Stable Isotope Labeling Research Articles

The Dynamic and Crucial Role of the Arginine Methylproteome in Myoblast Cell Differentiation.

Protein arginine methylation is an extensive and functionally significant post-translational modification. However, little is known about its role in differentiation at the systems level. Using stable isotope labeling by amino acids in cell culture (SILAC) proteomics of whole proteome analysis in proliferating or five-day differentiated mouse C2C12 myoblasts, followed by high-resolution mass spectrometry, biochemical assays, and specific immunoprecipitation of mono- or dimethylated arginine peptides, we identified several protein families that were differentially methylated on arginine. Our study is the first to reveal global changes in the arginine mono- or dimethylation of proteins in proliferating myoblasts and differentiated myocytes and to identify enriched protein domains and novel short linear motifs (SLiMs). Our data may be crucial for dissecting the links between differentiation and cancer growth.

Mycorrhiza-feeding soil invertebrates in two coniferous forests traced with 13C labelling.

Mycorrhizal fungi represent a potentially abundant carbon resource for soil animals, but their role in soil food webs remains poorly understood. To detect taxa that are trophically linked to the extraradical mycelium of mycorrhizal fungi, we used stable isotope (13C) labelling of whole trees in combination with the in-growth mesh bag technique in two coniferous forests. This allowed us to detect the flux of carbon in the mycelium of mycorrhizal fungi, and consequently in the tissues of soil invertebrates. The mycorrhizal fungal genera constituted 93.5% of reads in mycelium samples from the in-growth mesh bags. All mycelium from in-growth mesh bags and about 32% of the invertebrates sampled (in total 11 taxa) received the 13C label after 45days of exposure. The extent of feeding of soil invertebrates on the mycelium of mycorrhizal fungi depended on the taxonomic affinity of the animals. The strongest trophic link to the mycorrhiza-derived carbon was detected in Isotomidae (Collembola) and Oppiidae (Oribatida). The label was also observed in the generalist predators, indicating the propagation of mycorrhiza-derived carbon into the higher trophic levels of the soil food web. Higher 13C labelling in the tissues of euedaphic Collembola and Oribatida compared to atmobiotic and hemiedaphic families indicates the importance of mycorrhizal fungi as a food resource for invertebrates in deeper soil horizons.

Gut microbiota promote liver regeneration through hepatic membrane phospholipid biosynthesis

Hepatocyte growth and proliferation depends on membrane phospholipid biosynthesis. Short-chain fatty acids (SCFAs) generated by bacterial fermentation, delivered through the gut-liver axis, significantly contribute to lipid biosynthesis. We therefore hypothesized that dysbiotic insults like antibiotic treatment not only affect gut microbiota, but also impair hepatic lipid synthesis and liver regeneration. Stable isotope labeling and 70% partial hepatectomy (PHx) was carried out in C57Bl/6J wild-type mice, in mice treated with broad-spectrum antibiotics, in germ-free mice and mice colonized with minimal microbiota. The microbiome was analyzed by 16S rRNA gene sequencing and microbial culture. Gut content, liver, blood and primary hepatocyte organoids were tested by mass spectrometry-based lipidomics, quantitative reverse-transcription PCR (qRT-PCR), immunoblot and immunohistochemistry for expression of proliferative and lipogenic markers. Matched biopsies from hyperplastic and hypoplastic liver tissue of patients subjected to surgical intervention to induce hyperplasia were analyzed by qRT-PCR for lipogenic enzymes. Three days of antibiotic treatment induced persistent dysbiosis with significantly decreased beta-diversity and richness, but a massive increase of Proteobacteria, accompanied by decreased colonic SCFAs. After PHx, antibiotic-treated mice showed delayed liver regeneration, increased mortality, impaired hepatocyte proliferation and decreased hepatic phospholipid synthesis. Expression of the lipogenic enzyme SCD1 was upregulated after PHx but delayed by antibiotic treatment. Germ-free mice essentially recapitulated the phenotype of antibiotic treatment. Phospholipid biosynthesis, hepatocyte proliferation, liver regeneration and survival were rescued in gnotobiotic mice colonized with a minimal SCFA-producing microbial community. SCFAs induced the growth of murine hepatocyte organoids and hepatic SCD1 expression in mice. Further, SCD1 was required for proliferation of human hepatoma cells and was associated with liver regeneration in human patients. Gut microbiota are pivotal for hepatic membrane phospholipid biosynthesis and liver regeneration. Gut microbiota affect hepatic lipid metabolism through the gut-liver axis, but the underlying mechanisms are poorly understood. Perturbations of the gut microbiome, e.g. by antibiotics, impair the production of bacterial metabolites, which normally serve as building blocks for membrane lipids in liver cells. As a consequence, liver regeneration and survival after liver surgery is severely impaired. Even though this study is preclinical, its results might allow physicians in the future to improve patient outcomes after liver surgery, by modulation of gut microbiota or their metabolites.

