Showing papers by "Steven P. Gygi published in 2018"
TL;DR: It is shown that pharmacological elevation of circulating succinate drives UCP1-dependent thermogenesis by brown adipose tissue in vivo, which stimulates robust protection against diet-induced obesity and improves glucose tolerance, and reveals an unexpected mechanism for control of thermogenesis.
Abstract: Thermogenesis by brown and beige adipose tissue, which requires activation by external stimuli, can counter metabolic disease1. Thermogenic respiration is initiated by adipocyte lipolysis through cyclic AMP–protein kinase A signalling; this pathway has been subject to longstanding clinical investigation2–4. Here we apply a comparative metabolomics approach and identify an independent metabolic pathway that controls acute activation of adipose tissue thermogenesis in vivo. We show that substantial and selective accumulation of the tricarboxylic acid cycle intermediate succinate is a metabolic signature of adipose tissue thermogenesis upon activation by exposure to cold. Succinate accumulation occurs independently of adrenergic signalling, and is sufficient to elevate thermogenic respiration in brown adipocytes. Selective accumulation of succinate may be driven by a capacity of brown adipocytes to sequester elevated circulating succinate. Furthermore, brown adipose tissue thermogenesis can be initiated by systemic administration of succinate in mice. Succinate from the extracellular milieu is rapidly metabolized by brown adipocytes, and its oxidation by succinate dehydrogenase is required for activation of thermogenesis. We identify a mechanism whereby succinate dehydrogenase-mediated oxidation of succinate initiates production of reactive oxygen species, and drives thermogenic respiration, whereas inhibition of succinate dehydrogenase supresses thermogenesis. Finally, we show that pharmacological elevation of circulating succinate drives UCP1-dependent thermogenesis by brown adipose tissue in vivo, which stimulates robust protection against diet-induced obesity and improves glucose tolerance. These findings reveal an unexpected mechanism for control of thermogenesis, using succinate as a systemically-derived thermogenic molecule.
325 citations
TL;DR: A streamlined TMT protocol is established that enables deep proteome and medium-scale phosphoproteome analysis and permits the seamless addition of a phosphopeptide enrichment step ("mini-phos") with little deviation from thedeep proteome analysis.
Abstract: Mass spectrometry (MS) coupled toisobaric labeling has developed rapidly into a powerful strategy for high-throughput protein quantification. Sample multiplexing and exceptional sensitivity allow for the quantification of tens of thousands of peptides and, by inference, thousands of proteins from multiple samples in a single MS experiment. Accurate quantification demands a consistent and robust sample-preparation strategy. Here, we present a detailed workflow for SPS-MS3-based quantitative abundance profiling of tandem mass tag (TMT)-labeled proteins and phosphopeptides that we have named the streamlined (SL)-TMT protocol. We describe a universally applicable strategy that requires minimal individual sample processing and permits the seamless addition of a phosphopeptide enrichment step ("mini-phos") with little deviation from the deep proteome analysis. To showcase our workflow, we profile the proteome of wild-type Saccharomyces cerevisiae yeast grown with either glucose or pyruvate as the carbon source. Here, we have established a streamlined TMT protocol that enables deep proteome and medium-scale phosphoproteome analysis.
207 citations
TL;DR: Temporal digital snapshots of ubiquitin signaling on the mitochondrial outer membrane in embryonic stem cell-derived neurons are developed and the power of this approach to quantify pathway modulators and to mechanistically define the role of PARKIN UBL phosphorylation in pathway activation in induced neurons is demonstrated.
129 citations
TL;DR: Test the ability of two common methods, a tandem mass tagging (TMT) method and a label-free quantitation method (LFQ), to achieve comprehensive quantitative coverage by benchmarking their capacity to measure 3 different levels of change across an entire data set.
