Isobaric Tags Research Articles

ITRAQ-Based Proteomic Analysis Unveils NCAM1 as a Novel Regulator in Doxorubicin-Induced Cardiotoxicity and DT-010-Exerted Cardioprotection

Background: Doxorubicin (DOX) causes lethal cardiotoxicity, which limits its clinical utility. The molecular mechanisms and effective strategies to combat its cardiotoxicity need further exploration. DT-010, a novel conjugate of danshensu (DSS) and tetramethylpyrazine( TMP), is considered a promising candidate for treating DOX-induced cardiotoxicity. In this study, we aimed to investigate the underlying molecular mechanisms of DOX-induced cardiotoxicity and the cardioprotective effects of DT-010. Methods: Isobaric tags for relative and absolute quantitation (iTRAQ) in proteomics analysis was employed to analyze the differentially expressed proteins in DOX-injuried hearts. Gene ontology (GO) enrichment analysis and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were carried out to evaluated the potential mechanisms of DOXinduced cardiotoxicity. The effects of NCAM1 on DOX-induced cardiotoxicity in H9c2 cells, as well as the cardioprotection of DT-010 were assessed through NACM1siRNA transfection, cell viability assay, cell apoptosis staining, reactive oxygen species measurement, and western blotting. Results: Proteomics analysis revealed that several signaling pathways, including the tricarboxylic acid (TCA) cycle and oxidative phosphorylation, were involved in DOX-induced cardiotoxicity. NCAM1 is one of the significantly changed proteins. DT-010 treatment regulated NCAM1 protein expression. Silencing NCAM1 in DOX-treated H9c2 cells decreased cell viability, increased cell apoptosis and reactive oxygen species (ROS) generation, and attenuated the cardioprotective effects of DT-010. Furthermore, NCAM1 knockdown promoted p38 activation and inhibited the expressions of peroxisome proliferator-activated receptor gamma coactivator- 1 alpha (PGC-1α) and heme oxygenase-1 (HO-1) in DOX-treated cells. Conclusion: These findings indicate a definite role of NCAM1 in DOX-induced cardiotoxicity and DT-010-exerted cardioprotection, which is mediated through the p38 and Sirt1/PGC- 1α/HO-1 pathway.

PhoXplex: Combining Phospho-enrichable Cross-Linking with Isobaric Labeling for Quantitative Proteome-Wide Mapping of Protein Interfaces.

Integrating cross-linking mass spectrometry (XL-MS) into structural biology workflows provides valuable information about the spatial arrangement of amino acid stretches, which can guide elucidation of protein assembly architecture. Additionally, the combination of XL-MS with peptide quantitation techniques is a powerful approach to delineate protein interface dynamics across diverse conditions. While XL-MS is increasingly effective with isolated proteins or small complexes, its application to whole-cell samples poses technical challenges related to analysis depth and throughput. The use of enrichable cross-linkers has greatly improved the detectability of protein interfaces in a proteome-wide scale, facilitating global protein-protein interaction mapping. Therefore, bringing together enrichable cross-linking and multiplexed peptide quantification is an appealing approach to enable comparative characterization of structural attributes of proteins and protein interactions. Here, we combined phospho-enrichable cross-linking with TMT labeling to develop a streamline workflow (PhoXplex) for the detection of differential structural features across a panel of cell lines in a global scale. We achieved deep coverage with quantification of over 9000 cross-links and long loop-links in total including potentially novel interactions. Overlaying AlphaFold predictions and disorder protein annotations enables exploration of the quantitative cross-linking data set, to reveal possible associations between mutations and protein structures. Lastly, we discuss current shortcomings and perspectives for deep whole-cell profiling of protein interfaces at large-scale.

Identification of FGG as a Biomarker in Early Gastric Cancer via Tissue Proteomics and Clinical Verification.