Primary Structure of Glycans by NMR Spectroscopy.

Glycans, carbohydrate molecules in the realm of biology, are present as biomedically important glycoconjugates and a characteristic aspect is that their structures in many instances are branched. In determining the primary structure of a glycan, the sugar components including the absolute configuration and ring form, anomeric configuration, linkage(s), sequence, and substituents should be elucidated. Solution state NMR spectroscopy offers a unique opportunity to resolve all these aspects at atomic resolution. During the last two decades, advancement of both NMR experiments and spectrometer hardware have made it possible to unravel carbohydrate structure more efficiently. These developments applicable to glycans include, inter alia, NMR experiments that reduce spectral overlap, use selective excitations, record tilted projections of multidimensional spectra, acquire spectra by multiple receivers, utilize polarization by fast-pulsing techniques, concatenate pulse-sequence modules to acquire several spectra in a single measurement, acquire pure shift correlated spectra devoid of scalar couplings, employ stable isotope labeling to efficiently obtain homo- and/or heteronuclear correlations, as well as those that rely on dipolar cross-correlated interactions for sequential information. Refined computer programs for NMR spin simulation and chemical shift prediction aid the structural elucidation of glycans, which are notorious for their limited spectral dispersion. Hardware developments include cryogenically cold probes and dynamic nuclear polarization techniques, both resulting in enhanced sensitivity as well as ultrahigh field NMR spectrometers with a 1H NMR resonance frequency higher than 1 GHz, thus improving resolution of resonances. Taken together, the developments have made and will in the future make it possible to elucidate carbohydrate structure in great detail, thereby forming the basis for understanding of how glycans interact with other molecules.

Quantitative proteomic analysis reveals the effects of mu opioid agonists on HT22 cells.

Introduction: At present, the mu opioid receptor is the most important neuroaesthetics receptor in anesthesiology research, and the damage that it does to the nervous system is unknown. Methods: We investigated the effects of loperamide, an agonist of the mu opioid receptor, on protein expression in HT22 cells using stable isotope labeling of amino acids in cell culture (SILAC), immobilized metal affinity chromatography (IMAC) enrichment, and high-resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS). A total of 7,823 proteins were identified. Results and Discussion: Bioinformatic analysis revealed that mu opioid receptor agonism can induce distinct changes in the proteome of HT22 cells. These findings improve our understanding of narcotic drugs.

Generation of an Aptamer Targeting Receptor-Type Tyrosine-Protein Phosphatase F

Cell-SELEX is a powerful tool to generate aptamers that specifically bind the native molecules on living cells. Here, we report an aptamer ZAJ4a generated by cell-SELEX. The molecular target of ZAJ4a was pulled down by the enriched cell-SELEX pool and identified to be the receptor-type tyrosine-protein phosphatase F (PTPRF) through a stable isotope labeling using amino acids in cell culture (SILAC)-based quantitative proteomic method. ZAJ4a showed high binding affinity with nanomolar range to cancer cells expressing PTPRF. Meanwhile, PTPRF was proven to highly express on several cancer cell lines using ZAJ4a as a molecular probe and to highly express in many kinds of cancer samples using gene expression profiling interactive analysis (GEPIA2) from the TCGA and GTEx databases. These results indicate that the aptamer generated by cell-SELEX showed good specificity at the molecular level. This cell-SELEX and target identification strategies show great potential for identifying biomarkers on the cell surface.

