Label-free Mass Spectrometry Analysis Research Articles

Study on the interaction protein of transcription factor Smad3 based on TurboID proximity labeling technology

TurboID is a highly efficient biotin-labelling enzyme, which can be used to explore a number of new intercalating proteins due to the very transient binding and catalytic functions of many proteins. TGF-β/Smad3 signaling pathway is involved in many diseases, especially in diabetic nephropathy and inflammation. In this paper, a stably cell line transfected with Smad3 were constructed by using lentiviral infection. To further investigate the function of TGF-β/Smad3, the protein labeling experiment was conducted to find the interacting protein with Smad3 gene. Label-free mass spectrometry analysis was performed to obtain 491 interacting proteins, and the interacting protein hnRNPM was selected for IP and immunofluorescence verification, and it was verified that the Smad3 gene had a certain promoting effect on the expression of hnRNPM gene, and then had an inhibitory effect on IL-6. It lays a foundation for further study of the function of Smad3 gene and its involved regulatory network.

Abstract 6965: Characterizing the proteome landscape of extracellular vesicles from prostate cancer cell lines by mass spectrometry

Abstract Multiple subtypes of prostate cancer can emerge in advanced stages of disease, such as tumors that maintain androgen receptor features (AR+), those without AR but with neuroendocrine features (AR-/NE+), and those without AR or NE features (AR-/NE-). These latter subtypes are particularly aggressive and incredibly lethal. Extracellular vesicles (EVs) are small particles that are mediators of cell-cell communication and have been nominated as potential biomarkers of disease state. We hypothesized that the proteome of EVs contains information about their originating cell that can distinguish between prostate cancer subtypes. To address this, we isolated EVs from cell lines of differing subtypes – AR+ (LNCaP, LNCaP95, C4-2, 22RV1), AR-/NE+(NCI-H660, EF1, LASCPC-01), and AR-/NE- (PC3, DU145) – and prepared the EV proteome for quantitative, label-free mass spectrometry analysis. This identified 2,000+ proteins in the EVs for each cell line where EVs from similar subtypes clustered together. Interestingly, we also found that EVs contained enough protein information to utilize developed gene signatures for AR or NE activity. EVs isolated from AR-/NE+ cells contained known NE signature proteins such as SYP, CHGA, and UCHL1. EVs from the AR+ cell lines displayed >50% AR activity driven by PSA and STEAP1 proteins in addition to other significant distinguishing protein markers. This supports the potential for using the proteome of tumor-derived EVs to identify novel signature molecular markers besides those already known to the field for developing liquid-based biomarker assays. In addition, our work characterizes the EV surfaceome which is critical for enriching signals from tumor-derived EVs feasible for clinical diagnostics assay development. While PSMA has been a reliable marker for cells reliant on AR signaling, less is known about protein markers in the AR- subset. Our work highlights TROP2 as especially enriched in the EVs from AR-/NE- cell lines, supporting its relevance as a target in patients without treatment options. To evaluate disease outcomes, further clinical studies are needed to develop specific and sensitive tests based on the EV proteome derived from advanced prostate cancer patients. Citation Format: Megan L. Ludwig, Abderrahman Day, Hannah E. Bergom, Zoi Sychev, Alec Horrmann, Justin Hwang, Justin M. Drake. Characterizing the proteome landscape of extracellular vesicles from prostate cancer cell lines by mass spectrometry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6965.

Mass spectrometry-based proteomic analysis to characterize cisplatin induced early signaling events in head and neck squamous cell carcinoma

ABSTRACT Cisplatin is the commonly used chemotherapeutic drug in treatment of various cancers. However, development of resistance towards cisplatin results in tumor recurrence. Here, we aim to understand the mechanisms of action of cisplatin and emergence of resistance to cisplatin using mass spectrometry-based proteomic approach. A panel of head and neck squamous cell carcinoma (HNSCC) cell lines were treated with cisplatin at respective IC50 for 24 h and label-free mass spectrometry analysis was carried out. Proteomic analysis of A253, FaDu, Det562 and CAL27 cell lines upon cisplatin treatment resulted in the identification of 5,060, 4,816, 4,537 and 4,142 proteins, respectively. Bioinformatics analysis of differentially regulated proteins revealed proteins implicated in DNA damage bypass pathway, translation and mRNA splicing to be enriched. Further, proteins associated with cisplatin resistance exhibited alterations following short-term cisplatin exposure. Among these, class III tubullin protein (TUBB3) was found to be upregulated in cisplatin-treated cells compared to untreated cells. Western blot analysis confirmed the elevated expression of TUBB3 in cells treated with cisplatin for 24 h, and also in cisplatin resistant HNSCC cell lines. This study delineates the early signaling events that enable HNSCC cells to counteract the cytotoxic effects of cisplatin and facilitate the development of resistance.

