Global Proteomic Profiling Research Articles

Proteomic profiling of murine biliary-derived hepatic organoids and their capacity for drug disposition, bioactivation and detoxification

Hepatic organoids are a recent innovation in in vitro modeling. Initial studies suggest that organoids better recapitulate the liver phenotype in vitro compared to pre-existing proliferative cell models. However, their potential for drug metabolism and detoxification remains poorly characterized, and their global proteome has yet to be compared to their tissue of origin. This analysis is urgently needed to determine what gain-of-function this new model may represent for modeling the physiological and toxicological response of the liver to xenobiotics. Global proteomic profiling of undifferentiated and differentiated hepatic murine organoids and donor-matched livers was, therefore, performed to assess both their similarity to liver tissue, and the expression of drug-metabolizing enzymes and transporters. This analysis quantified 4405 proteins across all sample types. Data are available via ProteomeXchange (PXD017986). Differentiation of organoids significantly increased the expression of multiple cytochrome P450, phase II enzymes, liver biomarkers and hepatic transporters. While the final phenotype of differentiated organoids is distinct from liver tissue, the organoids contain multiple drug metabolizing and transporter proteins necessary for liver function and drug metabolism, such as cytochrome P450 3A, glutathione-S-transferase alpha and multidrug resistance protein 1A. Indeed, the differentiated organoids were shown to exhibit increased sensitivity to midazolam (10–1000 µM) and irinotecan (1–100 µM), when compared to the undifferentiated organoids. The predicted reduced activity of HNF4A and a resulting dysregulation of RNA polymerase II may explain the partial differentiation of the organoids. Although further experimentation, optimization and characterization is needed relative to pre-existing models to fully contextualize their use as an in vitro model of drug-induced liver injury, hepatic organoids represent an attractive novel model of the response of the liver to xenobiotics. The current study also highlights the utility of global proteomic analyses for rapid and accurate evaluation of organoid-based test systems.

Serum proteomic profiling in patients with advanced Schistosoma japonicum-induced hepatic fibrosis

BackgroundSchistosoma japonicum is a parasitic flatworm that is the aetiological agent of human schistosomiasis, an important cause of hepatic fibrosis. Schistosomiasis-induced hepatic fibrosis is a consequence of the highly fibrogenic nature of egg-induced granulomatous lesions, which are the main pathogenic features of schistosomiasis. Although global awareness of the association between schistosomiasis-induced hepatic fibrosis and S. japonicum infection is increasing, little is known about the molecular differences associated with rapid progression to schistosomiasis in cirrhotic patients.MethodsWe systematically used data-independent acquisition (DIA)-based liquid chromatography-mass spectrometry to identify differentially expressed proteins in serum samples from patients with advanced S. japonicum-induced hepatic fibrosis.ResultsOur analysis identified 1144 proteins, among which 66 were differentially expressed between the healthy control group and the group of patients with advanced S. japonicum-induced hepatic fibrosis stage F2 (SHF-F2) and 214 were differentially expressed between the SHF-F2 and SHF-F4 groups (up- or downregulation of at least 1.5-fold in serum samples). The results also indicated that two selected proteins (C1QA and CFD) are potential biomarkers for distinguishing between patients with SHF-F2 and those with SHF-F4 due to S. japonicum infection.ConclusionsWe provide here the first global proteomic profile of serum samples from patients with advanced S. japonicum-induced hepatic fibrosis. The proteins C1QA and CFD are potential diagnostic markers for patients with SHF-F2 and SHF-F4 due to S. japonicum infection, although further large-scale studies are needed. Our DIA-based quantitative proteomic analysis revealed molecular differences among individuals at different stages of advanced S. japonicum-induced hepatic fibrosis and may provide fundamental information for further detailed investigations.Graphical

Lactate Dehydrogenase B and Pyruvate Oxidation Pathway Associated With Carfilzomib-Related Cardiotoxicity in Multiple Myeloma Patients: Result of a Multi-Omics Integrative Analysis