Early and accurate diagnosis of gastric cancer (GC) is essential for reducing mortality and improving patient well-being. However, methods for the early diagnosis of GC are still lacking. In this study, by isobaric tagging for relative and absolute quantitation (iTRAQ), we identified 336 proteins that overlapped among the upregulated differentially expressed proteins (DEPs) in early gastric cancer (EGC) versus progressive gastric cancer (PGC), upregulated DEPs in EGC versus nongastric cancer (NGC), and nonsignificant proteins in EGC versus NGC. These DEPs were involved primarily in the neutrophil-related immune response. Network analysis of proteins and pathways revealed that fibrinogen α (FGA), β (FGB), and γ (FGG) are candidates for distinguishing EGC. Furthermore, parallel reaction monitoring (PRM), immunohistochemistry (IHC), and Western blot (WB) assays of clinical samples confirmed that, compared with that in PGC and NGC, only FGG was uniquely and significantly upregulated in the gastric mucosa of EGC. Our results demonstrated that FGG in the gastric mucosa could be a novel biomarker to diagnose EGC patients via endoscopy.

Achieving a 35-Plex Tandem Mass Tag Reagent Set through Deuterium Incorporation.

Mass spectrometry-based sample multiplexing with isobaric tags permits the development of high-throughput and precise quantitative biological assays with proteome-wide coverage and minimal missing values. Here, we nearly doubled the multiplexing capability of the TMTpro reagent set to a 35-plex through the incorporation of one deuterium isotope into the reporter group. Substituting deuterium frequently results in suboptimal peak coelution, which can compromise the accuracy of reporter ion-based quantification. To counteract the deuterium effect on quantitation, we implemented a strategy that necessitated the segregation of nondeuterium and deuterium-containing channels into distinct subplexes during normalization procedures, with reassembly through a common bridge channel. This multiplexing strategy of "design independent sub-plexes but acquire together" (DISAT) was used to compare protein expression differences between human cell lines and in a cysteine-profiling (i.e., chemoproteomics) experiment to identify compounds binding to cysteine-113 of Pin1.

DiDBiT-TMT: A Novel Method to Quantify Changes in the Proteomic Landscape Induced by Neural Plasticity.

Direct detection of biotinylated proteins (DiDBiT) is a proteomic method that can enrich and detect newly synthesized proteins (NSPs) labeled with bio-orthogonal amino acids with 20-fold improved detectability compared to conventional methods. However, DiDBiT has currently been used to compare only two conditions per experiment. Here, we present DiDBiT-TMT, a method that can be used to quantify NSPs across many conditions and replicates in the same experiment by combining isobaric tandem mass tagging (TMT) with DiDBiT. We applied DiDBiT-TMT to brain slices to determine changes in the de novo proteome that occur after inducing chemical long-term potentiation (cLTP) or treatment with the neuromodulator norepinephrine. We successfully demonstrated DiDBiT-TMT's capacity to quantitatively compare up to 9 samples in parallel. We showed that there is a minimal overlap among NSPs that are differentially expressed in cLTP-treated organotypic brain slices, norepinephrine-treated organotypic brain slices, and organotypic slices undergoing combinatorial treatment with norepinephrine and cLTP. Our results point to the possible divergence of the molecular mechanisms underlying these treatments and showcase the applicability of DiDBiT-TMT for studying neurobiology.

