High-resolution Proteomic Analysis Research Articles

Integrin α6-containing extracellular vesicles promote lymphatic remodelling for pre-metastatic niche formation in lymph nodes via interplay with CD151.

Heterogeneous extracellular vesicles (EVs) from various types of tumours are acknowledged for inducing the formation of pre-metastatic "niches" in draining lymph nodes (LNs) to promote lymphatic metastasis. In order to identify the specific subpopulations of EVs involved, we performed high-resolution proteomic analysis combined with nanoflow cytometry of bladder cancer (BCa) tissue-derived EVs to identify a novel subset of tumour-derived EVs that contain integrin α6 (ITGA6+EVs) and revealed the positive correlation of ITGA6+EVs with the formation of pre-metastatic niche in draining LNs and lymphatic metastasis in multicentre clinical analysis of 820-case BCa patients. BCa-derived ITGA6+EVs induced E-selectin (SELE)-marked lymphatic remodelling pre-metastatic niche and promoted metastasis in draining LNs through delivering cargo circRNA-LIPAR to lymphatic endothelial cells in vivo and in vitro. Mechanistically, LIPAR linked ITGA6 to the switch II domain of RAB5A and sustained RAB5A GTP-bound activated state, thus maintaining the production of ITGA6+EVs loaded with LIPAR through endosomal trafficking. ITGA6+EVs targeted lymphatic vessels through ITGA6-CD151 interplay and released LIPAR to induce SELE overexpression-marked lymphatic remodelling pre-metastatic niche. Importantly, we constructed engineered-ITGA6 EVs to inhibit lymphatic pre-metastatic niche, which suppressed lymphatic metastasis and prolonged survival in preclinical models. Collectively, our study uncovers the mechanism of BCa-derived ITGA6+EVs mediating pre-metastatic niche and provides an engineered-EV-based strategy against BCa lymphatic metastasis.

Abstract A018: Pathways to enzalutamide resistance: An analysis of proteomic changes in enzalutamide-resistant prostate cancer cells

Abstract Introduction: Enzalutamide is indicated for treatment of castrate-resistant prostate cancer (CRPC), but therapeutic effect is transient, with improvement in survival of approximately 4 months. The mechanisms by which prostate cancer (PCa) cells survive Enzalutamide therapy are poorly understood, but androgen receptor (AR) and non-AR mediated mechanisms have been reported from transcriptome studies. We performed unbiased high resolution proteomic analyses to identify proteins and pathways that are involved in survival and progression in Enzalutamide treatment failure. Materials & Methods: Enzalutamide-resistant MR49F and MR42D cell lines were maintained in standard medium (DMEM with 5% FBS) with Enzalutamide 10µM. Parental LNCaP cells were grown in standard medium. For perturbation, MR49F and MR42D cells were then changed to standard medium with either Enzalutamide 10µM (ENZA) or DMSO (control) for 7 days in. Medium was changed every 48 hours. Protein lysates from 4 biological replicates were labeled using a Tandem Mass Tag (TMT) assay and subjected to mass spectrometry (MS). Protein abundance was measured and significant differences in expression were analysed using a 2-way t-test with a permutation based false detection rate (FDR) of 0.05 and 250 randomisations in the Perseus software suite. MR49F and MR42D were compared to parental LNCaP, and MR49F and MR42D were compared on and off Enzalutamide treatment pressure. Functional biological pathways involved were identified using the DAVID and WebGestalt online pathway analysis tools. Results: MS identified 5364 proteins. Of these, 247 (180 up, 67 down) were differentially expressed by at least 50% in both MR49F and MR42D cell lines relative to parental LNCaP cells. When MR49F and MR42D cell lines were grown with or without Enzalutamide pressure, 5 proteins (4 up, 1 down) were regulated by at least 50% in both cell lines (ANLN, NIN, UQCRB, HIST1H3A and S100P). Pathway analyses with the differentially expressed and regulated proteins identified perturbations in biological pathways with a high representation of pathways related to metabolism, including amino acid and fatty acid metabolism, ascorbate and propanoate metabolism, drug transport and metabolism, and cell adhesion molecules. Conclusions: This unbiased proteomic analysis of Enzalutamide-resistant PCa cells identified proteins and pathways that may be essential for PCa cell survival and progression during Enzalutamide therapy failure. Enzalutamide therapy failure appears to involve some non-AR mediated mechanisms which may be targeted for therapeutic benefit. A better understanding of the mechanisms of Enzalutamide resistance could lead to development of novel therapies for post-Enzalutamide disease progression. Citation Format: Chidi N. Molokwu, Anders Kristensen, Chris Sutton. Pathways to enzalutamide resistance: An analysis of proteomic changes in enzalutamide-resistant prostate cancer cells [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr A018.