Multiple myeloma (MM) is the second most frequent hematologic cancer in the United States. Carfilzomib (CFZ), an irreversible proteasome inhibitor being used to treat relapsed and refractory MM, has been associated with cardiotoxicity, including heart failure. We hypothesized that a multi-omics approach integrating data from different omics would provide insights into the mechanisms of CFZ-related cardiovascular adverse events (CVAEs). Plasma samples were collected from 13 MM patients treated with CFZ (including 7 with CVAEs and 6 with no CVAEs) at the University of Florida Health Cancer Center. These samples were evaluated in global metabolomic profiling, global proteomic profiling, and microRNA (miRNA) profiling. Integrative pathway analysis was performed to identify genes and pathways differentially expressed between patients with and without CVAEs. The proteomics analysis identified the up-regulation of lactate dehydrogenase B (LDHB) [fold change (FC) = 8.2, p = 0.01] in patients who experienced CVAEs. The metabolomics analysis identified lower plasma abundance of pyruvate (FC = 0.16, p = 0.0004) and higher abundance of lactate (FC = 2.4, p = 0.0001) in patients with CVAEs. Differential expression analysis of miRNAs profiling identified mir-146b to be up-regulatein (FC = 14, p = 0.046) in patients with CVAE. Pathway analysis suggested that the pyruvate fermentation to lactate pathway is associated with CFZ-CVAEs. In this pilot multi-omics integrative analysis, we observed the down-regulation of pyruvate and up-regulation of LDHB among patients who experienced CVAEs, suggesting the importance of the pyruvate oxidation pathway associated with mitochondrial dysfunction. Validation and further investigation in a larger independent cohort are warranted to better understand the mechanisms of CFZ-CVAEs.

RRBP1 rewires cisplatin resistance in oral squamous cell carcinoma by regulating Hippo pathway.

Chemoresistance is one of the major factors for treatment failure in OSCC. Identifying key resistance triggering molecules will be useful strategy for developing novel treatment methods. To identify the causative factors of chemoresistance, we performed RNA sequencing and global proteomic profiling of human OSCC lines presenting with sensitive, early and late cisplatin-resistance patterns. From the common set of dysregulated genes from both the analysis, RRBP1 was identified to be upregulated in both early and late cisplatin-resistant cells with respect to the sensitive counterpart. Analysis of OSCC patient sample indicates that RRBP1 expression is upregulated in chemotherapy-non-responder tumours as compared to chemotherapy-responder tumours. Genetic (knockout) or pharmacological (Radezolid, represses expression of RRBP1) inhibition of RRBP1 restores cisplatin-mediated cell death in chemo-resistant OSCC. Mechanistically, RRBP1 regulates Yes-associated protein1 (YAP1), a key protein in the Hippo pathway to induce chemoresistance. The PDC xenograft data suggests that knockout of RRBP1 induces cisplatin-mediated cell death and facilitates a significant reduction of tumour burden. Overall, our data suggests that (I) RRBP1 is a major driver of cisplatin-resistance in OSCC, (II) RRBP1 regulates YAP1 expression to mediate cisplatin-resistance, (III) Radezolid represses RRBP1 expression and (IV) targeting RRBP1 reverses cisplatin-induced chemoresistance in advanced OSCC.

An RNA tagging approach for system-wide RNA-binding proteome profiling and dynamics investigation upon transcription inhibition.

RNA-protein interactions play key roles in epigenetic, transcriptional and posttranscriptional regulation. To reveal the regulatory mechanisms of these interactions, global investigation of RNA-binding proteins (RBPs) and monitor their changes under various physiological conditions are needed. Herein, we developed a psoralen probe (PP)-based method for RNA tagging and ribonucleic-protein complex (RNP) enrichment. Isolation of both coding and noncoding RNAs and mapping of 2986 RBPs including 782 unknown candidate RBPs from HeLa cells was achieved by PP enrichment, RNA-sequencing and mass spectrometry analysis. The dynamics study of RNPs by PP enrichment after the inhibition of RNA synthesis provides the first large-scale distribution profile of RBPs bound to RNAs with different decay rates. Furthermore, the remarkably greater decreases in the abundance of the RBPs obtained by PP-enrichment than by global proteome profiling suggest that PP enrichment after transcription inhibition offers a valuable way for large-scale evaluation of the candidate RBPs.

Global Proteomic Profile Integrated to Quantitative and Morphometric Assessment of Enteric Neurons: Investigation of the Mechanisms Involved in the Toxicity Induced by Acute Fluoride Exposure in the Duodenum.