189 Association of the liver phosphoproteome with divergent dry matter intake in finishing beef steers

Abstract As the need for improving the sustainability of the beef industry increases, optimizing feed efficiency is of the utmost importance. The liver has a central role in nutrient metabolism, and for ruminants particularly, it is essential for providing glucose through gluconeogenesis via substrates such as propionate, lactate, and glycerol. Further, through cell signaling and vagal afferent signals, the liver acts to stimulate and inhibit dry matter intake (DMI). Thus, the objective of this study was to investigate alterations in the phosphoproteome of finishing beef steers in relation to divergent DMI to identify changes in the abundance of signaling proteins. Angus steers [n = 57; initial body weight (BW) = 518 ± 27 kg] underwent a 63-d intake trial utilizing an Insentec Roughage Intake Control System (Hokofarm Group, Emmeloord, The Netherlands). Prior to beginning the trial, liver biopsy samples were collected from all steers. Samples from steers with the greatest and least DMI (n = 8 each) were utilized for phosphoproteomic analysis. Liver protein was isolated and labeled with isobaric tandem mass tagging reagents. Samples were enriched for phosphopeptides using immobilized metal affinity chromatography and subsequently analyzed using high-performance liquid chromatography tandem mass spectrometry. Spectral data were searched in the National Center for Biotechnology Information Bos taurus protein database using MaxQuant (version 2.2.0.0; Max Planck Inst., Munich, Germany), and the data were analyzed using Perseus (version 2.0.10.0; Max Planck Inst.). Two sample t-tests were conducted to determine the difference in phosphopeptide abundance between steers with high and low DMI. A total of 16,365 phosphopeptides were identified, and 287 were differentially expressed (False Discovery Rate < 0.05). In addition, 2,777 phosphopeptides were differentially expressed based on an unadjusted P < 0.05 which were used for enrichment analysis of Gene Ontology processes and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways using the Database for Annotation, Visualization, and Integrated Discovery. The background for this analysis was all identified phosphopeptides. Gene ontology terms enriched included processes related to phosphorylation activity, intracellular signal transduction, response to insulin, kinase activity, and signaling protein binding. Enriched KEGG pathways included pathways related to insulin signaling, such as AMPK signaling, mTOR signaling, and MAPK signaling. Further, pathways related to glycolysis and gluconeogenesis, glucagon signaling, insulin resistance, carbon metabolism, and various other signaling pathways were also enriched. These results suggest a link between the liver phosphoproteome and DMI, indicating that divergent DMI is associated with alterations in signaling pathways related to nutrient uptake and metabolic processes.

Main metabolic pathways of Solanum nigrum L. hyperaccumulating cadmium except of copper simultaneously through differentially expressed proteins analysis

Determining the hyperaccumulation mechanism of Solanum nigrum L., which exclusively accumulates cadmium (Cd), presents significant challenges due to the difficulty in identifying its unique characteristics. While some metabolic pathways related to Cd accumulation can be explored, there are no methods to ascertain if other heavy metals may share the same pathways. Isobaric tags for relative and absolute quantitation (iTRAQ) were employed to investigate the metabolic pathways associated with Cd hyperaccumulation and Cu accumulation (non-Cu hyperaccumulator) by comparing differentially expressed proteins (DEPs). The results showed that 27 intersecting DEPs reflecting relative metabolic pathways related to Cd accumulation were identified by comparing DEPs in leaves and roots, including carbon metabolism, aminoacyl-tRNA biosynthesis, phagosome, peroxisome, as well as starch and sucrose metabolism. These pathways might be responsible for the values of Cd enrichment factor (EF) and translocation factor (TF) exceeding 1, associated with key proteins participated in phosphoenolpyruvate, carboxylase, chloroplastic catalytic activity, and granule-bound starch synthase I. The combined metabolic pathways identified by 2 intersecting DEPs related to Cu accumulation could result in Cu EF >1 in the 0.2 Cu mg kg−1 treatment, EF <1 in the 5 mg kg−1 treatment, and TF<1 in both treatments, associated with key proteins, which might concern photosynthesis-antenna proteins and hydroxymethylbilane synthase. No metabolic pathways related to simultaneous accumulation of Cd and Cu has been identified. The identified DEPs were validated using Western blotting with five key proteins. Additionally, Western blotting and yeast mutant confirmed the presence of proteins related to carbon fixation in photosynthetic organisms, carbon metabolism, peroxisome, as well as starch and sucrose metabolism. Photosynthetic, O2•−, H2O2 and non-enzymatic antioxidants indices reflecting protein-related differences indirectly supported the above results. These findings are crucial for further exploration of the Cd hyperaccumulation mechanism.

The Potential of Eight Plasma Proteins as Biomarkers in Redefining Leptospirosis Diagnosis.