Quantitative label-free proteomic analysis of excretory-secretory proteins in different developmental stages of Trichinella spiralis

Trichinella spiralis (T. spiralis) is a zoonotic parasitic nematode with a unique life cycle, as all developmental stages are contained within a single host. Excretory-secretory (ES) proteins are the main targets of the interactions between T. spiralis and the host at different stages of development and are essential for parasite survival. However, the ES protein profiles of T. spiralis at different developmental stages have not been characterized. The proteomes of ES proteins from different developmental stages, namely, muscle larvae (ML), intestinal infective larvae (IIL), preadult (PA) 6 h, PA 30 h, adult (Ad) 3 days post-infection (dpi) and Ad 6 dpi, were characterized via label-free mass spectrometry analysis in combination with bioinformatics. A total of 1217 proteins were identified from 9341 unique peptides in all developmental stages, 590 of which were quantified and differentially expressed. GO classification and KEGG pathway analysis revealed that these proteins were important for the growth of the larvae and involved in energy metabolism. Moreover, the heat shock cognate 71 kDa protein was the centre of protein interactions at different developmental stages. The results of this study provide comprehensive proteomic data on ES proteins and reveal that these ES proteins were differentially expressed at different developmental stages. Differential proteins are associated with parasite survival and the host immune response and may be potential early diagnostic antigen or antiparasitic vaccine candidates.

Unveiling Distinct Proteomic Signatures in Complicated Crohn’s Disease That Could Predict the Disease Course

Crohn’s disease (CD) is characterized by a chronic, progressive inflammation of the gastrointestinal tract often leading to complications, such as strictures and fistulae. Currently, there are no validated tools anticipating short- and long-term outcomes at an early stage. This investigation aims to elucidate variations in protein abundance across distinct CD phenotypes with the objective of uncovering potential biomarkers implicated in disease advancement. Serum samples collected from 30 CD patients and 15 healthy age-matched controls (HC) were subjected to depletion of highly abundant proteins and to a label-free mass spectrometry analysis. Twenty-four proteins were shown to be significantly different when comparing CD with HC. Of these, WD repeat-containing protein 31 (WDR31), and proteins involved in the acute inflammatory response, leucine-rich alpha-2-glycoprotein (LRG1) and serum amyloid A1 (SAA1), were more abundant in the aggressive subgroup. Against standard biomarkers, a positive correlation between SAA1 and WDR31 and C-reactive protein (CRP) was found. In this study, a unique serum biomarker panel for aggressive CD was identified, which could aid in predicting the disease course.

Study of FOXO1-interacting proteins using TurboID-based proximity labeling technology

BackgroundProtein‒protein interactions (PPIs) are the foundation of the life activities of cells. TurboID is a biotin ligase with higher catalytic efficiency than BioID or APEX that reduces the required labeling time from 18 h to 10 min. Since many proteins participate in binding and catalytic events that are very short-lived, it is theoretically possible to find relatively novel binding proteins using the TurboID technique. Cell proliferation, apoptosis, autophagy, oxidative stress and metabolic disorders underlie many diseases, and forkhead box transcription factor 1 (FOXO1) plays a key role in these physiological and pathological processes.ResultsThe FOXO1-TurboID fusion gene was transfected into U251 astrocytes, and a cell line stably expressing FOXO1 was constructed. While constructing the FOXO1 overexpression plasmid, we also added the gene sequence of TurboID to perform biotin labeling experiments in the successfully fabricated cell line to look for FOXO1 reciprocal proteins. Label-free mass spectrometry analysis was performed, and 325 interacting proteins were found. A total of 176 proteins were identified in the FOXO1 overexpression group, and 227 proteins were identified in the Lipopolysaccharide -treated group (Lipopolysaccharide, LPS). Wild-type U251 cells were used to exclude interference from nonspecific binding. The FOXO1-interacting proteins hnRNPK and RBM14 were selected for immunoprecipitation and immunofluorescence verification.ConclusionThe TurboID technique was used to select the FOXO1-interacting proteins, and after removing the proteins identified in the blank group, a large number of interacting proteins were found in both positive groups. This study lays a foundation for further study of the function of FOXO1 and the regulatory network in which it is involved.