Monitoring mitochondrial translation by pulse SILAC

Mitochondrial ribosomes are specialized to translate the 13 membrane proteins encoded in the mitochondrial genome, which shapes the oxidative phosphorylation complexes essential for cellular energy metabolism. Despite the importance of mitochondrial translation (MT) control, it is challenging to identify and quantify the mitochondrial-encoded proteins because of their hydrophobic nature and low abundance. Here, we introduce a mass spectrometry-based proteomic method that combines biochemical isolation of mitochondria with pulse stable isotope labeling by amino acids in cell culture. Our method provides the highest protein identification rate with the shortest measurement time among currently available methods, enabling us to quantify 12 of the 13 mitochondrial-encoded proteins. We applied this method to uncover the global picture of (post-)translational regulation of both mitochondrial- and nuclear-encoded subunits of oxidative phosphorylation complexes. We found that inhibition of MT led to degradation of orphan nuclear-encoded subunits that are considered to form subcomplexes with the mitochondrial-encoded subunits. This method should be readily applicable to study MT programs in many contexts, including oxidative stress and mitochondrial disease.

Determination of 25-hydroxyvitamin D in serum by pre-column derivatization-stable isotope labeling-ultra performance liquid chromatography-quadrupole electrostatic field orbitrap high-resolution mass spectrometry

To establish a rapid and accurate method for the determination of 25-hydroxyvitamin D_3(25(OH)D_3) and 25-hydroxyvitamin D_2(25(OH)D_2)in serum by pre-column derivatization with stable isotope labeling and ultra performance liquid chromatography-quadrupole electrostatic field orbitrap high-resolution mass spectrometry, which could be used to diagnose vitamin deficiency and to assess the nutritional status of vitamin D in the population. The serum samples with isotopic internal standard were extracted by mixed extraction solvent(ethyl acetate∶hexane = 2∶1, V/V), centrifuged and dried by the nitrogen blowing, and derivatized with 4-phenyl-1, 2, 4-triazoline-3, 5-dione(PTAD). The reaction products were separated on a BEH C_(18) column(2.1 mm×50 mm, 1.7 μm), and eluted with a 0.1% formic acid/water solution-acetonitrile gradient. The mass spectrometry was performed in positive electrospray ionization(ESI~+) and parallel reaction monitoring(PRM) for the detection of the targets, and quantified by isotope internal standard. The limits of detection both for 25(OH)D_3 and 25(OH)D_2 were 0.01 ng/mL and the limits of quantification were 0.03 ng/mL. The concentration series of 25(OH)D_2 ranged from 0.5 to 40.0 ng/mL and the concentration series of 25(OH)D_3 ranged from 2.5 to 200.0 ng/mL. The recoveries(n=5) were 95.7%-101.3% for 25(OH)D_3, and 98.7%-108.6% for 25(OH)D_2, with the relative standard deviations(RSDs) of 0.5%-4.9% and 2.2%-3.4%, respectively. The accuracy of the determination of 25(OH)D_3 and 25(OH)D_2 in Level 2 serum of the 25(OH)D standard reference material NIST SRM 972a was 104.8% and 94.9%, respectively. The M(P25, P75)serum levels of 25(OH)D_3 and 25(OH)D_2 for 116 pregnant women at the first trimester from Beijing were 25.7(20.8, 32.6) ng/mL and 0.8(0.4, 1.1) ng/mL, respectively. This method is highly sensitive, qualitatively accurate and suitable for evaluation and monitoring the nutritional status of vitamin D in the population.

Identification of potential HDAC11 deacylase substrates by affinity pulldown MS.