Abstract: Proteomics experiments commonly aim to estimate and detect differential abundance across all expressed proteins. Within this experimental design, some of the most challenging measurements are small fold changes for lower abundance proteins. While bottom-up proteomics methods are approaching comprehensive coverage of even complex eukaryotic proteomes, failing to reliably quantify lower abundance proteins can limit the precision and reach of experiments to much less than the identified—let alone total—proteome. Here we test the ability of two common methods, a tandem mass tagging (TMT) method and a label-free quantitation method (LFQ), to achieve comprehensive quantitative coverage by benchmarking their capacity to measure 3 different levels of change (3-, 2-, and 1.5-fold) across an entire data set. Both methods achieved comparably accurate estimates for all 3-fold-changes. However, the TMT method detected changes that reached statistical significance three times more often due to higher precision and fewe...
115 citations
TL;DR: The RNA-processing factor Nudt21 is identified as a novel post-transcriptional regulator of cell fate change using transcription-factor-induced reprogramming as a screening assay and a direct, previously unappreciated link between alternative polyadenylation and chromatin signaling is established.
106 citations
TL;DR: The structure-based evolution of OGT inhibitors culminating in compounds with low nanomolar inhibitory potency and on-target cellular activity are reported, providing insight into how to inhibit glycosyltransferases, a family of enzymes that has been notoriously refractory to inhibitor development.
Abstract: Reversible glycosylation of nuclear and cytoplasmic proteins is an important regulatory mechanism across metazoans. One enzyme, O-linked N-acetylglucosamine transferase (OGT), is responsible for all nucleocytoplasmic glycosylation and there is a well-known need for potent, cell-permeable inhibitors to interrogate OGT function. Here we report the structure-based evolution of OGT inhibitors culminating in compounds with low nanomolar inhibitory potency and on-target cellular activity. In addition to disclosing useful OGT inhibitors, the structures we report provide insight into how to inhibit glycosyltransferases, a family of enzymes that has been notoriously refractory to inhibitor development.
97 citations
TL;DR: It is shown that synthesis of the mitochondrial phospholipid cardiolipin is indispensable for stimulating and sustaining thermogenicfat function and has a powerful impact on organismal energy homeostasis through thermogenic fat bioenergetics.
97 citations
TL;DR: Mechanisms of skewed metabolic remodeling in aged T cells are defined and evidence that modulation of metabolism has the potential to promote immune function in aged individuals is provided.
Abstract: T cell-mediated immune responses are compromised in aged individuals, leading to increased morbidity and reduced response to vaccination. While cellular metabolism tightly regulates T cell activation and function, metabolic reprogramming in aged T cells has not been thoroughly studied. Here, we report a systematic analysis of metabolism during young versus aged naive T cell activation. We observed a decrease in the number and activation of naive T cells isolated from aged mice. While young T cells demonstrated robust mitochondrial biogenesis and respiration upon activation, aged T cells generated smaller mitochondria with lower respiratory capacity. Using quantitative proteomics, we defined the aged T cell proteome and discovered a specific deficit in the induction of enzymes of one-carbon metabolism. The activation of aged naive T cells was enhanced by addition of products of one-carbon metabolism (formate and glycine). These studies define mechanisms of skewed metabolic remodeling in aged T cells and provide evidence that modulation of metabolism has the potential to promote immune function in aged individuals.
77 citations
TL;DR: It is demonstrated that the acylated form of ACP is an acetyl-CoA-dependent allosteric activator of the LYR protein family used to stimulate ETC biogenesis and could provide an elegant mechanism for coordinating the assembly of ETC complexes with one another and with substrate availability.
69 citations
TL;DR: Modifications to standard culture conditions are described that permit the growth of naive human pluripotent stem cells with reduced genomic instability and provide a simple modification to current methods that can enable robust growth and reduced genomic stability in naive hESCs.