Diagnosis of T-cell-mediated kidney rejection by biopsy-based proteomic biomarkers and machine learning.

Biopsy-based diagnosis is essential for maintaining kidney allograft longevity by ensuring prompt treatment for graft complications. Although histologic assessment remains the gold standard, it carries significant limitations such as subjective interpretation, suboptimal reproducibility, and imprecise quantitation of disease burden. It is hoped that molecular diagnostics could enhance the efficiency, accuracy, and reproducibility of traditional histologicmethods. Quantitative label-free mass spectrometry analysis was performed on a set of formalin-fixed, paraffin-embedded (FFPE) biopsies from kidney transplant patients, including five samples each with diagnosis of T-cell-mediated rejection (TCMR), polyomavirus BK nephropathy (BKPyVN), and stable (STA) kidney function control tissue. Using the differential protein expression result as a classifier, three different machine learning algorithms were tested to build a molecular diagnostic model for TCMR. The label-free proteomics method yielded 800-1350 proteins that could be quantified with high confidence per sample by single-shot measurements. Among these candidate proteins, 329 and 467 proteins were defined as differentially expressed proteins (DEPs) for TCMR in comparison with STA and BKPyVN, respectively. Comparing the FFPE quantitative proteomics data set obtained in this study using label-free method with a data set we previously reported using isobaric labeling technology, a classifier pool comprised of features from DEPs commonly quantified in both data sets, was generated for TCMR prediction. Leave-one-out cross-validation result demonstrated that the random forest (RF)-based model achieved the best predictive power. In a follow-up blind test using an independent sample set, the RF-based model yields 80% accuracy for TCMR and 100% for STA. When applying the established RF-based model to two public transcriptome datasets, 78.1%-82.9% sensitivity and 58.7%-64.4% specificity was achieved respectively. This proof-of-principle study demonstrates the clinical feasibility of proteomics profiling for FFPE biopsies using an accurate, efficient, and cost-effective platform integrated of quantitative label-free mass spectrometry analysis with a machine learning-based diagnostic model. It costs less than 10 dollars per test.

Abstract PO011: Proteomic profiling of childhood liver cancer: identification of novel diagnostic and prognostic biomarkers