The enteric nervous system is responsible for controlling the gastrointestinal tract (GIT) functions. Enteric neuropathies are highly correlated to the development of several intestinal disturbances. Fluoride (F) is extensively applied for dental health improvement and its ingestion can promote systemic toxicity with mild to severe GIT symptomatology and neurotoxicity. Although F harmful effects have been published, there is no information regarding noxiousness of a high acute F exposure (25mg F/kg) on enteric neurons and levels of expression of intestinal proteins in the duodenum. Quantitative proteomics of the duodenum wall associated to morphometric and quantitative analysis of enteric neurons displayed F effects of a high acute exposure. F-induced myenteric neuroplasticity was characterized by a decrease in the density of nitrergic neurons and morphometric alterations in the general populations of neurons, nitrergic neurons, and substance P varicosities. Proteomics demonstrated F-induced alterations in levels of expression of 356 proteins correlated to striated muscle cell differentiation; generation of precursor metabolites and energy; NADH and glutathione metabolic process and purine ribonucleoside triphosphate biosynthesis. The neurochemical role of several intestinal proteins was discussed specially related to the modulation of enteric neuroplasticity. The results provide a new perspective on cell signaling pathways of gastrointestinal symptomatology promoted by acute F toxicity.

Surfactant-assisted one-pot sample preparation for label-free single-cell proteomics

Large numbers of cells are generally required for quantitative global proteome profiling due to surface adsorption losses associated with sample processing. Such bulk measurement obscures important cell-to-cell variability (cell heterogeneity) and makes proteomic profiling impossible for rare cell populations (e.g., circulating tumor cells (CTCs)). Here we report a surfactant-assisted one-pot sample preparation coupled with mass spectrometry (MS) method termed SOP-MS for label-free global single-cell proteomics. SOP-MS capitalizes on the combination of a MS-compatible nonionic surfactant, n-Dodecyl-β-D-maltoside, and hydrophobic surface-based low-bind tubes or multi-well plates for ‘all-in-one’ one-pot sample preparation. This ‘all-in-one’ method including elimination of all sample transfer steps maximally reduces surface adsorption losses for effective processing of single cells, thus improving detection sensitivity for single-cell proteomics. This method allows convenient label-free quantification of hundreds of proteins from single human cells and ~1200 proteins from small tissue sections (close to ~20 cells). When applied to a patient CTC-derived xenograft (PCDX) model at the single-cell resolution, SOP-MS can reveal distinct protein signatures between primary tumor cells and early metastatic lung cells, which are related to the selection pressure of anti-tumor immunity during breast cancer metastasis. The approach paves the way for routine, precise, quantitative single-cell proteomics.

Abstract PS18-06: Proteomic analysis of breast cancer formalin-fixed paraffin-embedded clinical specimens identifies biologically-important subtypes with distinct clinical outcomes

Abstract Background: Genomic classification of breast cancer has advanced breast cancer diagnosis and outcomes. However, extensive heterogeneity still exists beyond their DNA or RNA profiles. Newer classifications based on protein profiling are being developed to investigate the molecular oncology of breast cancers at the level where most drugs act. Using a recently-developed technology, we performed global proteomic profiling of 300 breast cancer specimens linked to outcome data. Methods: Sections of 75 samples from each PAM50 intrinsic subtype (Luminal A, Luminal B, Her2-enriched, Basal-like; n = 300) were macrodissected and analyzed using the Single-Pot Solid-Phase enhanced Sample Preparation Clinical Tissue Proteomics, a highly sensitive 11-sample multiplex massspectrometry protocol applicable to formalin-fixed, paraffin embedded (FFPE) specimens. This methodology enables comprehensive quantification of protein expression for classifier and biomarker discovery. Patients were diagnosed during 2008-2013 (n = 178, dataset I) and 1986-1992 (n = 122, dataset II). Results: In-depth proteomic analysis measured 9088 proteins in total, including 4214 proteins quantified in every sample. Consensus clustering of 174 evaluable cases in dataset I identified four distinct groups based on expression values for 1054 highly variant proteins. Cluster 3 (n = 47, mostly basal-like with HER2-Enriched) displayed the most favorable recurrence free survival (RFS) when compared to other clusters (HR = 0.22, 95%CI [0.08-0.63], p = 0.005). This cluster was enriched for immune related pathways including antigen processing and presentation and type I & II interferon signaling, and displayed high tumor infiltrating lymphocyte counts, characterizing this cluster as “immune hot”. In contrast, cluster 2 (n = 50, mostly basal-like) exhibited the poorest RFS (HR = 2.88, 95%CI [1.45-5.70], p = 0.002) and was enriched for proteins related to stromal and extracellular matrix with few immune related peptides. Cluster 1 (n = 34, luminal B and HER2-Enriched) was associated with lipid metabolism, whereas cluster 4 (n = 43, mostly HER2-Enriched with luminal A and luminal B) had a profile enriched for extracellular matrix, blood coagulation and complement activation. Conclusions: Global proteomic analysis on FFPE specimens can characterize the heterogeneity of breast cancer in a reliable and clinically-applicable high throughput manner. Our methodology identifies protein candidates that potentially serve as therapeutic targets and could be adapted to archived clinical specimens from other tumors. Citation Format: Karama Asleh, Gian Luca Negri, Sandra E. Spencer Miko, Shane Colborne, Christopher S. Hughes, Xiu Q. Wang, Dongxia Gao, C. Blake Gilks, Stephen K.L. Chia, Torsten O. Nielsen, Gregg B. Morin. Proteomic analysis of breast cancer formalin-fixed paraffin-embedded clinical specimens identifies biologically-important subtypes with distinct clinical outcomes [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS18-06.