Leptospirosis, a notifiable endemic disease in Malaysia, has higher mortality rates than regional dengue fever. Diverse clinical symptoms and limited diagnostic methods complicate leptospirosis diagnosis. The demand for accurate biomarker-based diagnostics is increasing. This study investigated the plasma proteome of leptospirosis patients with leptospiraemia and seroconversion compared with dengue patients and healthy subjects using isobaric tags for relative and absolute quantitation (iTRAQ)-mass spectrometry (MS). The iTRAQ analysis identified a total of 450 proteins, which were refined to a list of 290 proteins through a series of exclusion criteria. Differential expression in the plasma proteome of leptospirosis patients compared to the control groups identified 11 proteins, which are apolipoprotein A-II (APOA2), C-reactive protein (CRP), fermitin family homolog 3 (FERMT3), leucine-rich alpha-2-glycoprotein 1 (LRG1), lipopolysaccharide-binding protein (LBP), myosin-9 (MYH9), platelet basic protein (PPBP), platelet factor 4 (PF4), profilin-1 (PFN1), serum amyloid A-1 protein (SAA1), and thrombospondin-1 (THBS1). Following a study on a verification cohort, a panel of eight plasma protein biomarkers was identified for potential leptospirosis diagnosis: CRP, LRG1, LBP, MYH9, PPBP, PF4, SAA1, and THBS1. In conclusion, a panel of eight protein biomarkers offers a promising approach for leptospirosis diagnosis, addressing the limitations of the "one disease, one biomarker" concept.

Conjugation of primary amine groups in targeted proteomics.

Primary amines, in the form of unmodified N-terminus of peptide/protein and unmodified lysine residue, are perhaps the most important functional groups that can serve as the starting points in proteomic analysis, especially via mass spectrometry-based approaches. A variety of multifunctional probes that conjugate primary amine groups through covalent bonds have been developed and employed to facilitate protein/protein complex characterization, including identification, quantification, structure and localization elucidation, protein-protein interaction investigation, and so forth. As an integral part of more accurate peptide quantification in targeted proteomics, isobaric stable isotope-coded primary amine labeling approaches eventually facilitated protein/peptide characterization at the single-cell level, paving the way for single-cell proteomics. The development and advances in the field can be reviewed in terms of key components of a multifunctional probe: functional groups and chemistry for primary amine conjugation; hetero-bifunctional moiety for separation/enrichment of conjugated protein/protein complex; and functionalized linker/spacer. Perspectives are primarily focused on optimizing primary amine conjugation under physiological conditions to improve characterization of native proteins, especially those associated with the surface of living cells/microorganisms.

Acetylation of PGK1 at lysine 323 promotes glycolysis, cell proliferation, and metastasis in luminal A breast cancer cells

BackgroundIn prior research employing iTRAQ (Isobaric Tags for Relative and Absolute Quantitation) technology, we identified a range of proteins in breast cancer tissues exhibiting high levels of acetylation. Despite this advancement, the specific functions and implications of these acetylated proteins in the context of cancer biology have yet to be elucidated. This study aims to systematically investigate the functional roles of these acetylated proteins with the objective of identifying potential therapeutic targets within breast cancer pathophysiology.MethodsAcetylated targets were identified through bioinformatics, with their expression and acetylation subsequently confirmed. Proteomic analysis and validation studies identified potential acetyltransferases and deacetylases. We evaluated metabolic functions via assays for catalytic activity, glucose consumption, ATP levels, and lactate production. Cell proliferation and metastasis were assessed through viability, cycle analysis, clonogenic assays, PCNA uptake, wound healing, Transwell assays, and MMP/EMT marker detection.ResultsAcetylated proteins in breast cancer were primarily involved in metabolism, significantly impacting glycolysis and the tricarboxylic acid cycle. Notably, PGK1 showed the highest acetylation at lysine 323 and exhibited increased expression and acetylation across breast cancer tissues, particularly in T47D and MCF-7 cells. Notably, 18 varieties acetyltransferases or deacetylases were identified in T47D cells, among which p300 and Sirtuin3 were validated for their interaction with PGK1. Acetylation at 323 K enhanced PGK1's metabolic role by boosting its activity, glucose uptake, ATP production, and lactate output. This modification also promoted cell proliferation, as evidenced by increased viability, S phase ratio, clonality, and PCNA levels. Furthermore, PGK1-323 K acetylation facilitated metastasis, improving wound healing, cell invasion, and upregulating MMP2, MMP9, N-cadherin, and Vimentin while downregulating E-cadherin.ConclusionPGK1-323 K acetylation was significantly elevated in T47D and MCF-7 luminal A breast cancer cells and this acetylation could be regulated by p300 and Sirtuin3. PGK1-323 K acetylation promoted cell glycolysis, proliferation, and metastasis, highlighting novel epigenetic targets for breast cancer therapy.