Distinct microRNA and protein profiles of extracellular vesicles secreted from myotubes from morbidly obese donors with type 2 diabetes in response to electrical pulse stimulation.

Lifestyle disorders like obesity, type 2 diabetes (T2D), and cardiovascular diseases can be prevented and treated by regular physical activity. During exercise, skeletal muscles release signaling factors that communicate with other organs and mediate beneficial effects of exercise. These factors include myokines, metabolites, and extracellular vesicles (EVs). In the present study, we have examined how electrical pulse stimulation (EPS) of myotubes, a model of exercise, affects the cargo of released EVs. Chronic low frequency EPS was applied for 24h to human myotubes isolated and differentiated from biopsy samples from six morbidly obese females with T2D, and EVs, both exosomes and microvesicles (MV), were isolated from cell media 24h thereafter. Size and concentration of EV subtypes were characterized by nanoparticle tracking analysis, surface markers were examined by flow cytometry and Western blotting, and morphology was confirmed by transmission electron microscopy. Protein content was assessed by high-resolution proteomic analysis (LC-MS/MS), non-coding RNA was quantified by Affymetrix microarray, and selected microRNAs (miRs) validated by real time RT-qPCR. The size and concentration of exosomes and MV were unaffected by EPS. Of the 400 miRs identified in the EVs, EPS significantly changed the level of 15 exosome miRs, of which miR-1233-5p showed the highest fold change. The miR pattern of MV was unaffected by EPS. Totally, about 1000 proteins were identified in exosomes and 2000 in MV. EPS changed the content of 73 proteins in exosomes, 97 in MVs, and of these four were changed in both exosomes and MV (GANAB, HSPA9, CNDP2, and ATP5B). By matching the EPS-changed miRs and proteins in exosomes, 31 targets were identified, and among these several promising signaling factors. Of particular interest were CNDP2, an enzyme that generates the appetite regulatory metabolite Lac-Phe, and miR-4433b-3p, which targets CNDP2. Several of the regulated miRs, such as miR-92b-5p, miR-320b, and miR-1233-5p might also mediate interesting signaling functions. In conclusion, we have used a combined transcriptome-proteome approach to describe how EPS affected the cargo of EVs derived from myotubes from morbidly obese patients with T2D, and revealed several new factors, both miRs and proteins, that might act as exercise factors.

An optimized approach and inflation media for obtaining complimentary mass spectrometry-based omics data from human lung tissue.

Human disease states are biomolecularly multifaceted and can span across phenotypic states, therefore it is important to understand diseases on all levels, across cell types, and within and across microanatomical tissue compartments. To obtain an accurate and representative view of the molecular landscape within human lungs, this fragile tissue must be inflated and embedded to maintain spatial fidelity of the location of molecules and minimize molecular degradation for molecular imaging experiments. Here, we evaluated agarose inflation and carboxymethyl cellulose embedding media and determined effective tissue preparation protocols for performing bulk and spatial mass spectrometry-based omics measurements. Mass spectrometry imaging methods were optimized to boost the number of annotatable molecules in agarose inflated lung samples. This optimized protocol permitted the observation of unique lipid distributions within several airway regions in the lung tissue block. Laser capture microdissection of these airway regions followed by high-resolution proteomic analysis allowed us to begin linking the lipidome with the proteome in a spatially resolved manner, where we observed proteins with high abundance specifically localized to the airway regions. We also compared our mass spectrometry results to lung tissue samples preserved using two other inflation/embedding media, but we identified several pitfalls with the sample preparation steps using this preservation method. Overall, we demonstrated the versatility of the inflation method, and we can start to reveal how the metabolome, lipidome, and proteome are connected spatially in human lungs and across disease states through a variety of different experiments.