Mass Spectrometry-Based Redox and Protein Profiling of Failing Human Hearts.

Oxidative stress contributes to detrimental functional decline of the myocardium, leading to the impairment of the antioxidative defense, dysregulation of redox signaling, and protein damage. In order to precisely dissect the changes of the myocardial redox state correlated with oxidative stress and heart failure, we subjected left-ventricular tissue specimens collected from control or failing human hearts to comprehensive mass spectrometry-based redox and quantitative proteomics, as well as glutathione status analyses. As a result, we report that failing hearts have lower glutathione to glutathione disulfide ratios and increased oxidation of a number of different proteins, including constituents of the contractile machinery as well as glycolytic enzymes. Furthermore, quantitative proteomics of failing hearts revealed a higher abundance of proteins responsible for extracellular matrix remodeling and reduced abundance of several ion transporters, corroborating contractile impairment. Similar effects were recapitulated by an in vitro cell culture model under a controlled oxygen atmosphere. Together, this study provides to our knowledge the most comprehensive report integrating analyses of protein abundance and global and peptide-level redox state in end-stage failing human hearts as well as oxygen-dependent redox and global proteome profiles of cultured human cardiomyocytes.

Abstract PR002: Global proteomic profiling of endometrial carcinomas identify prognostic markers

Abstract While endometrial cancer (EC) has an overall good prognosis, some patients do poorly and there is room for refinement within current classification systems. Using the TCGA prognostic grouping of EC, our group developed the Proactive Molecular Risk Classifier for Endometrial Cancer (ProMisE), which reliably and reproducibly stratifies ECs into four prognostic groups: POLEmut, p53wt/NSMP, MMRd (mismatch repair deficient), and p53abn, with the best prognosis for POLEmut to worst prognosis for p53abn. In the current study, global proteomic analysis was performed using the clinical SP3-CTP workflow on archival tissues from 151 patients including 40 MMRd, 34 POLEmut, 34 p53abn and 43 p53wt/NSMP, with clinical follow up data. Included in the cohort were 11 replicate samples (different parts of the same tumor) to examine spatial heterogeneity in the proteomic profiles. Replicate samples were highly correlated to each other, with the exception of three POLEmut cases with very poor correlation in the proteome in different parts of the tumor. As POLEmut tumors have an exceedingly high mutation burden, it is not surprising that this translates to heterogeneity at the proteomic level. Disease specific survival was examined to determine prognostic significance within the whole cohort and within individual molecular subgroups. High TOMM34, PLTP or TSFM expression was correlated to poor disease specific survival in the whole cohort and independently prognostic when molecular subtype, grade and histotype are considered. High MGST, NCL or XPNPEP3 were associated with poor outcomes within the p53wt/NSMP group. POLD2 and ENAH were prognostic within the MMRd group. Within the p53abn group, ACADVL and BABAM1 were found to be prognostic, and GRB7 was found to be enriched in the p53abn group compared to other molecular subtypes. As the group with the worst prognosis, p53abn group could benefit from novel therapeutic avenues. ACADVL, BABAM1 and GRB7 all lie within pathways that are potentially targetable. Our proteomic analysis has identified prognostic markers that may be useful in further refining current molecular classification to help guide treatment decisions. Furthermore, new therapeutic interventions could be developed to target proteins and pathways identified by this proteomics screen. Citation Format: Dawn R. Cochrane, Gian Negri, Jutta Huvila, David A. Farnell, Emily Thompson, Winnie Yang, Genny Trigo-Gonzales, Amy Lum, Sandra Spencer, Ryan Riley, Samuel Leung, Christine Chow, Jamie Lim, Martin Koebel, Stefan Kommoss, Friedrich Kommoss, Lien Hoang, David G. Huntsman, Gregg Morin, Jessica N. McAlpine. Global proteomic profiling of endometrial carcinomas identify prognostic markers [abstract]. In: Proceedings of the AACR Virtual Special Conference: Endometrial Cancer: New Biology Driving Research and Treatment; 2020 Nov 9-10. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(3_Suppl):Abstract nr PR002.

