virus in vitro. We hypothesise that increased ORF9b in B.1.1.7 is a direct consequence of a triple nucleotide mutation in nucleocapsid (28280:GAT > CAT, D3L) creating a transcription regulatory-like sequence complementary to a region 3' of the genomic leader. These findings provide a unique insight into the biology of B.1.1.7 and support monitoring of sgRNA profiles to evaluate emerging potential variants of concern.

Developmental disruption to the cortical transcriptome and synaptosome in a model of SETD1A loss-of-function.

Large-scale genomic studies of schizophrenia implicate genes involved in the epigenetic regulation of transcription by histone methylation and genes encoding components of the synapse. However, the interactions between these pathways in conferring risk to psychiatric illness are unknown. Loss-of-function (LoF) mutations in the gene encoding histone methyltransferase, SETD1A, confer substantial risk to schizophrenia. Among several roles, SETD1A is thought to be involved in the development and function of neuronal circuits. Here, we employed a multi-omics approach to study the effects of heterozygous Setd1a LoF on gene expression and synaptic composition in mouse cortex across five developmental timepoints from embryonic day 14 to postnatal day 70. Using RNA sequencing, we observed that Setd1a LoF resulted in the consistent downregulation of genes enriched for mitochondrial pathways. This effect extended to the synaptosome, in which we found age-specific disruption to both mitochondrial and synaptic proteins. Using large-scale patient genomics data, we observed no enrichment for genetic association with schizophrenia within differentially expressed transcripts or proteins, suggesting they derive from a distinct mechanism of risk from that implicated by genomic studies. This study highlights biological pathways through which SETD1A LOF may confer risk to schizophrenia. Further work is required to determine whether the effects observed in this model reflect human pathology.

Low expression of EXOSC2 protects against clinical COVID-19 and impedes SARS-CoV-2 replication.

New therapeutic targets are a valuable resource in the struggle to reduce the morbidity and mortality associated with the COVID-19 pandemic, caused by the SARS-CoV-2 virus. Genome-wide association studies (GWAS) have identified risk loci, but some loci are associated with co-morbidities and are not specific to host-virus interactions. Here, we identify and experimentally validate a link between reduced expression of EXOSC2 and reduced SARS-CoV-2 replication. EXOSC2 was one of 332 host proteins examined, all of which interact directly with SARS-CoV-2 proteins; EXOSC2 interacts with Nsp8 which forms part of the viral RNA polymerase. Lung-specific eQTLs were identified from GTEx (v7) for each of the 332 host proteins. Aggregating COVID-19 GWAS statistics for gene-specific eQTLs revealed an association between increased expression of EXOSC2 and higher risk of clinical COVID-19 which survived stringent multiple testing correction. EXOSC2 is a component of the RNA exosome and indeed, LC-MS/MS analysis of protein pulldowns demonstrated an interaction between the SARS-CoV-2 RNA polymerase and the majority of human RNA exosome components. CRISPR/Cas9 introduction of nonsense mutations within EXOSC2 in Calu-3 cells reduced EXOSC2 protein expression, impeded SARS-CoV-2 replication and upregulated oligoadenylate synthase ( OAS) genes, which have been linked to a successful immune response against SARS-CoV-2. Reduced EXOSC2 expression did not reduce cellular viability. OAS gene expression changes occurred independent of infection and in the absence of significant upregulation of other interferon-stimulated genes (ISGs). Targeted depletion or functional inhibition of EXOSC2 may be a safe and effective strategy to protect at-risk individuals against clinical COVID-19.

Transcriptional programs regulating neuronal differentiation are disrupted in DLG2 knockout human embryonic stem cells and enriched for schizophrenia and related disorders risk variants.