A slit-diaphragm-associated protein network for dynamic control of renal filtration

The filtration of blood in the kidney which is crucial for mammalian life is determined by the slit-diaphragm, a cell-cell junction between the foot processes of renal podocytes. The slit-diaphragm is thought to operate as final barrier or as molecular sensor of renal filtration. Using high-resolution proteomic analysis of slit-diaphragms affinity-isolated from rodent kidney, we show that the native slit-diaphragm is built from the junction-forming components Nephrin, Neph1 and Podocin and a co-assembled high-molecular weight network of proteins. The network constituents cover distinct classes of proteins including signaling-receptors, kinases/phosphatases, transporters and scaffolds. Knockout or knock-down of either the core components or the selected network constituents tyrosine kinase MER (MERTK), atrial natriuretic peptide-receptor C (ANPRC), integral membrane protein 2B (ITM2B), membrane-associated guanylate-kinase, WW and PDZ-domain-containing protein1 (MAGI1) and amyloid protein A4 resulted in target-specific impairment or disruption of the filtration process. Our results identify the slit-diaphragm as a multi-component system that is endowed with context-dependent dynamics via a co-assembled protein network.

PLCG1 is required for AML1-ETO leukemia stem cell self-renewal

AbstractIn an effort to identify novel drugs targeting fusion-oncogene–induced acute myeloid leukemia (AML), we performed high-resolution proteomic analysis. In AML1-ETO (AE)-driven AML, we uncovered a deregulation of phospholipase C (PLC) signaling. We identified PLCgamma 1 (PLCG1) as a specific target of the AE fusion protein that is induced after AE binding to intergenic regulatory DNA elements. Genetic inactivation of PLCG1 in murine and human AML inhibited AML1-ETO dependent self-renewal programs, leukemic proliferation, and leukemia maintenance in vivo. In contrast, PLCG1 was dispensable for normal hematopoietic stem and progenitor cell function. These findings are extended to and confirmed by pharmacologic perturbation of Ca++-signaling in AML1-ETO AML cells, indicating that the PLCG1 pathway poses an important therapeutic target for AML1-ETO+ leukemic stem cells.

Branched Chain Amino Acid Catabolism Restricts Alpha-Ketoglutarate Levels Resembling IDHmut-Driven DNA Hypermethylation in AML Stem Cells