Abstract Childhood liver cancers hepatoblastoma (HB) and hepatocellular carcinoma (HCC) are rare diseases but with a rising incidence. HEMNOS (Hepatocellular malignant neoplasm, not otherwise specified) is a recent entity with histopathological features of HB and HCC. Current chemotherapy treatments are effective to shrink tumor before surgery, nonetheless can cause severe lifelong adverse effects and are not effective for patients with aggressive and metastatic HB or HCC (~20% die due to the disease). The HB and HCC diagnosis is key to assign therapeutic regimens in the ongoing Pediatric Hepatic International Tumor Trial (PHITT); however, differential diagnosis for some patients is challenging due to the lack of specific biomarkers. Nowadays, there is an urgent need to identify diagnostic and prognostic biomarkers to improve the clinical management of childhood liver cancer. Herein, we aimed to uncover the proteomic profiles of different types of childhood liver cancer and identify new biomarkers for improving tumor diagnosis and early detection of the most aggressive cases. For this, 99 frozen tissue samples from 71 patients (mean age: 54.5 months, 60.6% boys, 40.9% metastasis, 23.7% deaths) including 70 primary tumors (57 HB, 8 HCC and 5 HEM-NOS), 22 non-tumors and 7 metastases were analyzed by label free mass spectrometry analysis. A total of 5,417 proteins were sequenced in the different samples. Through supervised analysis, we identified a total of 1302 differential expressed proteins in tumors as compared to non-tumor livers (FC +2 and FDR <0.01). A total of 246 proteins of them (19%) were commonly differently expressed in all tumors types; having HEMNOS a mixture of HB and HCC protein profiling. A panel of potential diagnostic biomarkers including 179, 120 and 81 upregulated proteins (FC >2) was defined for HB, HCC and HEM-NOS, respectively. Hierarchical unsupervised clustering and principal component analysis showed two main cluster of tumors: one including HB and HEM-NOS and another one with a mixture of HB, HEM-NOS and HCC. Interestingly, this second cluster was associated with clinical and molecular features of aggressive tumors such as multifocality (p = 0.029), Epigenetic Epi-CB subclass (p = 0.006), high-risk Molecular Risk Stratification (p = 0.009) and poor survival (log rank test=0.012). A total of 2082 proteins were found to be differently expressed between the two prognostic clusters (FC +2 and FDR < 0.01); the GEO enrichment analysis revealed that these proteins were significantly enriched in post-transcriptional gene silencing and RNA splicing mechanisms (FDR <10−7). In addition, a 25-protein signature associated to liver cancer prognosis was defined (FC>6 and FDR < 10−14). In conclusion, we identified a list of potential diagnostic and prognostic protein biomarkers that after a validation in large patient cohort, could be used to improve the clinical management of childhood liver cancer. Citation Format: Álvaro Del Río-Álvarez, Juan Carrillo-Reixach, Laura Royo, Montse Domingo-Sàbat, Mikel Azkargorta, Roland Kapler, Stefano Cairo, Christian Vokuhl, Ronald de Krijger, Rita Alaggio, Marta Garrido, Gabriela Guillen, Constantino Sábado, Laura Guerra, Francisco Hernandez, Maria Elena Mateos, Manuel López-Satamaría, Barbara Torres, Maria Pilar Abad, Bajčiová Viera, Piotr Czauderna, Marie Annick Buendia, Felix Elortza, Keith Wheatley, Bruce Morland, Carolina Armengol. Proteomic profiling of childhood liver cancer: identification of novel diagnostic and prognostic biomarkers [abstract]. In: Proceedings of the AACR Special Conference: Advances in the Pathogenesis and Molecular Therapies of Liver Cancer; 2022 May 5-8; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(17_Suppl):Abstract nr PO011.

Quantitative Proteomics of Uukuniemi Virus-host Cell Interactions Reveals GBF1 as Proviral Host Factor for Phleboviruses

Novel tick-borne phleboviruses in the Phenuiviridae family, which are highly pathogenic in humans and all closely related to Uukuniemi virus (UUKV), have recently emerged on different continents. How phleboviruses assemble, bud, and exit cells remains largely elusive. Here, we performed high-resolution, label-free mass spectrometry analysis of UUKV immunoprecipitated from cell lysates and identified 39 cellular partners interacting with the viral envelope glycoproteins. The importance of these host factors for UUKV infection was validated by silencing each host factor by RNA interference. This revealed Golgi-specific brefeldin A-resistance guanine nucleotide exchange factor 1 (GBF1), a guanine nucleotide exchange factor resident in the Golgi, as a critical host factor required for the UUKV life cycle. An inhibitor of GBF1, Golgicide A, confirmed the role of the cellular factor in UUKV infection. We could pinpoint the GBF1 requirement to UUKV replication and particle assembly. When the investigation was extended to viruses from various positive and negative RNA viral families, we found that not only phleboviruses rely on GBF1 for infection, but also Flavi-, Corona-, Rhabdo-, and Togaviridae In contrast, silencing or blocking GBF1 did not abrogate infection by the human adenovirus serotype 5 and immunodeficiency retrovirus type 1, the replication of both requires nuclear steps. Together our results indicate that UUKV relies on GBF1 for viral replication, assembly and egress. This study also highlights the proviral activity of GBF1 in the infection by a broad range of important zoonotic RNA viruses.