Abstract: Human embryonic stem cells (hESCs) can be captured in a primed state in which they resemble the postimplantation epiblast, or in a naive state where they resemble the preimplantation epiblast. Naive-cell-specific culture conditions allow the study of preimplantation development ex vivo but reportedly lead to chromosomal abnormalities, which compromises their utility in research and potential therapeutic applications. Although MEK inhibition is essential for the naive state, here we show that reduced MEK inhibition facilitated the establishment and maintenance of naive hESCs that retained naive-cell-specific features, including global DNA hypomethylation, HERVK expression, and two active X chromosomes. We further show that hESCs cultured under these modified conditions proliferated more rapidly; accrued fewer chromosomal abnormalities; and displayed changes in the phosphorylation levels of MAPK components, regulators of DNA damage/repair, and cell cycle. We thus provide a simple modification to current methods that can enable robust growth and reduced genomic instability in naive hESCs.
66 citations
TL;DR: It is demonstrated that the deubiquitinase OTUD4 is phosphorylated near its catalytic domain, activating a latent K63-specific deUBiquit inase, and the Toll-like receptor (TLR)-associated factor MyD88 is revealed as a target of this K63 deubiqueitin enzyme activity.
TL;DR: BioPlex Display is developed, which integrates individual protein querying, access to empirical data, and on-the-fly annotation of networks within an easy-to-use and mobile web application that enables exploration and dissemination of the growing BioPlex interaction network.
Abstract: The development of large-scale data sets requires a new means to display and disseminate research studies to large audiences. Knowledge of protein–protein interaction (PPI) networks has become a principle interest of many groups within the field of proteomics. At the confluence of technologies, such as cross-linking mass spectrometry, yeast two-hybrid, protein cofractionation, and affinity purification mass spectrometry (AP–MS), detection of PPIs can uncover novel biological inferences at a high-throughput. Thus new platforms to provide community access to large data sets are necessary. To this end, we have developed a web application that enables exploration and dissemination of the growing BioPlex interaction network. BioPlex is a large-scale interactome data set based on AP–MS of baits from the human ORFeome. The latest BioPlex data set release (BioPlex 2.0) contains 56 553 interactions from 5891 AP–MS experiments. To improve community access to this vast compendium of interactions, we developed BioPle...
TL;DR: An autoinhibitory conformation in the conserved H3K9 methyltransferase Clr4 of the fission yeast Schizosaccharomyces pombe helps to prevent aberrant heterochromatin formation and maintains epigenetic stability.
Abstract: Histone H3 lysine 9 methylation (H3K9me) mediates heterochromatic gene silencing and is important for genome stability and the regulation of gene expression1–4. The establishment and epigenetic maintenance of heterochromatin involve the recruitment of H3K9 methyltransferases to specific sites on DNA, followed by the recognition of pre-existing H3K9me by the methyltransferase and methylation of proximal histone H35–11. This positive feedback loop must be tightly regulated to prevent deleterious epigenetic gene silencing. Extrinsic anti-silencing mechanisms involving histone demethylation or boundary elements help to limit the spread of inappropriate H3K9me12–15. However, how H3K9 methyltransferase activity is locally restricted or prevented from initiating random H3K9me—which would lead to aberrant gene silencing and epigenetic instability—is not fully understood. Here we reveal an autoinhibited conformation in the conserved H3K9 methyltransferase Clr4 (also known as Suv39h) of the fission yeast Schizosaccharomyces pombe that has a critical role in preventing aberrant heterochromatin formation. Biochemical and X-ray crystallographic data show that an internal loop in Clr4 inhibits the catalytic activity of this enzyme by blocking the histone H3K9 substrate-binding pocket, and that automethylation of specific lysines in this loop promotes a conformational switch that enhances the H3K9me activity of Clr4. Mutations that are predicted to disrupt this regulation lead to aberrant H3K9me, loss of heterochromatin domains and inhibition of growth, demonstrating the importance of the intrinsic inhibition and auto-activation of Clr4 in regulating the deposition of H3K9me and in preventing epigenetic instability. Conservation of the Clr4 autoregulatory loop in other H3K9 methyltransferases and the automethylation of a corresponding lysine in the human SUV39H2 homologue16 suggest that the mechanism described here is broadly conserved. An autoinhibitory conformation of the histone H3K9 methyltransferase Clr4 of Schizosaccharomyces pombe helps to prevent aberrant heterochromatin formation and maintains epigenetic stability.