Lysine fatty acylation is a protein posttranslational modification (PTM) that has been linked to various important biological processes. HDAC11, the sole member of class IV of histone deacetylases (HDACs), has been shown to have high lysine defatty-acylase activity. In order to better understand the functions of lysine fatty acylation and its regulation by HDAC11, it is important to identify the physiological substrates of HDAC11. This can be achieved through profiling the interactome of HDAC11 using a stable isotope labeling with amino acids in cell culture (SILAC) proteomics strategy. Here we describe a detailed method on using SILAC to identify the interactome of HDAC11. This method can be similarly used to identify the interactome, and thus potential substrates, of other PTM enzymes.

Nonribosomal peptides protect Pseudomonas nunensis 4A2e from amoebal and nematodal predation.

The rhizosphere is a highly competitive environment forcing bacteria to evolve strategies to oppose their enemies. The production of toxic secondary metabolites allows bacteria to counteract predators. In this study, we describe the anti-predator armamentarium of the soil-derived bacterium Pseudomonas nunensis 4A2e. Based on a genome mining approach, we identified several biosynthetic gene clusters coding for nonribosomal peptide synthetases. Generation of gene deletion mutants of the respective clusters shows a loss of defense capabilities. We isolated the novel lipopeptides keanumycin D and nunapeptins B and C, and fully elucidated their structures by a combination of in-depth mass spectrometry experiments, stable isotope labelling, and chemical synthesis. Additionally, investigation of the quorum sensing-dependent biosynthesis allowed us to elucidate parts of the underlying regulation of the biosynthetic machinery. Ecology-inspired bioassays highlight the role of these peptides as a defence strategy against protozoans and led us to find a previously unknown function against the bacterivorous nematode Oscheius myriophilus.

A feasible protocol to profile bile acids in dried blood spots from rats using a UHPLC-MS/MS method combining a surrogate matrix.

Dried blood spot (DBS) sampling is a promising method for microliter blood sample collection with the advantages of convenient transportation, storage and clinical operations. However, it is challenging to develop an analytical protocol to determine endogenous metabolites, such as bile acids (BAs) in DBSs, due to the low-blood-volume character of DBSs and the complex features of filter paper. Herein, we developed a method of fast ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) to profile and quantify BAs in DBSs. The pretreatment methods were optimized and a two-step solvent addition method, where a small amount of water was firstly added to moisten the DBS and then methanol was added, showed high extraction efficiency for multiple BAs in DBSs. The UHPLC-MS/MS conditions were optimized and 35BAs in different types could be profiled with good resolution and quantified with acceptable precision and accuracy. Preparation of a DBS surrogate matrix without endogenous BAs has been well developed using rat erythrocytes in BSA solution and showed good performance on both the signal suppression/enhancement percentage and parallelism assessment evaluation of three different stable-isotope-labeled (SIL) BAs. The established protocol was successfully applied to profile BAs in DBSs of intrahepatic cholestasis model and healthy control rats with good repeatability. To our knowledge, it is the first time that 35 BAs in DBSs could be well profiled and an appropriate DBS surrogate matrix has been developed. This protocol presents future-oriented applications of DBSs for relevant preclinical studies to profile BAs and probe biomarkers.

13CO2-labelling and Sampling in Algae for Flux Analysis of Photosynthetic and Central Carbon Metabolism.