751 A comparative analysis of skin aging-associated secreted proteins (SAASP) produced by dermal fibroblasts from intrinsically versus extrinsically aged human skin

Skin aging results to a large extent from the accumulation of senescent dermal fibroblasts. In a previous systematic molecular analysis of normal human dermal fibroblasts (NHDF) isolated from intrinsically aged human skin of donors aged 18-25 versus 35-49 versus ≥ 60 years we observed that intrinsic aging of NHDF is associated with the development of a protein secretion pattern referred to as SAASP. In the present study we asked if and how this secretory phenotype might differ between NHDF isolated from intrinsically versus extrinsically aged human skin. We again obtained NHDF from young versus middle aged versus old donors. In this study, however, for each donor NHDF were isolated not only from intrinsically aged, i.e. buttock skin, but also from extrinsically aged, i.e. neck skin. Quantitative, label-free mass spectrometry analysis of the proteome of these cells revealed an age-dependent progressive loss of protein diversity, which was significantly more pronounced in extrinsically aged NHDF. This was not reflected by changes in the secretome of the same cells. Accordingly, quantitative analysis of the entire secretome present in conditioned cell culture supernatants derived from these cells showed that the diversity of proteins in the secretome was not reduced if compared between the three age groups for intrinsic and extrinsic aging, respectively. In fact, secretome diversity even increased for intrinsically versus extrinsically aged NHDF of old donors. Subsequent network and enrichment analysis revealed that the secretomes of extrinsically versus intrinsically aged NHDF differ from each other also at a functional level. As an example, in old donors, secreted proteins relevant for extracellular matrix organization were significantly less abundant in extrinsically aged NHDF. These results demonstrate that the SAASP produced by NHDF profoundly differs between intrinsically versus extrinsically aged human skin, indicating that the relationship between intrinsic and extrinsic skin aging is highly complex in nature.

Label-free based comparative proteomic analysis of whey proteins between different milk yields of Dezhou donkey

Donkey milk, similar to human milk in compositions, has been suggested as the best potential hypoallergenic replacement diet for babies suffering from cow milk protein allergens and a promising nutraceutical for aged people. In this study, label-free mass spectrometry analysis was conducted to quantitatively identify the whey proteins differentially expressed in high-milk-yield samples compared with low-milk-yield samples. A total of 216 whey proteins were identified, and 19 of them showed significant differences in high-milk-yield samples. Of these proteins, 16 were upregulated and 3 were downregulated. Differentially expressed proteins (DEPs) were subjected to intensive bioinformatic analysis. Results revealed that the majority of DEPs participated in protein processing in endoplasmic reticulum, estrogen signaling pathway, progesterone-mediated oocyte maturation, and PI3K-Akt signaling pathway. Functional protein analysis suggested that proteins functioned in binding, catalytic activity, molecular function regulation, structural molecule activity, and transporter activity. Our study was the first to analyze the whey protein profile of different samples of donkey milk and to identify candidate proteins that could be used to explore the molecular mechanism related to the yield traits of Dezhou donkey milk. This study provided the biomarkers for the selection of high-milk-yielding donkey and obtained valuable information for future studies.

Proposed mechanism for understanding the dose- and time-dependency of the effects of fluoride in the liver

Fluoride (F) can induce changes in the expression of several liver proteins. It is suggested that these changes are dose- and time-dependent. The objective of this study was to analyze the effect of different F concentrations and exposure times to this ion on the pattern of protein expression in the liver of rats. Thirt-six 21-day-old male Wistar rats were divided into 2 groups (n = 18) according to the treatment duration (20 or 60 days). Each of these groups was then divided into 3 subgroups (n = 6) according to the concentration of F administered in drinking water, as follows: 0 mg/L (control), 15 mg/L or 50 mg/L. After the experiment periods, the animals were anesthetized and the liver and blood were collected. F was analyzed in plasma and liver. Part of the liver was fixed for histological analysis. Liver proteins were extracted and prepared for quantitative label-free mass spectrometry analysis. F concentrations in plasma and liver were significantly higher in the group treated with 50 mg /L in comparison with control, regardless the time of exposure. Histological alterations in the liver were more evident in the subgroups treated for 20 days. The proteomic analysis revealed changes in proteins related to endoplasmic reticulum and mitochondrial oxidative stress, mitochondrial alteration, apoptosis and cellular respiration upon exposure to F. The results reinforce previous findings showing that the effects of F in the liver are dose- and time-dependent and provide the molecular basis for understanding the evolution of these effects.

Differential proteomic analysis of endometrial fluid suggests increased inflammation and impaired glucose metabolism in non-implantative IVF cycles and pinpoints PYGB as a putative implantation marker.