Coordinated programs of gene expression drive brain development. It is unclear which transcriptional programs, in which cell-types, are affected in neuropsychiatric disorders such as schizophrenia. Here we integrate human genetics with transcriptomic data from differentiation of human embryonic stem cells into cortical excitatory neurons. We identify transcriptional programs expressed during early neurogenesis in vitro and in human foetal cortex that are down-regulated in DLG2-/- lines. Down-regulation impacted neuronal differentiation and maturation, impairing migration, morphology and action potential generation. Genetic variation in these programs is associated with neuropsychiatric disorders and cognitive function, with associated variants predominantly concentrated in loss-of-function intolerant genes. Neurogenic programs also overlap schizophrenia GWAS enrichment previously identified in mature excitatory neurons, suggesting that pathways active during prenatal cortical development may also be associated with mature neuronal dysfunction. Our data from human embryonic stem cells, when combined with analysis of available foetal cortical gene expression data, de novo rare variants and GWAS statistics for neuropsychiatric disorders and cognition, reveal a convergence on transcriptional programs regulating excitatory cortical neurogenesis.

Proximity-dependent biotin identification (BioID) reveals a dynamic LSD1-CoREST interactome during embryonic stem cell differentiation.

Lysine specific demethylase 1 (LSD1) regulates gene expression as part of the CoREST complex, along with co-repressor of REST (CoREST) and histone deacetylase 1 (HDAC1). CoREST is recruited to specific genomic loci by core components and numerous transient interactions with chromatin-associated factors and transcription factors. We hypothesise that many of these weaker and transient associations may be difficult to identify using traditional co-immunoprecipitation methods. We have therefore employed proximity-dependent biotin-identification (BioID) with four different members of the CoREST complex, in three different cell types, to identify a comprehensive network of LSD1/CoREST associated proteins. In HEK293T cells, we identified 302 CoREST-associated proteins. Among this group were 16 of 18 known CoREST components and numerous novel associations, including readers (CHD3, 4, 6, 7 and 8), writers (KMT2B and KMT2D) and erasers (KDM2B) of histone methylation. However, components of other HDAC1 containing complexes (e.g. Sin3) were largely absent. To examine the dynamic nature of the CoREST interactome in a primary cell type, we replaced endogenous LSD1 with BirA*-LSD1 in embryonic stem (ES) cells and performed BioID in pluripotent, early- and late-differentiating environments. We identified 156 LSD1-associated proteins of which 67 were constitutively associated across all three time-points (43%), including novel associations with the MMB and ChAHP complexes, implying that the majority of interactors are both dynamic and cell type dependent. In total, we have performed 16 independent BioID experiments for LSD1 in three different cell types, producing a definitive network of LSD1-assoicated proteins that should provide a major resource for the field.

PGFinder, a novel analysis pipeline for the consistent, reproducible, and high-resolution structural analysis of bacterial peptidoglycans.

Many software solutions are available for proteomics and glycomics studies, but none are ideal for the structural analysis of peptidoglycan (PG), the essential and major component of bacterial cell envelopes. It icomprises glycan chains and peptide stems, both containing unusual amino acids and sugars. This has forced the field to rely on manual analysis approaches, which are time-consuming, labour-intensive, and prone to error. The lack of automated tools has hampered the ability to perform high-throughput analyses and prevented the adoption of a standard methodology. Here, we describe a novel tool called PGFinder for the analysis of PG structure and demonstrate that it represents a powerful tool to quantify PG fragments and discover novel structural features. Our analysis workflow, which relies on open-access tools, is a breakthrough towards a consistent and reproducible analysis of bacterial PGs. It represents a significant advance towards peptidoglycomics as a full-fledged discipline.

Proteomic Approaches to Study Cysteine Oxidation: Applications in Neurodegenerative Diseases.

Oxidative stress appears to be a key feature of many neurodegenerative diseases either as a cause or consequence of disease. A range of molecules are subject to oxidation, but in particular, proteins are an important target and measure of oxidative stress. Proteins are subject to a range of oxidative modifications at reactive cysteine residues, and depending on the level of oxidative stress, these modifications may be reversible or irreversible. A range of experimental approaches has been developed to characterize cysteine oxidation of proteins. In particular, mass spectrometry-based proteomic methods have emerged as a powerful means to identify and quantify cysteine oxidation sites on a proteome scale; however, their application to study neurodegenerative diseases is limited to date. Here we provide a guide to these approaches and highlight the under-exploited utility of these methods to measure oxidative stress in neurodegenerative diseases for biomarker discovery, target engagement and to understand disease mechanisms.