The flux in photosynthesis can be studied by performing 13CO2 pulse labelling and analysing the temporal labelling kinetics of metabolic intermediates using gas or liquid chromatography linked to mass spectrometry. Metabolic flux analysis (MFA) is the primary approach for analysing metabolic network function and quantifying intracellular metabolic fluxes. Different MFA approaches differ based on the metabolic state (steady vs. non-steady state) and the use of stable isotope tracers. The main methodology used to investigate metabolic systems is metabolite steady state associated with stable isotope labelling experiments. Specifically, in biological systems like photoautotrophic organisms, isotopic non-stationary 113C metabolic flux analysis at metabolic steady state with transient isotopic labelling (13C-INST-MFA) is required. The common requirement for metabolic steady state, alongside its very short half-timed reactions, complicates robust MFA of photosynthetic metabolism. While custom gas chambers design has addressed these challenges in various model plants, no similar tools were developed for liquid photosynthetic cultures (e.g., algae, cyanobacteria), where diffusion and equilibration of inorganic carbon species in the medium entails a new dimension of complexity. Recently, a novel tailor-made microfluidics labelling system has been introduced, supplying short 13CO2 pulses at steady state, and resolving fluxes across most photosynthetic metabolic pathways in algae. The system involves injecting algal cultures and medium containing pre-equilibrated inorganic 13C into a microfluidic mixer, followed by rapid metabolic quenching, enabling precise seconds-level label pulses. This was complemented by a 13CO2-bubbling-based open labelling system (photobioreactor), allowing long pulses (minutes-hours) required for investigating fluxes into central C metabolism and major products. This combined labelling procedure provides a comprehensive fluxome cover for most algal photosynthetic and central C metabolism pathways, thus allowing comparative flux analyses across algae and plants.

HBV X Protein Induces Degradation of UBXN7, a Novel Negative Regulator of NF-κB Signaling, to Promote HBV Replication.

Chronic hepatitis B virus (HBV) infection is a leading cause of hepatocellular carcinoma. However, the function and mechanism of the effect of HBV on host protein ubiquitination remain largely unknown. We aimed at characterizing whether and how HBV promotes self-replication by affecting host protein ubiquitination. In this study, we identified UBXN7, a novel inhibitor for nuclear factor kappa B (NF-κB) signaling, was degraded via interaction with HBV X protein (HBx) to activate NF-κB signaling and autophagy, thereby affecting HBV replication. The expression of UBXN7 was analyzed by Western blot and quantitative reverse transcription polymerase chain reaction in HBV-transfected hepatoma cells and HBV-infected primary human hepatocytes (PHHs). The effects of UBXN7 on HBV replication were analyzed by using invitro and invivo assays, including stable isotope labeling by amino acids in cell culture (SILAC) analysis. Changes in HBV replication and the associated molecular mechanisms were analyzed in hepatoma cell lines. SILAC analyses showed that the ubiquitination of UBXN7 was significantly increased in HepG2.2.15 cells compared with control cells. After HBV infection, HBx protein interacted with UBXN7 to promote K48-linked ubiquitination of UBXN7 at K99, leading to UBXN7 degradation. On the other hand, UBXN7 interacted with the ULK domain of IκB kinase β through its ubiquitin-associating domain to facilitate its degradation. This in turn reduced NF-κB signaling, leading to reduced autophagy and consequently decreased HBV replication.

CYP46A1 activation by low-dose efavirenz enhances brain cholesterol metabolism in subjects with early Alzheimer’s disease

BackgroundEfavirenz is an anti-HIV drug, and cytochrome P450 46A1 (CYP46A1) is a CNS-specific enzyme that metabolizes cholesterol to 24-hydroxycholesterol (24HC). We have previously shown that allosteric CYP46A1 activation by low-dose efavirenz in a transgenic mouse model of Alzheimer’s disease (AD) enhanced both cholesterol elimination and turnover in the brain and improved animal performance in memory tests. Here, we sought to determine whether CYP46A1 could be similarly activated by a low-dose efavirenz in human subjects. MethodsThis pilot study enrolled 5 subjects with early AD. Participants were randomized to placebo (n = 1) or two daily efavirenz doses (50 mg and 200 mg, n = 2 for each) for 20 weeks and evaluated for safety and CYP46A1 target engagement (plasma 24HC levels). A longitudinal mixed model was used to ascertain the statistical significance of target engagement. We also measured 24HC in CSF and conducted a unique stable isotope labeling kinetics (SILK) study with deuterated water to directly measure CYP46A1 activity changes in the brain.ResultsIn subjects receiving efavirenz, there was a statistically significant within-group increase (P ≤ 0.001) in the levels of plasma 24HC from baseline. The levels of 24HC in the CSF of subjects on the 200-mg dose of efavirenz were also increased. Target engagement was further supported by the labeling kinetics of 24HC by deuterated water in the SILK study. There were no serious adverse effects in any subjects.ConclusionsOur findings suggest efavirenz target engagement in human subjects with early AD. This supports the pursuit of a larger trial for further determination and confirmation of the efavirenz dose that exerts maximal enzyme activation, as well as evaluation of this drug’s effects on AD biomarkers and clinical symptomatology.Trial registrationClinicalTrials.gov, NCT03706885.