Proteomic Characterization of Human Neural Stem Cells and Their Secretome During in vitro Differentiation.

Cell therapies represent a promising approach to slow down the progression of currently untreatable neurodegenerative diseases (e.g., Alzheimer's and Parkinson's disease or amyotrophic lateral sclerosis), as well as to support the reconstruction of functional neural circuits after spinal cord injuries. In such therapies, the grafted cells could either functionally integrate into the damaged tissue, partially replacing dead or damaged cells, modulate inflammatory reaction, reduce tissue damage, or support neuronal survival by secretion of cytokines, growth, and trophic factors. Comprehensive characterization of cells and their proliferative potential, differentiation status, and population purity before transplantation is crucial to preventing safety risks, e.g., a tumorous growth due to the proliferation of undifferentiated stem cells. We characterized changes in the proteome and secretome of human neural stem cells (NSCs) during their spontaneous (EGF/FGF2 withdrawal) differentiation and differentiation with trophic support by BDNF/GDNF supplementation. We used LC-MS/MS in SWATH-MS mode for global cellular proteome profiling and quantified almost three thousand cellular proteins. Our analysis identified substantial protein differences in the early stages of NSC differentiation with more than a third of all the proteins regulated (including known neuronal and NSC multipotency markers) and revealed that the BDNF/GDNF support affected more the later stages of the NSC differentiation. Among the pathways identified as activated during both spontaneous and BDNF/GDNF differentiation were the HIF-1 signaling pathway, Wnt signaling pathway, and VEGF signaling pathway. Our follow-up secretome analysis using Luminex multiplex immunoassay revealed significant changes in the secretion of VEGF and IL-6 during NSC differentiation. Our results further demonstrated an increased expression of neuropilin-1 as well as catenin β-1, both known to participate in the regulation of VEGF signaling, and showed that VEGF-A isoform 121 (VEGF121), in particular, induces proliferation and supports survival of differentiating cells.

CMTM6 drives cisplatin resistance by regulating Wnt signaling through the ENO-1/AKT/GSK3β axis.

Rewiring tumor cells to undergo drug-induced apoptosis is a promising way to overcome chemoresistance. Therefore, identifying causative factors for chemoresistance is of high importance. Unbiased global proteome profiling of sensitive, early, and late cisplatin-resistant oral squamous cell carcinoma (OSCC) lines identified CMTM6 as a top-ranked upregulated protein. Analyses of OSCC patient tumor samples demonstrated significantly higher CMTM6 expression in chemotherapy (CT) nonresponders as compared with CT responders. In addition, a significant association between higher CMTM6 expression and poorer relapse-free survival in esophageal squamous cell carcinoma, head and neck squamous cell carcinoma, and lung squamous cell carcinoma was observed from Kaplan-Meier plot analysis. Stable knockdown (KD) of CMTM6 restored cisplatin-mediated cell death in chemoresistant OSCC lines. Upon CMTM6 overexpression in CMTM6-KD lines, the cisplatin-resistant phenotype was rescued. The patient-derived cell xenograft model of chemoresistant OSCC displaying CMTM6 depletion restored the cisplatin-induced cell death and tumor burden substantially. The transcriptome analysis of CMTM6-KD and control chemoresistant cells depicted enrichment of the Wnt signaling pathway. We demonstrated that CMTM6 interaction with membrane-bound Enolase-1 stabilized its expression, leading to activation of Wnt signaling mediated by AKT–glycogen synthase kinase-3β. CMTM6 has been identified as a stabilizer of programmed cell death ligand 1. Therefore, as CMTM6 facilitates tumor cells for immune evasion and mediates cisplatin resistance, it could be a promising therapeutic target for treating therapy-resistant OSCC.