Is there any difference in the protein composition of the endometrial fluid aspirate (EFA) obtained the day of embryo transfer in in vitro fertilization (IVF) cycles achieving and not achieving pregnancy? Comparative analysis identified a differential protein expression pattern in 'implantative' and 'non-implantative' IVF cycles. EFA allows non-invasive characterization of the endometrium, and may contain important information on its receptivity when performing (IVF) cycles. Endometrial side of implantation has usually been studied with endometrial biopsy in an IVF cycle prior to embryo transfer, focusing on 'receptive/non-receptive' endometria and with low-throughput proteomic techniques. We have compared the protein expression patterns in EFA from a total of 110 women undergoing IVF, corresponding to 50 implantative and 60 non-implantative IVF cycles. Discovery (38 patients) and Validation (42 patients) sample cohorts were analyzed using a high-throughput differential proteomic approach. Then, the differential expression of glycogen phosphorylase B (PYGB) was validated by western blotting in an additional cohort (30 patients). The study period was 18 months. The population under study consisted of 110 women aged 18-40 years old, undergoing their first or second IVF/ intracytoplasmic sperm injection cycle, with normal uterus and endometrium, and 1-2 good quality embryos, and embryo transfer being performed on Day 3. Endometrial fluid aspiration was performed immediately before the embryo transfer. Samples (80) were initially distributed in two independent cohorts and analyzed by liquid chromatography-mass spectrometry. The first cohort was used for the discovery and the second for the validation of the results. Filter-aided sample preparation was used for the in-solution tryptic digestion of the proteins present in the samples, followed by label-free mass spectrometry analysis. In order to unravel the molecular features of receptivity, the lists of differential proteins were thoroughly analyzed using different bioinformatic tools, including GSEA, IPA and GO analysis. A false discovery rate-based correction of the t-test P-values was carried out in order to strengthen the reliability of the results. Functional analyses denoted the deregulation of important processes governing receptivity, such as antimicrobial response, cell-cell interaction, immune response and inflammatory signaling, among others. Overall eight proteins were commonly deregulated in both studied datasets and brain form glycogen phosphorylase (PYGB) was selected for confirmatory analysis. Our results were obtained from patients with normal uterus and endometrium and with good quality embryos, who had fresh Day-3 embryo transfer, in stimulated cycles. Therefore, our observations may not be applicable to poor prognosis cases or non-stimulated cycles. This work provides insights into the molecular features of implantative IVF cycles using non-invasive methods. It reveals that EFA may reflect an increased inflammatory state in non-implantative endometrium. Additionally, it proposes PYGB as a potential biomarker for endometrial receptivity or implantation success. This knowledge opens a new avenue for developing embryo transfer strategies, through the improvement of embryo culture media or modifying endometrial fluid composition to increase pregnancy rates. This study was partially funded by a Grant for Fertility Innovation (GFI, 2011) from Merck (Darmstadt, Germany). Authors declare no competing interests. Not applicable.

PdtaS Deficiency Affects Resistance of Mycobacteria to Ribosome Targeting Antibiotics.

Two-component regulatory systems (TCSSs) are key regulatory elements responsible for the adaptation of bacteria to environmental stresses. A classical TCSS is typically comprised of a sensory histidine kinase and a corresponding response regulator. Here, we used homologous recombination to construct a Mycobacterium smegmatis mutant defective in the synthesis of cytosolic histidine kinase PdtaS (Msmeg_1918). The resulting ΔpdtaS mutant strain was tested in the Phenotype Microarray screening system, which allowed us to identify aminoglycoside antibiotic sensitivity, tetracyclines antibiotic resistance as well as membrane transport and respiration, as the main processes affected by removal of pdtaS. The antibiotic sensitivity profiles were confirmed by survival assessment and complementation studies. To gain insight into the molecular mechanisms responsible for the observed phenotype, we compared ribosomal RNA and protein profiles of the mutant and wild-type strains. We carried out Northern blotting and qRT-PCR to compare rRNA levels and analyzed ribosome sedimentation patterns of the wild-type and mutant strains on sucrose gradients. Isolated ribosomes were further used to estimate relative abundance of individual proteins in the ribosomal subunits using label free mass spectrometry analysis. Additionally, the ΔpdtaS mutant revealed lower activity of the respiratory chain as measured by the rate of TTC (triphenyltetrazolium chloride) reduction, while at the same time showing only insignificant changes in the uptake of aminoglycosides. We postulate that deficiency of PdtaS affects the oxidative respiration rates and ribosomal composition causing relevant changes to intrinsic resistance or susceptibility to antibiotics targeting ribosomes, which are commonly used to treat mycobacterial infections.