TL;DR: The data indicate that the RNAP II/U1 snRNP machinery functions in a wide variety of molecular pathways, and these pathways are candidates for playing roles in ALS/SMA pathogenesis.
Abstract: Mutations in multiple RNA/DNA binding proteins cause Amyotrophic Lateral Sclerosis (ALS). Included among these are the three members of the FET family (FUS, EWSR1 and TAF15) and the structurally similar MATR3. Here, we characterized the interactomes of these four proteins, revealing that they largely have unique interactors, but share in common an association with U1 snRNP. The latter observation led us to analyze the interactome of the U1 snRNP machinery. Surprisingly, this analysis revealed the interactome contains ~220 components, and of these, >200 are shared with the RNA polymerase II (RNAP II) machinery. Among the shared components are multiple ALS and Spinal muscular Atrophy (SMA)-causative proteins and numerous discrete complexes, including the SMN complex, transcription factor complexes, and RNA processing complexes. Together, our data indicate that the RNAP II/U1 snRNP machinery functions in a wide variety of molecular pathways, and these pathways are candidates for playing roles in ALS/SMA pathogenesis.
TL;DR: It is suggested that ERK activation in adipocytes and subsequent phosphorylation of the β3AR on S247 are critical regulatory steps in the enhanced adipocyte lipolysis of obesity.
Abstract: Objective The inappropriate release of free fatty acids from obese adipose tissue stores has detrimental effects on metabolism, but key molecular mechanisms controlling FFA release from adipocytes remain undefined. Although obesity promotes systemic inflammation, we find activation of the inflammation-associated Mitogen Activated Protein kinase ERK occurs specifically in adipose tissues of obese mice, and provide evidence that adipocyte ERK activation may explain exaggerated adipose tissue lipolysis observed in obesity. Methods and Results We provide genetic and pharmacological evidence that inhibition of the MEK/ERK pathway in human adipose tissue, mice, and flies all effectively limit adipocyte lipolysis. In complementary findings, we show that genetic and obesity-mediated activation of ERK enhances lipolysis, whereas adipose tissue specific knock-out of ERK2, the exclusive ERK1/2 protein in adipocytes, dramatically impairs lipolysis in explanted mouse adipose tissue. In addition, acute inhibition of MEK/ERK signaling also decreases lipolysis in adipose tissue and improves insulin sensitivity in obese mice. Mice with decreased rates of adipose tissue lipolysis in vivo caused by either MEK or ATGL pharmacological inhibition were unable to liberate sufficient White Adipose Tissue (WAT) energy stores to fuel thermogenesis from brown fat during a cold temperature challenge. To identify a molecular mechanism controlling these actions, we performed unbiased phosphoproteomic analysis of obese adipose tissue at different time points following acute pharmacological MEK/ERK inhibition. MEK/ERK inhibition decreased levels of adrenergic signaling and caused de-phosphorylation of the β3-adrenergic receptor (β3AR) on serine 247. To define the functional implications of this phosphorylation, we showed that CRISPR/Cas9 engineered cells expressing wild type β3AR exhibited β3AR phosphorylation by ERK2 and enhanced lipolysis, but this was not seen when serine 247 of β3AR was mutated to alanine. Conclusion Taken together, these data suggest that ERK activation in adipocytes and subsequent phosphorylation of the β3AR on S247 are critical regulatory steps in the enhanced adipocyte lipolysis of obesity.