The branched chain amino acid (BCAA) pathway and high levels of BCAA transaminase 1 (BCAT1) have recently been associated with aggressiveness in several cancer entities. However, the mechanistic role of BCAT1 in this process remains largely uncertain. By performing high-resolution proteomic analysis of human acute myeloid leukaemia (AML) stem cell (LSC) and non-LSC populations, we found the BCAA pathway enriched and BCAT1 protein and transcripts overexpressed in LSCs. We show that BCAT1, which transfers α-amino groups from BCAAs to α-ketoglutarate (αKG), is a critical regulator of intracellular αKG homeostasis. Next to its role in the tricarboxylic acid (TCA) cycle, αKG is an essential co-factor for αKG-dependent dioxygenases such as EGLN1 and the TET family of DNA demethylases. Knockdown of BCAT1 in leukaemia cells caused accumulation of αKG leading to EGLN1-mediated HIF1α protein degradation. This resulted in a growth and survival defect and abrogated leukaemia-initiating potential. In contrast, overexpression (OE) of BCAT1 in leukaemia cells decreased intracellular αKG levels and caused DNA hypermethylation via altered TET activity. BCAT1high AMLs displayed a DNA hypermethylation phenotype similar to cases carrying mutant isocitrate dehydrogenase (IDHmut), in which TET2 is inhibited by the oncometabolite 2-hydroxyglutarate. High levels of BCAT1 strongly correlate with shorter overall survival in IDHwtTET2wt, but not IDHmut or TET2mut AMLs. Gene sets characteristic for IDHmut AMLs were enriched in IDHwtTETwtBCAT1high patient samples. BCAT1high AMLs showed robust enrichment for LSC signatures, and paired sample analysis revealed a significant increase in BCAT1 levels upon disease relapse. In summary, by limiting intracellular αKG, BCAT1 links BCAA catabolism to HIF1α stability and regulation of the epigenomic landscape mimicking the effects of IDH mutations. In essence, our data strongly suggest that suppression of αKG-dependent dioxygenases constitutes a common feature of AML and, next to mutations in IDH and TET2, we identify the BCAA-BCAT1-αKG axis as alternative, non-genetic mechanism in LSCs to control the activity of these enzymes. Strategies to increase αKG levels, i.e. by inhibition of BCAT1 in order to compromise cancer stem cell function may lead to improved clinical outcomes of patients suffering from AML or other tumour entities expressing high levels of BCAT1 in the absence of IDH or TET mutations. DisclosuresWuchter:Hexal AG: Membership on an entity's Board of Directors or advisory committees, Other: Travel grants; Sanofi-Genzyme: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Thiede:Bayer: Consultancy, Speakers Bureau; Roche: Consultancy; Agendix: Employment; Novartis: Consultancy, Speakers Bureau.

A Biomimetic Glue Protein Modulates Hepatic Gene Expression.

Glue protein as secretion from fruit fly larva plays a significant role in metamorphosis as cementing material for pupation sites. However, the biochemical composition of this macromolecule remains obscure. This study takes the advantage of high-resolution proteomic analysis to unveil the protein compositions. A glue protein group is identified as chitin-binding motifs by bioinformatic analysis. Computational modeling analysis of representative proteins illustrates the binding site between protein and chitin. A biosynthetic approach is used to fabricate a glue protein by a modified Escherichia coli recombinant system. The as-biosynthesized biomimetic glue protein is applied as an extracellular matrix to investigate its biocompatibility and functionality. It is found that the purified recombinant protein shows enhanced performance to cellular viability. This finding provides a potential biomacromolecule candidate as an extracellular matrix for cell culture.

White adipose remodeling during browning in mice involves YBX1 to drive thermogenic commitment

ObjectiveIncreasing adaptive thermogenesis by stimulating browning in white adipose tissue is a promising method of improving metabolic health. However, the molecular mechanisms underlying this transition remain elusive. Our study examined the molecular determinants driving the differentiation of precursor cells into thermogenic adipocytes. MethodsIn this study, we conducted temporal high-resolution proteomic analysis of subcutaneous white adipose tissue (scWAT) after cold exposure in mice. This was followed by loss- and gain-of-function experiments using siRNA-mediated knockdown and CRISPRa-mediated induction of gene expression, respectively, to evaluate the function of the transcriptional regulator Y box-binding protein 1 (YBX1) during adipogenesis of brown pre-adipocytes and mesenchymal stem cells. Transcriptomic analysis of mesenchymal stem cells following induction of endogenous Ybx1 expression was conducted to elucidate transcriptomic events controlled by YBX1 during adipogenesis. ResultsOur proteomics analysis uncovered 509 proteins differentially regulated by cold in a time-dependent manner. Overall, 44 transcriptional regulators were acutely upregulated following cold exposure, among which included the cold-shock domain containing protein YBX1, peaking after 24 h. Cold-induced upregulation of YBX1 also occurred in brown adipose tissue, but not in visceral white adipose tissue, suggesting a role of YBX1 in thermogenesis. This role was confirmed by Ybx1 knockdown in brown and brite preadipocytes, which significantly impaired their thermogenic potential. Conversely, inducing Ybx1 expression in mesenchymal stem cells during adipogenesis promoted browning concurrent with an increased expression of thermogenic markers and enhanced mitochondrial respiration. At a molecular level, our transcriptomic analysis showed that YBX1 regulates a subset of genes, including the histone H3K9 demethylase Jmjd1c, to promote thermogenic adipocyte differentiation. ConclusionOur study mapped the dynamic proteomic changes of murine scWAT during browning and identified YBX1 as a novel factor coordinating the genomic mechanisms by which preadipocytes commit to brite/beige lineage.