Abstract 1527: BRD3 as a specific vulnerable therapeutic target in neuroblastoma

Abstract Introduction: BET inhibitors have raised high expectations for cancer treatment given their anti-proliferative effect by inhibiting BRD4 controlled enhancer activity of highly transcribed genes such as MYC(N). However, current inhibitors also target BRD2 and BRD3 which are functionally nonredundant with BRD4. In neuroblastoma only MYCN amplified tumors respond well to these drugs. Methods: We performed an integrated bioinformatics approach to scrutinize BET family genes as well as further candidate epigenetic regulators as targets for novel therapies in neuroblastoma. Results: First we performed a time-resolved expression data analysis of week 1 and 2 hyperplastic lesions and tumors derived from the TH-MYCN transgenic mouse model and confirmed dynamic regulation during tumor development for established neuroblastoma oncogenes and tumor suppressor genes. Next, we filtered within the highest upregulated genes for Cancer Gene Census (CGC) genes and identified 21 upregulated CGC genes mainly involved in chromatin remodeling and DNA repair. Finally, after further selection based on expression in CCLE and survival in neuroblastoma patients, BRD3 was identified as the top-ranked candidate. BRD3 exhibits drastic upregulation during tumor formation. Elevated BRD3 expression is the highest expressed gene in neuroblastoma cell lines upon analysis of the CCLE panel and associated with very poor prognosis. To explore the nonredundant functions of BRD3 in relation to BRD4, we performed RNA-sequencing after stable knockdown of BRD3 in neuroblastoma cell lines and compared the downstream effects on the transcriptome as well as the impact on cell viability to knockdown of BRD4 and pharmacological treatment with BET-inhibitors (JQ1, OTX015). In addition, we dissected the BRD3 protein complex by means of label-free mass spectrometry analysis to gain further insights into the BRD3 specific functions in relation to control of gene transcription and putative interaction with transcription factors such as MYCN. Current efforts are ongoing to test cooperative interaction of BRD3 versus BRD4 in dbh-MYCN driven neuroblastoma formation in zebrafish as well as BRD3 and BRD4 ChIP-sequencing in neuroblastoma cells. Conclusion: We identified BRD3 as a candidate novel driver gene in neuroblastoma and will present differential transcriptional control and protein interactions of BRD3 versus BRD4. This study can open the way towards developing BRD3 specific inhibitors for neuroblastoma and other BRD3 overexpressing cancers such as T-ALL and small cell lung carcinoma. Citation Format: Kaat Durinck, Jolien Dewyn, Anneleen Beckers, Siebe Loontiens, Suzanne Vanhauwaert, Daniel Carter, Belamy Chueng, Glenn Marshall, Katleen Depreter, Frank Westermann, Frank Speleman. BRD3 as a specific vulnerable therapeutic target in neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1527. doi:10.1158/1538-7445.AM2017-1527

Proteomic analysis of bronchoalveolar lavage fluid (BALF) from lung cancer patients using label-free mass spectrometry

BackgroundLung cancer is the leading cause of cancer-related mortality in both men and women throughout the world. The need to detect lung cancer at an early, potentially curable stage, is essential and may reduce mortality by 20%. The aim of this study was to identify distinct proteomic profiles in bronchoalveolar fluid (BALF) and plasma that are able to discriminate individuals with benign disease from those with non-small cell lung cancer (NSCLC). MethodsUsing label-free mass spectrometry analysis of BALF during discovery-phase analysis, a significant number of proteins were found to have different abundance levels when comparing control to adenocarcinoma (AD) or squamous cell lung carcinoma (SqCC). Validation of candidate biomarkers identified in BALF was performed in a larger cohort of plasma samples by detection with enzyme-linked immunoassay. ResultsFour proteins (Cystatin-C, TIMP-1, Lipocalin-2 and HSP70/HSPA1A) were selected as a representative group from discovery phase mass spectrometry BALF analysis. Plasma levels of TIMP-1, Lipocalin-2 and Cystatin-C were found to be significantly elevated in AD and SqCC compared to control. ConclusionThe results presented in this study indicate that BALF is an important proximal biofluid for the discovery and identification of candidate lung cancer biomarkers. General significanceThere is good correlation between the trend of protein abundance levels in BALF and that of plasma which validates this approach to develop a blood biomarker to aid lung cancer diagnosis, particularly in the era of lung cancer screening. The protein signatures identified also provide insight into the molecular mechanisms associated with lung malignancy.