TL;DR: It is shown experimentally that isobaric tag proteomics data are inherently compositional and highlighted the implications for data analysis and interpretation, and a unique compositional effect on proteins with infinite changes is demonstrated.
Abstract: Mass spectrometry (MS) has become an accessible tool for whole proteome quantitation with the ability to characterize protein expression across thousands of proteins within a single experiment. A subset of MS quantification methods (e.g., SILAC and label-free) monitor the relative intensity of intact peptides, where thousands of measurements can be made from a single mass spectrum. An alternative approach, isobaric labeling, enables precise quantification of multiple samples simultaneously through unique and sample specific mass reporter ions. Consequently, in a single scan, the quantitative signal comes from a limited number of spectral features (≤11). The signal observed for these features is constrained by automatic gain control, forcing codependence of concurrent signals. The study of constrained outcomes primarily belongs to the field of compositional data analysis. We show experimentally that isobaric tag proteomics data are inherently compositional and highlight the implications for data analysis a...
TL;DR: It is found that Selenof deficiency in mice leads to elevated levels of non-functional circulating plasma immunoglobulins and increased secretion of IgM during in vitro splenic B cell differentiation, and proteomic analyses suggest thatSelenof functions as a gatekeeper of immunoglobs and, likely, other client proteins that exit the ER, thereby supporting redox quality control of these proteins.
TL;DR: It is demonstrated that mutations in highly conserved residues of this domain affect protein stability, by increasing its ubiquitin-mediated degradation, and cell-proliferation rates, and mutated RPS15 can be loaded into the ribosomes, directly impacting on global protein synthesis and/or translational fidelity in a mutation-specific manner.
TL;DR: It is found that inhibiting the NAD+ salvage pathway depletes serine biosynthesis from glucose by impeding the NAD-dependent protein, 3-phosphoglycerate dehydrogenase (PHGDH), and these findings suggest that they may be effective for PHGDH-dependent cancers.
TL;DR: The analysis suggests that the proposed Bayesian selection model, compared with various imputation strategies and complete-case analyses, can increase accuracy and provide substantial improvements to interval coverage.
Abstract: An idealized version of a label-free discovery mass spectrometry proteomics experiment would provide absolute abundance measurements for a whole proteome, across varying conditions. Unfortunately, this ideal is not realized. Measurements are made on peptides requiring an inferential step to obtain protein level estimates. The inference is complicated by experimental factors that necessitate relative abundance estimation and result in widespread non-ignorable missing data. Relative abundance on the log scale takes the form of parameter contrasts. In a complete-case analysis, contrast estimates may be biased by missing data and a substantial amount of useful information will often go unused. To avoid problems with missing data, many analysts have turned to single imputation solutions. Unfortunately, these methods often create further difficulties by hiding inestimable contrasts, preventing the recovery of interblock information and failing to account for imputation uncertainty. To mitigate many of the problems caused by missing values, we propose the use of a Bayesian selection model. Our model is tested on simulated data, real data with simulated missing values, and on a ground truth dilution experiment where all of the true relative changes are known. The analysis suggests that our model, compared with various imputation strategies and complete-case analyses, can increase accuracy and provide substantial improvements to interval coverage.
TL;DR: It is demonstrated that E cyclins are dispensable for adult mouse physiology, but essential for liver cancer progression, and found that the function of the cyclin E catalytic partner, CDK2, is dispensable in liver cancer cells.