Coupling Microdroplet-Based Sample Preparation, Multiplexed Isobaric Labeling, and Nanoflow Peptide Fractionation for Deep Proteome Profiling of the Tissue Microenvironment.

There is increasing interest in developing in-depth proteomic approaches for mapping tissue heterogeneity in a cell-type-specific manner to better understand and predict the function of complex biological systems such as human organs. Existing spatially resolved proteomics technologies cannot provide deep proteome coverage due to limited sensitivity and poor sample recovery. Herein, we seamlessly combined laser capture microdissection with a low-volume sample processing technology that includes a microfluidic device named microPOTS (microdroplet processing in one pot for trace samples), multiplexed isobaric labeling, and a nanoflow peptide fractionation approach. The integrated workflow allowed us to maximize proteome coverage of laser-isolated tissue samples containing nanogram levels of proteins. We demonstrated that the deep spatial proteomics platform can quantify more than 5000 unique proteins from a small-sized human pancreatic tissue pixel (∼60,000 μm2) and differentiate unique protein abundance patterns in pancreas. Furthermore, the use of the microPOTS chip eliminated the requirement for advanced microfabrication capabilities and specialized nanoliter liquid handling equipment, making it more accessible to proteomic laboratories.

ITRAQ-Based Serum Proteomic Analysis Reveals Multifactorial Cellular Function Impairment and Aggravated Systematic Inflammation in Drug-free Obsessive-Compulsive Disorders.

Obsessive-compulsive disorder (OCD) is a debilitating mental disorder with obvious difficulties in treatment. Its pathogenesis has not been fully elucidated. Further understanding of etiology and mechanism needs to be explored further. We employed the isobaric tag for relative and absolute quantitation (iTRAQ)-based proteomic analysis to compare serum proteome profile between OCD patients and healthy controls, in order to find out the possible mechanism of OCD in the downstream biological process. Eighty-one drug-free OCD patients and 78 healthy controls were enrolled. A total of 475 proteins were identified. Totally, 80 proteins with p < 0.05 were selected for gene set enrichment analysis (GSEA), and only those with a fold change ≥1.2 and q value <0.2 between groups were accepted as differentially expressed proteins (DEPs). We observed a significant enrichment of immuno-inflammation-related pathways, along with intriguing expression trends that immuno-inflammation-related proteins were upregulated in GSEA. After that, 2 up-regulated proteins and 13 down-regulated ones were accepted as DEP. According to the available literature, most of the DEPs have not been reported in OCD. These DEPs were enriched in 121 gene ontology (GO) terms, including hepatocyte growth factor receptor activity, angiogenin-PRI complex, and so on. DEPs were enriched in pathways including adherens junction in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Alterations in DEPs including STXBP5L, GRN, and ANG were validated in OCD animal models. Our study suggested that OCD patients manifested multifactorial impairment in neuronal or non-neuronal cellular function under the inflammatory background. Further research employing larger sample sizes, longitudinal design, stratified analysis, and multiomics methodology will be needed. Experiments in laboratories were essential in illuminating the mechanism.

Massively parallel sample preparation for multiplexed single-cell proteomics using nPOP.

Single-cell proteomics by mass spectrometry (MS) allows the quantification of proteins with high specificity and sensitivity. To increase its throughput, we developed nano-proteomic sample preparation (nPOP), a method for parallel preparation of thousands of single cells in nanoliter-volume droplets deposited on glass slides. Here, we describe its protocol with emphasis on its flexibility to prepare samples for different multiplexed MS methods. An implementation using the plexDIA MS multiplexing method, which uses non-isobaric mass tags to barcode peptides from different samples for data-independent acquisition, demonstrates accurate quantification of ~3,000-3,700 proteins per human cell. A separate implementation with isobaric mass tags and prioritized data acquisition demonstrates analysis of 1,827 single cells at a rate of >1,000 single cells per day at a depth of 800-1,200 proteins per human cell. The protocol is implemented by using a cell-dispensing and liquid-handling robot-the CellenONE instrument-and uses readily available consumables, which should facilitate broad adoption. nPOP can be applied to all samples that can be processed to a single-cell suspension. It takes 1 or 2 d to prepare >3,000 single cells. We provide metrics and software (the QuantQC R package) for quality control and data exploration. QuantQC supports the robust scaling of nPOP to higher plex reagents for achieving reliable and scalable single-cell proteomics.