Regulation and function of the palmitoyl-acyltransferase ZDHHC5.

Protein palmitoylation (S-acylation) has emerged as an important player in a range of cellular processes, and as a result, the palmitoyl-acyltransferase (PAT) enzymes which mediate this modification have entered into the spotlight. Palmitoyltransferase ZDHHC5 (ZDHHC5) is among the more unique members of the PAT family as it is mainly localised to the plasma membrane and contains an extended cytoplasmic domain with several regulatory features. ZDHHC5 plays a vital role in a wide range of processes in different cell types. In this review, we offer a summary of the functions of ZDHHC5 in synaptic plasticity, cardiac function, cell adhesion and fatty acid uptake, among other processes. We also explore recent work has revealed several mechanisms to control the activity, localisation and function of ZDHHC5.

Cell-type-specific visualisation and biochemical isolation of endogenous synaptic proteins in mice.

In recent years, the remarkable molecular complexity of synapses has been revealed, with over 1,000 proteins identified in the synapse proteome. Although it is known that different receptors and other synaptic proteins are present in different types of neurons, the extent of synapse diversity across the brain is largely unknown. This is mainly due to the limitations of current techniques. Here, we report an efficient method for the purification of synaptic protein complexes, fusing a high-affinity tag to endogenous PSD95 in specific cell types. We also developed a strategy, which enables the visualisation of endogenous PSD95 with fluorescent-protein tag in Cre-recombinase-expressing cells. We demonstrate the feasibility of proteomic analysis of synaptic protein complexes and visualisation of these in specific cell types. We find that the composition of PSD95 complexes purified from specific cell types differs from those extracted from tissues with diverse cellular composition. The results suggest that there might be differential interactions in the PSD95 complexes in different brain regions. We have detected differentially interacting proteins by comparing data sets from the whole hippocampus and the CA3 subfield of the hippocampus. Therefore, these novel conditional PSD95 tagging lines will not only serve as powerful tools for precisely dissecting synapse diversity in specific brain regions and subsets of neuronal cells, but also provide an opportunity to better understand brain region- and cell-type-specific alterations associated with various psychiatric/neurological diseases. These newly developed conditional gene tagging methods can be applied to many different synaptic proteins and will facilitate research on the molecular complexity of synapses.

S-acylated Golga7b stabilises DHHC5 at the plasma membrane to regulate cell adhesion.

S-acylation (palmitoylation) is the only fully reversible lipid modification of proteins; however, little is known about how protein S-acyltransferases (PATs) that mediate it are regulated. DHHC5 is a PAT that is mainly localised at the plasma membrane with roles in synaptic plasticity, massive endocytosis and cancer cell growth/invasion. Here, we demonstrate that DHHC5 binds to and palmitoylates a novel accessory protein Golga7b. Palmitoylation of Golga7b prevents clathrin-mediated endocytosis of DHHC5 and stabilises it at the plasma membrane. Proteomic analysis of the composition of DHHC5/Golga7b-associated protein complexes reveals a striking enrichment in adhesion proteins, particularly components of desmosomes. We show that desmoglein-2 and plakophilin-3 are substrates of DHHC5 and that DHHC5 and Golga7b are required for localisation of desmoglein-2 to the plasma membrane and for desmosomal patterning. Loss of DHHC5/Golga7b causes functional impairments in cell adhesion, suggesting these proteins have a wider role in cell adhesion beyond desmosome assembly. This work uncovers a novel mechanism of DHHC5 regulation by Golga7b and demonstrates a role for the DHHC5/Golga7b complex in the regulation of cell adhesion.

Apoptotic signalling targets the post-endocytic sorting machinery of the death receptor Fas/CD95.