Abstract: E-type cyclins (cyclins E1 and E2) are components of the core cell cycle machinery and are overexpressed in many human tumor types. E cyclins are thought to drive tumor cell proliferation by activating the cyclin-dependent kinase 2 (CDK2). The cyclin E1 gene represents the site of recurrent integration of the hepatitis B virus in the pathogenesis of hepatocellular carcinoma, and this event is associated with strong up-regulation of cyclin E1 expression. Regardless of the underlying mechanism of tumorigenesis, the majority of liver cancers overexpress E-type cyclins. Here we used conditional cyclin E knockout mice and a liver cancer model to test the requirement for the function of E cyclins in liver tumorigenesis. We show that a ubiquitous, global shutdown of E cyclins did not visibly affect postnatal development or physiology of adult mice. However, an acute ablation of E cyclins halted liver cancer progression. We demonstrated that also human liver cancer cells critically depend on E cyclins for proliferation. In contrast, we found that the function of the cyclin E catalytic partner, CDK2, is dispensable in liver cancer cells. We observed that E cyclins drive proliferation of tumor cells in a CDK2- and kinase-independent mechanism. Our study suggests that compounds which degrade or inhibit cyclin E might represent a highly selective therapeutic strategy for patients with liver cancer, as these compounds would selectively cripple proliferation of tumor cells, while sparing normal tissues.
TL;DR: In this article, the authors employed CRISPR knockout (KO) to investigate the functions of four ALS-causative RNA/DNA binding proteins (FUS, EWSR1, TAF15 and MATR3) within the RNAP II/U1 snRNP machinery.
Abstract: Understanding the molecular pathways disrupted in motor neuron diseases is urgently needed. Here, we employed CRISPR knockout (KO) to investigate the functions of four ALS-causative RNA/DNA binding proteins (FUS, EWSR1, TAF15 and MATR3) within the RNAP II/U1 snRNP machinery. We found that each of these structurally related proteins has distinct roles with FUS KO resulting in loss of U1 snRNP and the SMN complex, EWSR1 KO causing dissociation of the tRNA ligase complex, and TAF15 KO resulting in loss of transcription factors P-TEFb and TFIIF. However, all four ALS-causative proteins are required for association of the ASC-1 transcriptional co-activator complex with the RNAP II/U1 snRNP machinery. Remarkably, mutations in the ASC-1 complex are known to cause a severe form of Spinal Muscular Atrophy (SMA), and we show that an SMA-causative mutation in an ASC-1 component or an ALS-causative mutation in FUS disrupts association between the ASC-1 complex and the RNAP II/U1 snRNP machinery. We conclude that ALS and SMA are more intimately tied to one another than previously thought, being linked via the ASC-1 complex.
03 May 2018
TL;DR: It is demonstrated that host ATP binding cassette transporter ABCB6, which encodes the Langereis blood group antigen, promotes erythrocyte invasion by the malaria parasite Plasmodium falciparum, and suggests that asymptomatic LangereIS null individuals may be better protected from malaria.
Abstract: The ATP-binding cassette transporter ABCB6 was recently discovered to encode the Langereis (Lan) blood group antigen. Lan null individuals are asymptomatic, and the function of ABCB6 in mature erythrocytes is not understood. Here, we assessed ABCB6 as a host factor for Plasmodium falciparum malaria parasites during erythrocyte invasion. We show that Lan null erythrocytes are highly resistant to invasion by P. falciparum, in a strain-transcendent manner. Although both Lan null and Jr(a-) erythrocytes harbor excess porphyrin, only Lan null erythrocytes exhibit a P. falciparum invasion defect. Further, the zoonotic parasite P. knowlesi invades Lan null and control cells with similar efficiency, suggesting that ABCB6 may mediate P. falciparum invasion through species-specific molecular interactions. Using tandem mass tag-based proteomics, we find that the only consistent difference in membrane proteins between Lan null and control cells is absence of ABCB6. Our results demonstrate that a newly identified naturally occurring blood group variant is associated with resistance to Plasmodium falciparum. Elizabeth Egan and colleagues demonstrate that host ATP binding cassette transporter ABCB6, which encodes the Langereis blood group antigen, promotes erythrocyte invasion by the malaria parasite Plasmodium falciparum. This study suggests that asymptomatic Langereis null individuals may be better protected from malaria.