The high-resolution proteomic analysis of protein composition of rat spleen lymphocytes stimulated by Concanavalin A; a comparison with morphine-treated cells

Morphine- and Concanavalin A-induced changes of protein composition of rat spleen lymphocytes were determined by high-resolution proteomic analysis, gel-free, label-free quantification, MaxLFQ. Stimulation by Con A resulted in a major reorganization of spleen cell protein composition evidenced by increased expression level of 94 proteins; 101 proteins were down-regulated (>2-fold). Interestingly, among proteins that were up-regulated to the largest extent were the prototypical brain proteins as a neuron specific enolase, synapsin-1, brain acid-soluble protein-1 and myelin basic protein. Morphine-induced change was limited to no more than 5 up-regulated and 18 down-regulated proteins (>2-fold).

Label-free high-resolution proteomic analysis of cerebrospinal fluid in Alzheimer's disease

Label-free high-resolution proteomic analysis of cerebrospinal fluid in Alzheimer's disease

Proteome-based classification of Nonmuscle Invasive Bladder Cancer.

DNA/RNA-based classification of bladder cancer (BC) supports the existence of multiple molecular subtypes, while investigations at the protein level are scarce. Here, we aimed to investigate if Nonmuscle Invasive Bladder Cancer (NMIBC) can be stratified to biologically meaningful groups based on the proteome. Tissue specimens from 117 patients at primary diagnosis (98 with NMIBC and 19 with MIBC), were processed for high-resolution proteomics analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The proteomics output was subjected to unsupervised consensus clustering, principal component analysis (PCA) and investigation of subtype-specific features, pathways, and gene sets. NMIBC patients were optimally stratified to three NMIBC proteomic subtypes (NPS), differing in size, clinicopathologic and molecular backgrounds: NPS1 (mostly high stage/grade/risk samples) was the smallest in size (17/98) and overexpressed proteins reflective of an immune/inflammatory phenotype, involved in cell proliferation, unfolded protein response and DNA damage response, whereas NPS2 (mixed stage/grade/risk composition) presented with an infiltrated/mesenchymal profile. NPS3 was rich in luminal/differentiation markers, in line with its pathological composition (mostly low stage/grade/risk samples). PCA revealed a close proximity of NPS1 and conversely, remoteness of NPS3 to the proteome of MIBC. Proteins distinguishing these two extreme subtypes were also found to consistently differ at the mRNA levels between high and low-risk subtypes of the UROMOL and LUND cohorts. Collectively, our study identifies three proteomic NMIBC subtypes and following a cross-omics validation in two independent cohorts, shortlists molecular features meriting further investigation for their biomarker or potentially therapeutic value.

Characteristic Amino Acid and Energy Metabolism in AML Stem Cells As Revealed By Quantitative Multiplex Proteomics