Calcium leakage involved in nematotoxic effects of the Conidiobolus obscurus CytCo protein on the pine wood nematode, Bursaphelenchus xylophilus.

The pine wood nematode Bursaphelenchus xylophilus, a severe invasive species, is responsible for causing widespread pine wilt disease. The CytCo protein, a pore-forming toxin derived from Conidiobolus obscurus, exhibits nematotoxicity towards B. xylophilus. Our present study reveals the expression variation of a range of gene products in B. xylophilus that respond to the effects of CytCo using the isobaric tags for relative and absolute quantification proteomics technology. Functional enrichment analysis indicates that many differentially expressed proteins are linked to calcium signaling system, proteasome, energy production and conversion, and the determination of adult lifespan. It suggests that the dysregulation of calcium homeostasis, energy metabolism, and apoptosis contribute to the CytCo nematotoxicity. Using the calcium ion (Ca2+)-indicator calcein, we detected changes in Ca2+ levels in B. xylophilus, with a significantly increase in fluorescence in the nematode's intestine and pseudocoelom following CytCo treatments. Meanwhile, the apoptosis and reactive oxygen species (ROS) assays showed an enhancement of fluorescence in B. xylophilus cells, with increased CytCo concentrations. The protein toxin CytCo triggers Ca2+ leakage, disrupts Ca2+ balance in B. xylophilus, and induces apoptosis and ROS outburst, thereby intensifying its nematotoxic effects. This finding facilitates our understanding of the modes of action of nematotoxic proteins, and contributes to the development of innovative nematode control strategies. © 2024 Society of Chemical Industry.

Mass Spectrometry Acquisition and Fractionation Recommendations for TMT11 and TMT16 Labeled Samples.

Proteome coverage and accurate protein quantification are both important for evaluating biological systems; however, compromises between quantification, coverage, and mass spectrometry (MS) resources are often necessary. Consequently, experimental parameters that impact coverage and quantification must be adjusted, depending on experimental goals. Among these parameters is offline prefractionation, which is utilized in MS-based proteomics to decrease sample complexity resulting in higher overall proteome coverage upon MS analysis. Prefractionation leads to increases in required MS analysis time, although this is often mitigated by isobaric labeling using tandem-mass tags (TMT), which allow samples to be multiplexed. Here we evaluate common prefractionation schemes, TMT variants, and MS acquisition methods and their impact on protein quantification and coverage. Furthermore, we provide recommendations for experimental design depending on the experimental goals.

6-Plex Tandem Phosphorus Tags (TPT) for Accurate Quantitative Proteomics.

Isobaric chemical labeling is a widely used strategy for high-throughput quantitative proteomics based on mass spectrometry. However, commercially available reagents have high costs in applications as well as the sensitivity limitations for detection of the trace protein samples. Previously, we developed a 2-plex isobaric labeling strategy based on phosphorus chemistry for ultrasensitive proteome quantification with high accuracy. In this work, 6-plex tandem phosphorus tags (TPT) were developed with 3-fold increase in the multiplexing quantitative capacity compared to the 2-plex isobaric phosphorus reagents introduced previously. High isotope enrichment of 18O labeling was incorporated into the phosphoryl group with three exchangeable oxygen atoms by using commercially available H218O. The combinational incorporations of 18O atom in reporter ions and balance group set up the low-cost foundation for development of multiplex TPT reagents. The novel 6-plex TPT reagents could produce phosphoramidate as unique reporter ions with approximately 1 Da mass difference and thus enable 6-plex quantitative analysis in high-resolution ESI-MS/MS analysis. Using HeLa cell tryptic peptides, we concluded that 6-plex TPT reagents could facilitate large-scale accurate quantitative proteomics with very high labeling efficiency.