Lead-Induced Motor Dysfunction Is Associated with Oxidative Stress, Proteome Modulation, and Neurodegeneration in Motor Cortex of Rats.

Lead (Pb) is a toxic metal with great neurotoxic potential. The aim of this study was to investigate the effects of a long-term Pb intoxication on the global proteomic profile, oxidative biochemistry and neuronal density in motor cortex of adult rats, and the possible outcomes related to motor functions. For this, Wistar rats received for 55 days a dose of 50 mg/Kg of Pb acetate by intragastric gavage. Then, the motor abilities were evaluated by open field and inclined plane tests. To investigate the possible oxidative biochemistry modulation, the levels of pro-oxidant parameters as lipid peroxidation and nitrites were evaluated. The global proteomic profile was evaluated by ultraefficiency liquid chromatography system coupled with mass spectrometry (UPLC/MS) followed by bioinformatic analysis. Moreover, it was evaluated the mature neuron density by anti-NeuN immunostaining. The statistical analysis was performed through Student's t-test, considering p < 0.05. We observed oxidative stress triggering by the increase in malonaldehyde and nitrite levels in motor cortex. In the proteomic analysis, the motor cortex presented alterations in proteins associated with neural functioning, morphological organization, and neurodegenerative features. In addition, it was observed a decrease in the number of mature neurons. These findings, associated with previous evidences observed in spinal cord, cerebellum, and hippocampus under the same Pb administration protocol, corroborate with the motor deficits in the rats towards Pb. Thus, we conclude that the long-term administration to Pb in young Wistar rats triggers impairments at several organizational levels, such as biochemical and morphological, which resulted in poor motor performance.

Liquid Chromatography and Tandem Mass Spectrometry in Label-Free Protein Quantification of Zebrafish (Danio rerio) Eggs.

Rapid innovations in core proteomic technologies and proteome-based bioinformatics fortified by recent genome sequencing allow the characterization and quantification of proteins on a global scale. These capabilities empower research to develop a more comprehensive understanding of how changes in protein expression and modification can affect complex signaling and regulatory networks. The consequences of these studies have significant implications for understanding how myriad activities are regulated in biological systems.Proteomic approaches have been applied to investigate the physiology, developmental biology, and impact of contaminants in fishes as model organisms. Here, we describe the use of label-free protein quantification and global proteome profiling to characterize eggs of different quality grades in the zebrafish .

Global Proteome Profiling to Assess Changes in Protein Abundance Using Isobaric Labeling and Liquid Chromatography-Tandem Mass Spectrometry.

Protein degradation is a critical component of all facets of cell biology, and recently methods have been developed to make use of targeted protein degradation as both an investigative tool and a potential therapeutic avenue. Mass spectrometry-based proteomic studies have allowed detailed characterization of changes in protein level and the biology underlying growth, development, and disease. Current methods and instrumentation allow identification and quantitative analysis of thousands of proteins in a single assay. The method described here involves cell lysis and digestion to peptides, labeling peptides with isobaric tagging TMT reagents, basic reversed phase fractionation, and liquid chromatography-tandem mass spectrometry analysis of the enriched peptides.

Screening of Potential Biomarkers for Gastric Cancer with Diagnostic Value Using Label-free Global Proteome Analysis