Acute Myeloid Leukemia (AML) is a rapidly progressing hematologic malignancy characterized by the accumulation of clonal myeloid progenitor cells arrested in their ability to differentiate into mature blood cells. While classic chemotherapy regimens lead to remission in the majority of patients, relapse rates are very high. Relapse and also refractoriness are caused by the hierarchical organization of AML with a minor fraction of leukemic stem cells (LSC) at the apex generating leukemic progeny, which make up the majority of leukemic cells. LSC harbor self-renewal activity, relative quiescence, resistance to apoptosis, and increased drug efflux that likely render them less susceptible to conventional therapies aimed at the bulk proliferative disease. From a clinical perspective, the leukemic stem cell model implies that in order to eradicate the disease and achieve long-term remissions, treatment courses must eliminate the LSC population.To identify putative therapeutic target structures and/or pathways selectively aiming at LSC we performed high-resolution proteomic analyses of LSC and non-LSC populations of primary AML patient samples and healthy control subjects. To identify functional LSCs, we fractionated AML patient samples according to CD34 and CD38 surface expression by fluorescent-activated cell sorting and transplanted the resulting four populations per sample into immunocompromized NSG mice. Leukemic populations, which were able to initiate the AML in mice were labeled LSCs, while engraftment failures were considered non-LSCs. Six AML patients containing both LSC and non-LSC fractions were chosen for proteomic analysis. As healthy controls, CD34+CD38- hematopoietic stem and progenitor cells (HSPC) populations obtained from elderly patients without hematologic conditions undergoing hip replacement surgery were analyzed. We performed in-depth quantitative multiplex proteomic analysis employing tandem mass tag labeling and high-resolution mass spectrometry and identified more than 7,200 proteins. Our analysis revealed between 1,097 and 1,937 differentially expressed proteins when comparing LSC with non-LSC populations for each AML sample. Gene set enrichment analyses (GSEA) showed a significant enrichment for DNA replication, cell cycle, ribosome biogenesis, and protein translation in non-LSC populations, in agreement with a more quiescent state of LSC.Next to Branched Chain Amino Acid degradation, which we have recently shown to control the activity of alpha-ketoglutarate dependent dioxygenases such as TET2 and EGLN1 in AML stem cells (Raffel et al., (2017). Nature 551(7680), 384-388) other metabolic processes including lipid metabolism and oxidative phosphorylation were enriched in LSC. When compared to healthy HSPCs, especially oxidative phosphorylation, RNA processing and cell adhesion were over-represented in LSC. We will present proteins differentially expressed and pathways enriched in LSCs versus non-LSCs. These may help to understand the mechanism of LSC self-renewal and function and simultaneously represent putative novel targets to eliminate leukemic stem cells in clinical settings. DisclosuresNo relevant conflicts of interest to declare.

BCAT1 restricts αKG levels in AML stem cells leading to IDHmut-like DNA hypermethylation.

The branched-chain amino acid (BCAA) pathway and high levels of BCAA transaminase 1 (BCAT1) have recently been associated with aggressiveness in several cancer entities. However, the mechanistic role of BCAT1 in this process remains largely uncertain. Here, by performing high-resolution proteomic analysis of human acute myeloid leukaemia (AML) stem-cell and non-stem-cell populations, we find the BCAA pathway enriched and BCAT1 protein and transcripts overexpressed in leukaemia stem cells. We show that BCAT1, which transfers α-amino groups from BCAAs to α-ketoglutarate (αKG), is a critical regulator of intracellular αKG homeostasis. Further to its role in the tricarboxylic acid cycle, αKG is an essential cofactor for αKG-dependent dioxygenases such as Egl-9 family hypoxia inducible factor 1 (EGLN1) and the ten-eleven translocation (TET) family of DNA demethylases. Knockdown of BCAT1 in leukaemia cells caused accumulation of αKG, leading to EGLN1-mediated HIF1α protein degradation. This resulted in a growth and survival defect and abrogated leukaemia-initiating potential. By contrast, overexpression of BCAT1 in leukaemia cells decreased intracellular αKG levels and caused DNA hypermethylation through altered TET activity. AML with high levels of BCAT1 (BCAT1high) displayed a DNA hypermethylation phenotype similar to cases carrying a mutant isocitrate dehydrogenase (IDHmut), in which TET2 is inhibited by the oncometabolite 2-hydroxyglutarate. High levels of BCAT1 strongly correlate with shorter overall survival in IDHWTTET2WT, but not IDHmut or TET2mut AML. Gene sets characteristic for IDHmut AML were enriched in samples from patients with an IDHWTTET2WTBCAT1high status. BCAT1high AML showed robust enrichment for leukaemia stem-cell signatures, and paired sample analysis showed a significant increase in BCAT1 levels upon disease relapse. In summary, by limiting intracellular αKG, BCAT1 links BCAA catabolism to HIF1α stability and regulation of the epigenomic landscape, mimicking the effects of IDH mutations. Our results suggest the BCAA-BCAT1-αKG pathway as a therapeutic target to compromise leukaemia stem-cell function in patients with IDHWTTET2WT AML.