In-depth phosphoproteomic profiling of the insulin signaling response in heart tissue and cardiomyocytes unveils canonical and specialized regulation

BackgroundInsulin signaling regulates cardiac substrate utilization and is implicated in physiological adaptations of the heart. Alterations in the signaling response within the heart are believed to contribute to pathological conditions such as type-2 diabetes and heart failure. While extensively investigated in several metabolic organs using phosphoproteomic strategies, the signaling response elicited in cardiac tissue in general, and specifically in the specialized cardiomyocytes, has not yet been investigated to the same extent.MethodsInsulin or vehicle was administered to male C57BL6/JRj mice via intravenous injection into the vena cava. Ventricular tissue was extracted and subjected to quantitative phosphoproteomics analysis to evaluate the insulin signaling response. To delineate the cardiomyocyte-specific response and investigate the role of Tbc1d4 in insulin signal transduction, cardiomyocytes from the hearts of cardiac and skeletal muscle-specific Tbc1d4 knockout mice, as well as from wildtype littermates, were studied. The phosphoproteomic studies involved isobaric peptide labeling with Tandem Mass Tags (TMT), enrichment for phosphorylated peptides, fractionation via micro-flow reversed-phase liquid chromatography, and high-resolution mass spectrometry measurements.ResultsWe quantified 10,399 phosphorylated peptides from ventricular tissue and 12,739 from isolated cardiomyocytes, localizing to 3,232 and 3,128 unique proteins, respectively. In cardiac tissue, we identified 84 insulin-regulated phosphorylation events, including sites on the Insulin Receptor (InsrY1351, Y1175, Y1179, Y1180) itself as well as the Insulin receptor substrate protein 1 (Irs1S522, S526). Predicted kinases with increased activity in response to insulin stimulation included Rps6kb1, Akt1 and Mtor. Tbc1d4 emerged as a major phosphorylation target in cardiomyocytes. Despite limited impact on the global phosphorylation landscape, Tbc1d4 deficiency in cardiomyocytes attenuated insulin-induced Glut4 translocation and induced protein remodeling. We observed 15 proteins significantly regulated upon knockout of Tbc1d4. While Glut4 exhibited decreased protein abundance consequent to Tbc1d4-deficiency, Txnip levels were notably increased. Stimulation of wildtype cardiomyocytes with insulin led to the regulation of 262 significant phosphorylation events, predicted to be regulated by kinases such as Akt1, Mtor, Akt2, and Insr. In cardiomyocytes, the canonical insulin signaling response is elicited in addition to regulation on specialized cardiomyocyte proteins, such as Kcnj11Y12 and DspS2597. Details of all phosphorylation sites are provided.ConclusionWe present a first global outline of the insulin-induced phosphorylation signaling response in heart tissue and in isolated adult cardiomyocytes, detailing the specific residues with changed phosphorylation abundances. Our study marks an important step towards understanding the role of insulin signaling in cardiac diseases linked to insulin resistance.Graphical

Comparative Proteomic Analysis of Floral Buds before and after Opening in Walnut (Juglans regia L.).