TL;DR: A peptide internal standard-based assay directed toward sample preparation strategies for mass spectrometry analysis to understand better phosphopeptide recovery from enrichment strategies, applicable to any enrichment protocol in a typical experimental workflow irrespective of sample origin or labeling strategy.
TL;DR: In this paper, the authors performed isobaric tag-based quantitative comparative phosphoproteomics of the schizont and segmenter stages from wild-type and pfpk7- parasite lines.
Abstract: PfPK7 is an “orphan” kinase displaying regions of homology to multiple protein kinase families. PfPK7 functions in regulating parasite proliferation/development as evident from the phenotype analysis of knockout parasites. Despite this regulatory role, the functions of PfPK7 in signaling pathways are not known. To better understand PfPK7-regulated phosphorylation events, we performed isobaric tag-based quantitative comparative phosphoproteomics of the schizont and segmenter stages from wild-type and pfpk7- parasite lines. This analysis identified 3,875 phosphorylation sites on 1,047 proteins. Among these phosphorylation events, 146 proteins with 239 phosphorylation sites displayed reduction in phosphorylation in the absence of PfPK7. Further analysis of the phosphopeptides revealed three motifs whose phosphorylation was down regulated in the pfpk7– cell line in both schizonts and segmenters. Decreased phosphorylation following loss of PfPK7 indicates that these proteins may function as direct substrates o...
TL;DR: A mechanism of ICL sensing by UHRF2, leading to FANCD2 recruitment and retention at ICLs, in turn facilitating activation of FAN CD2 by monoubiquitination is uncovered.
Abstract: The Fanconi Anemia (FA) pathway is important for repairing interstrand crosslinks (ICLs) between the Watson-Crick strands of the DNA double helix. An initial and essential stage in the repair process is the detection of the ICL. Here, we report the identification of UHRF2, a paralogue of UHRF1, as an ICL sensor protein. UHRF2 is recruited to ICLs in the genome within seconds of their appearance. We show that UHRF2 cooperates with UHRF1, to ensure recruitment of FANCD2 to ICLs. A direct protein-protein interaction is formed between UHRF1 and UHRF2, and between either UHRF1 and UHRF2, and FANCD2. Importantly, we demonstrate that the essential monoubiquitination of FANCD2 is stimulated by UHRF1/UHRF2. The stimulation is mediating by a retention of FANCD2 on chromatin, allowing for its monoubiquitination by the FA core complex. Taken together, we uncover a mechanism of ICL sensing by UHRF2, leading to FANCD2 recruitment and retention at ICLs, in turn facilitating activation of FANCD2 by monoubiquitination.
TL;DR: It is concluded that the detergent-free FPD strategy, particularly using the faster-flowing 30 kDa filter, is a seamless alteration to high-throughput TMT workflows.
Abstract: High-throughput proteome profiling requires thorough optimization to achieve comprehensive analysis We developed a filter aided sample preparation (FASP)-like, detergent-free method, termed Filter-Based Protein Digestion (FPD) We compared FPD to protein extraction methods commonly used in isobaric tag-based proteome profiling, namely trichloroacetic acid (TCA) and chloroform–methanol (C–M) precipitation We divided a mammalian whole cell lysate from the SH-SY5Y neuroblastoma cell line for parallel protein processing with TCA (n = 3), C–M (n = 2), and FPD using either 10 kDa (n = 3) or 30 kDa (n = 3) molecular weight cutoff membranes We labeled each sample with tandem mass tag (TMT) reagents to construct a TMT11-plex experiment In total, 8654 proteins were quantified across all samples Pairwise comparisons showed very little deviation for individual protein abundance measurements between the two FPD methods, whereas TCA and FPD showed the most difference Specifically, membrane proteins were more read
TL;DR: The authors hypothesize that both tissue‐specific and global protein abundance alterations result from nicotine exposure, and present the first proteomic profiling of multiple tissues from mice treated orally with nicotine.