High Resolution Proteomic Analysis of the Cervical Cancer Cell Lines Secretome Documents Deregulation of Multiple Proteases.

Oncogenic infection by HPV, eventually leads to cervical carcinogenesis, associated by deregulation of specific pathways and protein expression at the intracellular and secretome level. Thus, secretome analysis can elucidate the biological mechanisms contributing to cervical cancer. In the present study we systematically analyzed its constitution in four cervical cell lines employing a highly sensitive proteomic technology coupled with bioinformatics analysis. LC/MS-MS proteomics and bioinformatics analysis were performed in the secretome of four informative cervical cell lines SiHa (HPV16+), HeLa (HPV18+), C33A (HPV-) and HCK1T (normal). The proteomic pattern of each cancer cell line compared to HCK1T was identified and a detailed bioinformatics analysis disclosed inhibition of matrix metalloproteases in cancer cell lines. This prediction was further confirmed via zymography for MMP-2 and MMP-9, western blot analysis for ADAM10 and by MRM for TIMP1. The differential expression of important secreted proteins such as CATD, FUCA1 and SOD2 was also confirmed by western blot analysis. MRM-targeted proteomics analysis confirmed the differential expression of CATD, CATB, SOD2, QPCT and NEU1. High resolution proteomics analysis of cervical cancer secretome revealed significantly deregulated biological processes and proteins implicated in cervical carcinogenesis.

P.241 - Comparative high resolution proteomic analysis of dystrophic mouse models reveals a core dystrophic proteome and the impact of aging

The most common form of childhood muscular dystrophy is Duchenne muscular dystrophy (DMD), which is a severe, X-linked muscle wasting disorder. The disease is caused by mutations in the DMD gene, leading to loss of the dystrophin protein which in turn causes progressive muscle degeneration and premature death. Various mouse models have been developed to study the disease, such as the commonly used mdx mouse, which has a nonsense mutation in exon 23, though this mutation is not characteristically present in humans. Another model is the mdx52 mouse, which has an exon 52 deletion in the mutation “hotspot” region of the DMD gene. To better understand the dystrophic phenotype and its progression with aging, we undertook for the first-time comparative proteomic analysis of both mdx and mdx52 models, using high-resolution isoelectric Focusing Liquid Chromatography-tandem Mass Spectrometry (HiRIEF-LC-MS/MS) with Tandem Mass Tag (TMT) isobaric labelling of tibialis anterior (TA) skeletal muscle to identify proteomic characteristics at 8, 16 and 80 weeks of age. Comparative proteomic analysis of mdx, mdx52 and C57BL/6 control mice identified 4,974 proteins in 27plex format and revealed a core set of proteomic changes reflective of the dystrophic phenotype. Proteomic analysis with age identified core proteomic features of normal muscle aging, overlap of these with progression of the dystrophic phenotype and features specific to the dystrophic phenotype with age. This study provides novel insight into specific proteomic changes characteristic of muscle aging and the relationship of these to dystrophic pathology over time.

Novel Antibiotic Resistance Determinants from Agricultural Soil Exposed to Antibiotics Widely Used in Human Medicine and Animal Farming.