The walnut (Juglans regia L.) is a typical and an economically important tree species for nut production with heterodichogamy. The absence of female and male flowering periods seriously affects both the pollination and fruit setting rates of walnuts, thereby affecting the yield and quality. Therefore, studying the characteristics and processes of flower bud differentiation helps in gaining a deeper understanding of the regularity of the mechanism of heterodichogamy in walnuts. In this study, a total of 3540 proteins were detected in walnut and 885 unique differentially expressed proteins (DEPs) were identified using the isobaric tags for the relative and absolute quantitation (iTRAQ)-labeling method. Among all DEPs, 12 common proteins were detected in all four of the obtained contrasts. GO and KEGG analyses of 12 common DEPs showed that their functions are distributed in the cytoplasm metabolic pathways, photosynthesis, glyoxylate and dicarboxylate metabolism, and the biosynthesis of secondary metabolites, which are involved in energy production and conversion, synthesis, and the breakdown of proteomes. In addition, a function analysis was performed, whereby the DEPs were classified as involved in photosynthesis, morphogenesis, metabolism, or the stress response. A total of eight proteins were identified as associated with the morphogenesis of stamen development, such as stamen-specific protein FIL1-like (XP_018830780.1), putative leucine-rich repeat receptor-like serine/threonine-protein kinase At2g24130 (XP_018822513.1), cytochrome P450 704B1-like isoform X2 (XP_018845266.1), ervatamin-B-like (XP_018824181.1), probable glucan endo-1,3-beta-glucosidase A6 (XP_018844051.1), pathogenesis-related protein 5-like (XP_018835774.1), GDSL esterase/lipase At5g22810-like (XP_018833146.1), and fatty acyl-CoA reductase 2 (XP_018848853.1). Our results predict several crucial proteins and deepen the understanding of the biochemical mechanism that regulates the formation of male and female flower buds in walnuts.

Analysis of Protein Cysteine Acylation Using a Modified Suspension Trap (Acyl-Trap).

Proteins undergo reversible S-acylation via a thioester linkage in vivo. S-palmitoylation, modification by C16:0 fatty acid, is a common S-acylation that mediates critical protein-membrane and protein-protein interactions. The most widely used S-acylation assays, including acyl-biotin exchange and acyl resin-assisted capture, utilize blocking of free Cys thiols, hydroxylamine-dependent cleavage of the thioester and subsequent labeling of nascent thiol. These assays generally require >500 μg of protein input material per sample and numerous reagent removal and washing steps, making them laborious and ill-suited for high throughput and low input applications. To overcome these limitations, we devised "Acyl-Trap", a suspension trap-based assay that utilizes a thiol-reactive quartz to enable buffer exchange and hydroxylamine-mediated S-acyl enrichment. We show that the method is compatible with protein-level detection of S-acylated proteins (e.g., H-Ras) as well as S-acyl site identification and quantification using "on trap" isobaric labeling and LC-MS/MS from as little as 20 μg of protein input. In mouse brain, Acyl-Trap identified 279 reported sites of S-acylation and 1298 previously unreported putative sites. Also described are conditions for long-term hydroxylamine storage, which streamline the assay. More generally, Acyl-Trap serves as a proof-of-concept for PTM-tailored suspension traps suitable for both traditional protein detection and chemoproteomic workflows.

ESF1 and MIPEP proteins promote estrogen receptor-positive breast cancer proliferation and are associated with patient prognosis

BackgroundEstrogen receptor-positive (ER+) breast cancer accounts for two-thirds of all breast cancers, and its early and late recurrences still threaten patients’ long-term survival and quality of life. Finding candidate tumor antigens and potential therapeutic targets is critical to addressing these unmet needs.MethodThe isobaric tags for relative and absolute quantitation (iTRAQ) proteomic analysis was employed to identify the differentially expressed proteins (DEPs) between ER + breast cancer and corresponding adjacent normal tissue. Candidate DEPs were screened by bioinformatic analyses, and their expression was confirmed by immunohistochemical (IHC) staining and western blot. A series of in vitro experiments, including wound healing assay, colony formation, and cell cycle assay, were performed to reveal the functions of selected DEPs. Additionally, their clinical significances were further analyzed.ResultA total of 369 DEPs (fold change ≥ 2.0 or ≤ 0.66, P < 0.05) were discovered. Compared with normal tissue, 358 proteins were up-regulated and 11 proteins were down-regulated in ER + breast cancer. GO and KEGG enrichment analysis showed that DEPs were closely associated with RNA regulation and metabolic pathways. STRING analysis found ESF1 and MIPEP were the hub genes in breast cancer, whose increased expressions were verified by the IHC staining and western blot. Knocking down ESF1 and MIPEP inhibited colony formation and increased cell apoptosis. Besides, knocking down ESF1 inhibited wound healing but not MIPEP. In addition, ESF1 and MIPEP expression were negatively associated with patient prognosis.ConclusionThe upregulation of ESF1 and MIPEP promoted ER + breast cancer proliferation, which might provide novel targets for the development of new therapies.