Abstract: Nicotine is a major addictive compound in tobacco and a component of smoking-related products, such as e-cigarettes. Once internalized, nicotine can perturb many cellular pathways and can induce alterations in proteins across different cell types, however the mechanisms thereof remain undetermined. We hypothesize that both tissue-specific and global protein abundance alterations result from nicotine exposure. As such, we present the first proteome analysis of multiple tissues from nicotine-exposed mice. We treat mice via oral administration of nicotine at 200μg/mL in drinking water for 21 days. We investigate 7 tissues (brain, heart, kidney, liver, lung, pancreas, and spleen) from treated (n=5) and untreated control (n=5) mice. For each tissue, a TMT10-plex experiment (5 versus 5) was assembled. We apply a minimalistic proteomics strategy was employed using TMT reagents efficiently and fractionating by centrifugation-based reversed-phase columns to streamline sample preparation. Combined, we quantified over 11,000 non-redundant proteins from over 138,000 different peptides in 7 TMT10-plex experiments. Between 7 and 126 proteins are significantly altered in tissues from nicotine-exposed mice. Among these proteins, only 11 are altered in two or more tissues, many classified as from extracellular exosomes or involved in lipid metabolism. Our data showcase the vast extent of nicotine exposure across murine tissue.
TL;DR: It is shown that the TKO6 standard is a valuable tool for assessing quantification accuracy in isobaric-tag-based analyses and is designed specifically for data acquired at low (unit) mass resolution.
Abstract: Protein abundance profiling using isobaric labeling is a well-established quantitative mass spectrometry technique. However, ratio distortion resulting from coisolated and cofragmented ions, commonly referred to as interference, remains a drawback of this strategy. Tribrid mass spectrometers, such as the Orbitrap Fusion and the Orbitrap Fusion Lumos with a triple mass analyzer configuration, facilitate methods (namely, SPS-MS3) that can help alleviate interference. However, few standards are available to measure interference and thereby aid in method development. Here we introduce the TKO6 standard that assesses ion interference and is designed specifically for data acquired at low (unit) mass resolution. We use TKO6 to compare interference in MS2- versus MS3-based quantitation methods, data acquisition methods of different lengths, and ion-trap-based tandem mass tag reporter ion analysis (IT-MS3) with conventional Orbitrap-based analysis (OT-MS3). We show that the TKO6 standard is a valuable tool for assessing quantification accuracy in isobaric-tag-based analyses.
TL;DR: This study, which is the first TMT‐based proteome profiling of HAP1 cells, defines the effects of nicotine on non‐neuronal cellular proteomes and revealed that membrane proteins and proteins commonly associated with neurons were predominant among those with altered abundance.
Abstract: Nicotinic acetylcholine receptors (nAChR), the primary cell surface targets of nicotine, have implications in various neurological disorders. Here we investigate the proteome-wide effects of nicotine on human haploid cell lines (wildtype HAP1 and α7KO-HAP1) to address differences in nicotine-induced protein abundance profiles between these cell lines. We performed an SPS-MS3-based TMT10-plex experiment arranged in a 2-3-2-3 design with two replicates of the untreated samples and three of the treated samples for each cell line. We quantified 8775 proteins across all ten samples, of which several hundred differed significantly in abundance. Comparing α7KO-HAP1 and HAP1wt cell lines to each other revealed significant protein abundance alterations; however, we also measured differences resulting from nicotine treatment in both cell lines. Among proteins with increased abundance levels due to nicotine treatment included those previously identified: APP, APLP2, and ITM2B. The magnitude of these changes was greater in HAP1wt compared to the α7KO-HAP1 cell line, implying a potential role for the α7 nAChR in HAP1 cells. Moreover, the data revealed that membrane proteins and proteins commonly associated with neurons were predominant among those with altered abundance. This study, which is the first TMT-based proteome profiling of HAP1 cells, defines further the effects of nicotine on non-neuronal cellular proteomes.