Gastric cancer (GC) is known as a top malignant type of tumors worldwide. Despite the recent decrease in mortality rates, the prognosis remains poor. Therefore, it is necessary to find novel biomarkers with early diagnostic value for GC. In this study, we present a large-scale proteomic analysis of 30 GC tissues and 30 matched healthy tissues using label-free global proteome profiling. Our results identified 537 differentially expressed proteins, including 280 upregulated and 257 downregulated proteins. The ingenuity pathway analysis (IPA) results indicated that the sirtuin signaling pathway was the most activated pathway in GC tissues whereas oxidative phosphorylation was the most inhibited. Moreover, the most activated molecular function was cellular movement, including tissue invasion by tumor cell lines. Based on IPA results, 15 hub proteins were screened. Using the receiver operating characteristic curve, most of hub proteins showed a high diagnostic power in distinguishing between tumors and healthy controls. A four-protein (ATP5B-ATP5O-NDUFB4-NDUFB8) diagnostic signature was built using a random forest model. The area under the curve (AUC) values of this model were 0.996 and 0.886 for the training and testing sets, respectively, suggesting that the four-protein signature has a high diagnostic power. This signature was further tested with independent datasets using plasma enzyme-linked immune sorbent assays, resulting in an AUC value of 0.778 for distinguishing GC tissues from healthy controls, and using immunohistochemical tissue microarray analysis, resulting in an AUC value of 0.805. In conclusion, this study identifies potential biomarkers and improves our understanding of the pathogenesis, providing novel therapeutic targets for GC.

Global Proteomic Profiling of Piscirickettsia salmonis and Salmon Macrophage-Like Cells during Intracellular Infection.

Piscirickettsiasalmonis is an intracellular bacterial fish pathogen that causes piscirickettsiosis, a disease with numerous negative impacts in the Chilean salmon farming industry. Although transcriptomic studies of P. salmonis and its host have been performed, dual host–pathogen proteomic approaches during infection are still missing. Considering that gene expression does not always correspond with observed phenotype, and bacteriological culture studies inadequately reflect infection conditions, to improve the existing knowledge for the pathogenicity of P. salmonis, we present here a global proteomic profiling of Salmon salar macrophage-like cell cultures infected with P. salmonis LF-89. The proteomic analyses identified several P. salmonis proteins from two temporally different stages of macrophages infection, some of them related to key functions for bacterial survival in other intracellular pathogens. Metabolic differences were observed in early-stage infection bacteria, compared to late-stage infections. Virulence factors related to membrane, lipopolysaccharide (LPS) and surface component modifications, cell motility, toxins, and secretion systems also varied between the infection stages. Pilus proteins, beta-hemolysin, and the type VI secretion system (T6SS) were characteristic of the early-infection stage, while fimbria, upregulation of 10 toxins or effector proteins, and the Dot/Icm type IV secretion system (T4SS) were representative of the late-infection stage bacteria. Previously described virulence-related genes in P. salmonis plasmids were identified by proteomic assays during infection in SHK-1 cells, accompanied by an increase of mobile-related elements. By comparing the infected and un-infected proteome of SHK-1 cells, we observed changes in cellular and redox homeostasis; innate immune response; microtubules and actin cytoskeleton organization and dynamics; alteration in phagosome components, iron transport, and metabolism; and amino acids, nucleoside, and nucleotide metabolism, together with an overall energy and ATP production alteration. Our global proteomic profiling and the current knowledge of the P. salmonis infection process allowed us to propose a model of the macrophage–P. salmonis interaction.

Abstract 15012: Lactate Dehydrogenase B and Pyruvate Oxidation Associated With Carfilzomib-Related Cardiotoxicity in Multiple Myeloma Patients: A Multi-omics Study

Introduction: Multiple Myeloma (MM) is the second most frequent hematologic cancer in the United States. Carfilzomib (CFZ), a proteasome inhibitor used to treat relapsed and refractory MM, has been associated with cardiotoxicity including heart failure (HF). Hypothesis: We hypothesized that a multi-omics approach integrating data from different omics will provide insights into the mechanisms of CFZ-induced HF. Methods: Plasma samples were collected from 13 MM patients treated with CFZ (including 7 with symptomatic HF and 6 with no HF) at the University of Florida Health Cancer Center. Global metabolomics profiling was performed using a LC/MS metabolomic platform. Global proteomic profiling was performed on LC/MS/MS platform with ~ 370 proteins quantified by iTRAQ analysis. TaqMan ® OpenArray ® Human MicroRNA Panel, containing 754 human microRNAs (miRNAs), was used for miRNA profiling. Pathway integrative analysis was performed to identify genes and pathways that are differentially expressed between those who developed HF versus those who did not. Features with p-value &lt; 0.05 were considered statistically significant. Results: The cohort mean age was 61 years and 38.5% were men. Proteomics data identified up-regulation of lactate dehydrogenase B (LDHB) (fold change [FC]=8.2, p=0.01) in those who developed HF compared to those who did not. Metabolomics data identified lower plasma abundance of pyruvate (FC=0.16, p=0.0004) and higher abundance of lactate (FC=2.4, p=0.0001) in HF patients. Using miRNA target gene prediction tools, we observed that LDHB regulated by miR-139 which downregulated in HF patients (FC=0.03). Multi-omics integrative analysis suggested that the pyruvate fermentation to lactate (pyruvate oxidation) pathway is associated with CFZ-related HF. Conclusion: In this pilot multi-omics integrative analysis, we observed down-regulation of pyruvate and up-regulation of LDHB among patients with CFZ-related HF, which suggests the importance of pyruvate oxidation pathway and mitochondrial dysfunction in CFZ-related HF and that this pathway might be regulated by miR-139. Further investigation in a larger independent cohort is warranted to better understand the mechanisms of CFZ-related HF.