Combining Metabolic Pulse Labeling and Quantitative Proteomics to Monitor Protein Synthesis Upon Viral Infection.

Viruses like influenza A virus (IAV) hijack host cells in order to replicate. To actively and abundantly synthesize viral proteins, they reprogram the cellular transcriptional and translational landscape. Here, we present a proteomic approach that allows us to quantify the differences in host and viral protein synthesis comparatively for different strains of IAV. The method is based on combining quantitative proteomics using stable isotope labelling by amino acids in cell culture (SILAC) and bioorthogonal labeling with methionine analogs. This methodology accurately quantifies synthesis of host and viral proteins with high temporal resolution and faithfully detects global changes in cellular translation capacity. It thus provides unique insights into the dynamics of protein synthesis as the infection progresses.

Pyridinium and ammonium stable isotope labeling agents and their performance in the analysis of alkylamines in food and food packaging materials

Two pairs of stable isotope labeling (SIL) agents, (4-carboxyphenyl)trimethylammonium iodide (d0-CPTA) and its deuterated counterpart d3-CPTA, 1-methyl-nicotinamide iodide (d0-MNA) and its deuterated counterpart d3-MNA, were designed and synthesized. Their mass spectrometry (MS) sensitivity enhancement effect was studied and compared with commercial dansyl chloride to provide inspiration for labeling agent design. CPTA with quaternary ammonium group showed much higher MS sensitivity enhancement effect and was applied to the SIL analysis of alkylamines in food and food packaging materials. The matrix effect was minimized due to the SIL strategy and the permanent charge of the CPTA. The limits of detection (LODs) were in the range of 2.9–5.1 ng/L, and the limits of quantitation (LOQs) were in the range of 9.6–16.8 ng/L. The recoveries ranged from 91.2 % to 97.1 % with relative standard deviations of less than 6.6 %, and the matrix effect ranged from −1.8 % to −4.9 %.

Stable isotope labeling for detection of ozonation byproducts in effluent organic matter with FT-ICR-MS

Despite effluent organic matter (EfOM) being a major consumer of ozone during wastewater treatment, little is known about ozonation byproducts (OBPs) produced from EfOM. To unambiguously identify OBPs, heavy ozone was used to ozonate EfOM, resulting in 18O labeled and unlabeled OBPs. Labeled OBPs mostly represent a single 18O transfer and were classified as either direct or indirect OBPs based on the 18O/16O intensity ratios of the isotopologues. Of the 929 labeled OBPs, 84 were unequivocally classified as direct OBPs. The remainder suggest a major contribution by indirect, hydroxyl radical induced formation of OBPs in EfOM. Overall, labelled OBPs possess a low degree of unsaturation and contributed most to OBP peak intensity – marking them as potential end products. A few direct and indirect OBPs with high peak intensity containing 18O and heteroatoms (N, S) were fragmented with CID FT-ICR-MS/MS and screened for indicative neutral losses carrying heavy oxygen. The neutral loss screening was used to detect the 18O location on the OBP and indicate the original functional group in EfOM based on known reaction mechanisms. We identified sulfoxide and sulfonic acid functional groups in selected OBPs – implying the presence of reduced sulfur in EfOM molecules – while no evidence for nitrogen containing functional groups reacting with ozone was found.

Toward a hypothesis-free understanding of how phosphorylation dynamically impacts protein turnover.