Antibiotic resistance has emerged globally as one of the biggest threats to human and animal health. Although the excessive use of antibiotics is recognized as accelerating the selection for resistance, there is a growing body of evidence suggesting that natural environments are "hot spots" for the development of both ancient and contemporary resistance mechanisms. Given that pharmaceuticals can be entrained onto agricultural land through anthropogenic activities, this could be a potential driver for the emergence and dissemination of resistance in soil bacteria. Using functional metagenomics, we interrogated the "resistome" of bacterial communities found in a collection of Canadian agricultural soil, some of which had been receiving antibiotics widely used in human medicine (macrolides) or food animal production (sulfamethazine, chlortetracycline, and tylosin) for up to 16 years. Of the 34 new antibiotic resistance genes (ARGs) recovered, the majority were predicted to encode (multi)drug efflux systems, while a few share little to no homology with established resistance determinants. We characterized several novel gene products, including putative enzymes that can confer high-level resistance against aminoglycosides, sulfonamides, and broad range of beta-lactams, with respect to their resistance mechanisms and clinical significance. By coupling high-resolution proteomics analysis with functional metagenomics, we discovered an unusual peptide, PPPAZI 4, encoded within an alternative open reading frame not predicted by bioinformatics tools. Expression of the proline-rich PPPAZI 4 can promote resistance against different macrolides but not other ribosome-targeting antibiotics, implicating a new macrolide-specific resistance mechanism that could be fundamentally linked to the evolutionary design of this peptide.IMPORTANCE Antibiotic resistance is a clinical phenomenon with an evolutionary link to the microbial pangenome. Genes and protogenes encoding specialized and potential resistance mechanisms are abundant in natural environments, but understanding of their identity and genomic context remains limited. Our discovery of several previously unknown antibiotic resistance genes from uncultured soil microorganisms indicates that soil is a significant reservoir of resistance determinants, which, once acquired and "repurposed" by pathogenic bacteria, can have serious impacts on therapeutic outcomes. This study provides valuable insights into the diversity and identity of resistance within the soil microbiome. The finding of a novel peptide-mediated resistance mechanism involving an unpredicted gene product also highlights the usefulness of integrating proteomics analysis into metagenomics-driven gene discovery.

Immune regulation: Immune cell social networks.

High-resolution proteomics analysis of human immune cells reveals new social networks and establishes a framework for future study of intercellular communication.

In-depth High-resolution Proteomic Analysis Using Nasal Secretions form Chronic Rhinosinusitis and Nasal Polyps

In-depth High-resolution Proteomic Analysis Using Nasal Secretions form Chronic Rhinosinusitis and Nasal Polyps

High Resolution Quantitative Synaptic Proteome Profiling of Mouse Brain Regions After Auditory Discrimination Learning

The molecular synaptic mechanisms underlying auditory learning and memory remain largely unknown. Here, the workflow of a proteomic study on auditory discrimination learning in mice is described. In this learning paradigm, mice are trained in a shuttle box Go/NoGo-task to discriminate between rising and falling frequency-modulated tones in order to avoid a mild electric foot-shock. The protocol involves the enrichment of synaptosomes from four brain areas, namely the auditory cortex, frontal cortex, hippocampus, and striatum, at different stages of training. Synaptic protein expression patterns obtained from trained mice are compared to naïve controls using a proteomic approach. To achieve sufficient analytical depth, samples are fractionated in three different ways prior to mass spectrometry, namely 1D SDS-PAGE/in-gel digestion, in-solution digestion and phospho-peptide enrichment. High-resolution proteomic analysis on a mass spectrometer and label-free quantification are used to examine synaptic protein profiles in phospho-peptide-depleted and phospho-peptide-enriched fractions of synaptosomal protein samples. A commercial software package is utilized to reveal proteins and phospho-peptides with significantly regulated relative synaptic abundance levels (trained/naïve controls). Common and differential regulation modes for the synaptic proteome in the investigated brain regions of mice after training were observed. Subsequently, meta-analyses utilizing several databases are employed to identify underlying cellular functions and biological pathways.