Patient-derived xenograft (PDX) models of colorectal carcinoma (CRC) as a platform for chemosensitivity and biomarker analysis in personalized medicine

Patient-derived xenograft (PDX) tumor models represent a valuable platform for identifying new biomarkers and novel targets, to evaluate therapy response and resistance mechanisms. This study aimed at establishment, characterization and therapy testing of colorectal carcinoma-derived PDX. We generated 49 PDX and validated identity between patient tumor and corresponding PDX. Sensitivity of PDX toward conventional and targeted drugs revealed that 92% of PDX responded toward irinotecan, 45% toward 5-FU, 65% toward bevacizumab, and 61% toward cetuximab. Expression of epidermal growth factor receptor (EGFR) ligands correlated to the sensitivity toward cetuximab. Proto-oncogene B-RAF, EGFR, Kirsten rat sarcoma virus oncogene homolog gene copy number correlated positively with cetuximab and erlotinib sensitivity. The mutational analyses revealed an individual mutational profile of PDX and mainly identical profiles of PDX from primary tumor vs corresponding metastasis. Mutation in PIK3CA was a determinant of accelerated tumor doubling time. PDX with wildtype Kirsten rat sarcoma virus oncogene homolog, proto-oncogene B-RAF, and phosphatidylinositol-4,5-bisphosphate 3-kinaseM catalytic subunit alfa showed higher sensitivity toward cetuximab and erlotinib. To study the molecular mechanism of cetuximab resistance, cetuximab resistant PDX models were generated, and changes in HER2, HER3, betacellulin, transforming growth factor alfa were observed. Global proteome and phosphoproteome profiling showed a reduction in canonical EGFR-mediated signaling via PTPN11 (SHP2) and AKT1S1 (PRAS40) and an increase in anti-apoptotic signaling as a consequence of acquired cetuximab resistance. This demonstrates that PDX models provide a multitude of possibilities to identify and validate biomarkers, signaling pathways and resistance mechanisms for clinically relevant improvement in cancer therapy.

HYpro16: A Two-Proteome Mixture to Assess Interference in Isobaric Tag-Based Sample Multiplexing Experiments.

Isobaric tagging is a powerful strategy for global proteome profiling. A caveat of isobaric-tag-based quantification is "interference", which may be caused by coeluting peptides that are coisolated, cofragmented, and coanalyzed, thereby confounding quantitative accuracy. Here, we present a two-proteome standard that challenges the mass spectrometer to measure a range of protein abundance ratios in a background of potential interference. The HYpro16 standard consists of tandem mass tag (TMT) pro16-labeled human peptides at a 1:1 ratio across all channels into which is spiked TMTpro16-labeled yeast peptides in triplicate at 20:1, 10:1, 4:1, and 2:1 ratios. We showcase the HYpro16 standard by (1) altering the MS2 isolation window width and (2) examining different data acquisition methods (hrMS2, SPS-MS3, RTS-MS3). Our data illustrate that wider isolation widths moderately increase the TMT signal, the benefits of which are offset by decreased ratio accuracy. We also show that using real-time database searching (RTS)-MS3 resulted in the most accurate ratios. Additionally, the number of quantified yeast proteins using RTS-MS3 approaches that of hrMS2 when using a yeast-specific database for real-time searching. In short, this quality control standard allows for the assessment of multiple quantitative measurements within a single run, which can be compared across instruments to benchmark and track performance.