The turnover measurement of proteins and proteoforms has been largely facilitated by workflows coupling metabolic labeling with mass spectrometry (MS), including dynamic stable isotope labeling by amino acids in cell culture (dynamic SILAC) or pulsed SILAC (pSILAC). Very recent studies including ours have integrated themeasurement of post-translational modifications (PTMs) at the proteome level (i.e., phosphoproteomics) with pSILAC experiments in steady state systems, exploring the link between PTMs and turnover at the proteome-scale. An open question in the field is how to exactly interpret these complex datasets in a biological perspective. Here, we present a novel pSILAC phosphoproteomic dataset which was obtained during a dynamic process of cell starvation using data-independent acquisition MS (DIA-MS). To provide an unbiased "hypothesis-free" analysis framework, we developed a strategy to interrogate how phosphorylation dynamically impacts protein turnover across the time series data. With this strategy, we discovered a complex relationship between phosphorylation and protein turnover that was previously underexplored. Our results further revealed a link between phosphorylation stoichiometry with the turnover of phosphorylated peptidoforms. Moreover, our results suggested that phosphoproteomic turnover diversity cannot directly explain the abundance regulation of phosphorylation during cell starvation, underscoring the importance of future studies addressing PTM site-resolved protein turnover.

Flotillin-2 regulates epidermal growth factor receptor activation, degradation by Cbl-mediated ubiquitination, and cancer growth

Epidermal growth factor receptor (EGFR) signaling is frequently dysregulated in various cancers. The ubiquitin ligase Casitas B-lineage lymphoma proto-oncogene (Cbl) regulates degradation of activated EGFR through ubiquitination and acts as an adaptor to recruit proteins required for trafficking. Here, we used stable isotope labeling with amino acids in cell culture mass spectrometry to compare Cbl complexes with or without epidermal growth factor (EGF) stimulation. We identified over a hundred novel Cbl interactors, and a secondary siRNA screen found that knockdown of Flotillin-2 (FLOT2) led to increased phosphorylation and degradation of EGFR upon EGF stimulation in HeLa cells. In PC9 and H441cells, FLOT2 knockdown increased EGF-stimulated EGFR phosphorylation, ubiquitination, and downstream signaling, reversible by EGFR inhibitor erlotinib. CRISPR knockout (KO) of FLOT2 in HeLa cells confirmed EGFR downregulation, increased signaling, and increased dimerization and endosomal trafficking. Furthermore, we determined that FLOT2 interacted with both Cbl and EGFR. EGFR downregulation upon FLOT2 loss was Cbl dependent, as coknockdown of Cbl and Cbl-b restored EGFR levels. In addition, FLOT2 overexpression decreased EGFR signaling and growth. Overexpression of wildtype (WT) FLOT2, but not the soluble G2A FLOT2 mutant, inhibited EGFR phosphorylation upon EGF stimulation in HEK293T cells. FLOT2 loss induced EGFR-dependent proliferation and anchorage-independent growth. Lastly, FLOT2 KO increased tumor formation and tumor volume in nude mice and NSG mice, respectively. Together, these data demonstrated that FLOT2 negatively regulated EGFR activation and dimerization, as well as its subsequent ubiquitination, endosomal trafficking, and degradation, leading to reduced proliferation invitro and invivo.

Protein-Peptide Turnover Profiling reveals the order of PTM addition and removal during protein maturation

Post-translational modifications (PTMs) regulate various aspects of protein function, including degradation. Mass spectrometric methods relying on pulsed metabolic labeling are popular to quantify turnover rates on a proteome-wide scale. Such data have traditionally been interpreted in the context of protein proteolytic stability. Here, we combine theoretical kinetic modeling with experimental pulsed stable isotope labeling of amino acids in cell culture (pSILAC) for the study of protein phosphorylation. We demonstrate that metabolic labeling combined with PTM-specific enrichment does not measure effects of PTMs on protein stability. Rather, it reveals the relative order of PTM addition and removal along a protein’s lifetime—a fundamentally different metric. This is due to interconversion of the measured proteoform species. Using this framework, we identify temporal phosphorylation sites on cell cycle-specific factors and protein complex assembly intermediates. Our results thus allow tying PTMs to the age of the modified proteins.