Fas plays a major role in regulating ligand-induced apoptosis in many cell types. It is well known that several cancers demonstrate reduced cell surface levels of Fas and thus escape a potential control system via ligand-induced apoptosis, although underlying mechanisms are unclear. Here we report that the endosome associated trafficking regulator 1 (ENTR1), controls cell surface levels of Fas and Fas-mediated apoptotic signalling. ENTR1 regulates, via binding to the coiled coil domain protein Dysbindin, the delivery of Fas from endosomes to lysosomes thereby controlling termination of Fas signal transduction. We demonstrate that ENTR1 is cleaved during Fas-induced apoptosis in a caspase-dependent manner revealing an unexpected interplay of apoptotic signalling and regulation of endolysosomal trafficking resulting in a positive feedback signalling-loop. Our data provide insights into the molecular mechanism of Fas post-endocytic trafficking and signalling, opening possible explanations on how cancer cells regulate cell surface levels of death receptors.

Proteomic Profiling, Transcription Factor Modeling, and Genomics of Evolved Tolerant Strains Elucidate Mechanisms of Vanillin Toxicity in Escherichia coli.

Vanillin (4-hydroxy-3-methoxybenzaldehyde) is an economically important flavor compound that can be made in bacterial cell factories, but toxicity is a major problem for cells producing this aromatic aldehyde. Using (i) a global proteomic analysis supported by multiple physiological experiments, mutant analyses, and inferred transcription factor modeling and (ii) adaptive laboratory evolution (ALE) of vanillin tolerance combined with genome-wide analysis of the underlying mutations, mechanisms of vanillin toxicity in Escherichia coli have been elucidated. We identified 147 proteins that exhibited a significant change in abundance in response to vanillin, giving the first detailed insight into the cellular response to this aldehyde. Vanillin caused accumulation of reactive oxygen species invoking adaptations coordinated by a MarA, OxyR, and SoxS regulatory network and increased RpoS/DksA-dependent gene expression. Differential fumarase C upregulation was found to prevent oxidative damage to FumA and FumB during growth with vanillin. Surprisingly, vanillin-dependent reduction pf copper (II) to copper (I) led to upregulation of the copA gene and growth in the presence of vanillin was shown to be hypersensitive to inhibition by copper ions. AcrD and AaeAB were identified as potential vanillin efflux systems. Vanillin-tolerant strains isolated by ALE had distinct nonsynonymous single nucleotide polymorphisms (SNPs) in gltA that led to increased citrate synthase activity. Strain-specific mutations in cpdA, rob, and marC were also present. One strain had a large (∼10-kb) deletion that included the marRAB region. Our data provide new understanding of bacterial vanillin toxicity and identify novel gene targets for future engineering of vanillin-tolerant strains of E. coli IMPORTANCE A particular problem for the biotechnological production of many of the valuable chemicals that we are now able to manufacture in bacterial cells is that these products often poison the cells producing them. Solutions to improve product yields or alleviate such toxicity using the techniques of modern molecular biology first require a detailed understanding of the mechanisms of product toxicity. Here we have studied the economically important flavor compound vanillin, an aromatic aldehyde that exerts significant toxic effects on bacterial cells. We used high-resolution protein abundance analysis as a starting point to determine which proteins are upregulated and which are downregulated by growth with vanillin, followed by gene expression and mutant studies to understand the mechanism of the response. In a second approach, we evolved bacterial strains with higher vanillin tolerance. Their genome sequences have yielded novel insights into vanillin tolerance that are complementary to the proteomics data set.

Quantitative Analysis of Protein S-Acylation Site Dynamics Using Site-Specific Acyl-Biotin Exchange (ssABE).

Protein S-acylation (palmitoylation) is a reversible lipid modification that is increasingly recognized as an important regulator of protein function, including membrane association, trafficking, and subcellular localization. Most proteomic methods to study palmitoylation allow characterization of putative palmitoylated proteins but do not permit identification of individual sites of palmitoylation. We have recently adapted the Acyl-Biotin Exchange (ABE) method that is routinely used for palmitoyl-proteome characterization, to permit global S-acylation site analysis. This site-specific ABE (ssABE) protocol, when combined with SILAC-based quantification, allows both the large-scale identification of palmitoylation sites and quantitative profiling of palmitoylation site changes. This approach enables palmitoylation to be studied at a systems level comparable to other more intensively studied post-translational modifications.