Quantitative label-free mass spectrometry analysis of formalin-fixed, paraffin-embedded tissue representing the invasive cutaneous malignant melanoma proteome

Understanding the events at a protein level that govern the progression from melanoma in situ to invasive melanoma are important areas of current research to be developed. Recent advances in the analysis of formalin-fixed, paraffin-embedded tissue by proteomics, particularly using the filter-aided sample preparation protocol, has opened up the possibility of studying vast archives of clinical material and associated medical records. In the present study, quantitative protein profiling was performed using tandem mass spectrometry, and the proteome differences between melanoma in situ and invasive melanoma were compared. Biological pathway analyses revealed several signalling pathways differing between melanoma in situ and invasive melanoma, including metabolic pathways and the phosphoinositide 3-kinase-Akt signalling pathway. Selected proteins of interest (14–3-3ε and fatty acid synthase) were subsequently investigated using immunohistochemical analysis of tissue microarrays. Identifying the key proteins that play significant roles in the establishment of a more invasive phenotype in melanoma may ultimately aid diagnosis and treatment decisions.

An optimised method for the proteomic profiling of full thickness human skin.

BackgroundThe skin is the largest organ of the human body and is the first line barrier defence against trauma, microbial infiltration and radiation. Skin diseases can be a result of multi-systemic disease or an isolated condition. Due to its proteolysis resistant properties there are relatively few human skin proteomic datasets published compared with other human organs or body fluids. Skin is a challenging tissue to analyse using traditional proteomic techniques due to its high lipid content, insolubility and extensive cross-linking of proteins. This can complicate the isolation and digestion of proteins for analysis using mass spectrometry techniques.ResultsWe have optimised a sample preparation procedure to improve solubilisation and mass spectral compatibility of full thickness skin samples. Using this technique, we were able to obtain data for the proteome profile of full thickness human skin using on-line two-dimensional liquid chromatography, followed by ultra-high definition label-free mass spectrometry analysis (UDMSE). We were able to identify in excess of 2000 proteins from a full thickness skin sample.ConclusionsThe adoption of on-line fractionation and optimised acquisition protocols utilising ion mobility separation (IMS) technology has significantly increased the scope for protein identifications ten-fold.Electronic supplementary materialThe online version of this article (doi:10.1186/s12575-016-0045-y) contains supplementary material, which is available to authorized users.

Syntaxin13 Expression Is Regulated by Mammalian Target of Rapamycin (mTOR) in Injured Neurons to Promote Axon Regeneration

Injured peripheral neurons successfully activate intrinsic signaling pathways to enable axon regeneration. We have previously shown that dorsal root ganglia (DRG) neurons activate the mammalian target of rapamycin (mTOR) pathway following injury and that this activity enhances their axon growth capacity. mTOR plays a critical role in protein synthesis, but the mTOR-dependent proteins enhancing the regenerative capacity of DRG neurons remain unknown. To identify proteins whose expression is regulated by injury in an mTOR-dependent manner, we analyzed the protein composition of DRGs from mice in which we genetically activated mTOR and from mice with or without a prior nerve injury. Quantitative label-free mass spectrometry analyses revealed that the injury effects were correlated with mTOR activation. We identified a member of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) family of proteins, syntaxin13, whose expression was increased by injury in an mTOR-dependent manner. Increased syntaxin13 levels in injured nerves resulted from local protein synthesis and not axonal transport. Finally, knockdown of syntaxin13 in cultured DRG neurons prevented axon growth and regeneration. Together, these data suggest that syntaxin13 translation is regulated by mTOR in injured neurons to promote axon regeneration.