Comprehensive Proteomic Profiling Research Articles

P052 Comprehensive proteomic profiling of stool from UC and CD patients reveals expected and novel mucosal pathobiology, enabling identification of candidate non-invasive biomarkers to monitor mucosal disease activity

Abstract Background Inflammatory bowel disease (IBD) is a multifactorial disorder characterized by chronic gastrointestinal (GI) inflammation. In clinical practice, physicians urgently need biomarkers to monitor changes in mucosal disease to manage treatment. Non-invasive tools such as fecal biomarkers could allow for frequent monitoring of mucosal disease activity and may reflect inflammation of the entire GI tract. Towards identification of novel non-invasive fecal biomarkers, we performed a comprehensive, unbiased, analysis of the human fecal proteome from ulcerative colitis (UC), Crohn’s disease (CD) patients, and healthy controls (HC), focusing on human secreted proteins using proteomic approaches. Methods Fecal extraction processes optimized for recovery of secreted proteins were applied to stool samples of mild to severe UC and CD patients and HCs, 20 samples per group. An unbiased survey of the human fecal proteome of the IBD and HC stool extracts was performed using both data-dependent acquisition and data-independent acquisition methods on QExactive HF and Fusion Lumos mass spectrometers. Data were analyzed in Spectronaut for peptide identification, followed by analysis in MS Stats for protein quantification. Fecal calprotectin (fCAL) levels were measured in these stool samples using the Buhlmann EK-CAL immunoassay kit to correlate fCAL levels to proteomic calprotectin (S100A8/S100A9 heterodimer). Results We identified and quantified 594 differentially expressed proteins in stool using proteomics, including known proteins perturbed in IBD pathobiology. Elevated levels of neutrophilic proteins, e.g. fCal, neutrophil elastase, lactoferrin and leucine-rich Α-2 glycoprotein 1 were detected as well as proteins linked to rectal bleeding and ulceration e.g. hemoglobin subunit alpha 1 and beta. Pancreatic enzymes, suggested to be associated with active disease, were also detected. Overall, UC and CD contained many proteins not detected in HC. Differential abundances were observed between UC and CD patients, possibly reflecting differences in mucosal pathophysiology. The abundance of proteomic calprotectin correlated highly with the fCAL levels as determined by immunoassay (Spearman rank order > 0.9 for S100A8 and S100A9 to fCAL), validating the fecal proteomic approach to uncover known and novel proteins of IBD biology. Conclusion We developed and qualified methods to perform fecal proteomics on IBD stool samples, enabling identification of known and novel proteins of mucosal UC and CD disease biology. In depth analysis of these proteomic datasets may reveal novel fecal biomarker candidates, targeted to reflect mucosal disease activity and potentially serve as non-invasive surrogates for endoscopy and help guide treatment in IBD patients.

Comprehensive Proteomic Profiling of Aqueous Humor Proteins in Proliferative Diabetic Retinopathy.

PurposeProliferative diabetic retinopathy (PDR) is a serious ocular disease that can lead to retinal microvascular complications in patients with diabetes mellitus. To date, no studies have explored PDR development by analyzing the aqueous humor (AH). Therefore we carried out tandem mass tag (TMT) proteomic quantification to compare AH protein profiles between PDR and non-PDR subjects.MethodsWe enrolled six PDR and six control (senile cataract) subjects. AH samples were collected during surgery and stored at –80°C. Proteins were extracted, trypsin-digested, and labeled with TMTs for mass spectrometric analysis.ResultsWe found 191 proteins to be changed with |log2 (fold change)| ≥1 (P < 0.05 and identification with at least two peptides per protein). Of them, 111 were downregulated, whereas 80 were upregulated in the PDR group. Proteomic bioinformatic analysis indicated that PDR development was related to complement and coagulation cascades, platelet activation, extracellular matrix–receptor interaction, focal adhesion, protein digestion and absorption, human papillomavirus infection, PI3K-Akt signaling pathway, cholesterol metabolism, peroxisome proliferator-activated receptor signaling pathways, fat digestion and absorption, and vitamin digestion and absorption pathways.ConclusionsComprehensive proteomic profiling of the AH revealed 191 differentially expressed proteins between the two groups. Most of these proteins belong to secretory pathways, and therefore can be used as biomarkers in clinical testing and basic research.Translational RelevancePathway analysis and a review of the literature enabled us to draw a novel biological map that will support further studies on the underlying mechanisms and therapeutic control of PDR development.

Modulation of Rumen Microbes Through Extracellular Vesicle Released by the Rumen Fluke Calicophoron daubneyi.

Parasite derived extracellular vesicles (EVs) have been proposed to play key roles in the establishment and maintenance of infection. Calicophoron daubneyi is a newly emerging parasite of livestock with many aspects of its underpinning biology yet to be resolved. This research is the first in-depth investigation of EVs released by adult C. daubneyi. EVs were successfully isolated using both differential centrifugation and size exclusion chromatography (SEC), and morphologically characterized though transmission electron microscopy (TEM). EV protein components were characterized using a GeLC approach allowing the elucidation of comprehensive proteomic profiles for both their soluble protein cargo and surface membrane bound proteins yielding a total of 378 soluble proteins identified. Notably, EVs contained Sigma-class GST and cathepsin L and B proteases, which have previously been described in immune modulation and successful establishment of parasitic flatworm infections. SEC purified C. daubneyi EVs were observed to modulate rumen bacterial populations by likely increasing microbial species diversity via antimicrobial activity. This data indicates EVs released from adult C. daubneyi have a role in establishment within the rumen through the regulation of microbial populations offering new routes to control rumen fluke infection and to develop molecular strategies to improve rumen efficiency.

Biogeographical venom variation in the Indian spectacled cobra (Naja naja) underscores the pressing need for pan-India efficacious snakebite therapy.

BackgroundSnake venom composition is dictated by various ecological and environmental factors, and can exhibit dramatic variation across geographically disparate populations of the same species. This molecular diversity can undermine the efficacy of snakebite treatments, as antivenoms produced against venom from one population may fail to neutralise others. India is the world’s snakebite hotspot, with 58,000 fatalities and 140,000 morbidities occurring annually. Spectacled cobra (Naja naja) and Russell’s viper (Daboia russelii) are known to cause the majority of these envenomations, in part due to their near country-wide distributions. However, the impact of differing ecologies and environment on their venom compositions has not been comprehensively studied.MethodsHere, we used a multi-disciplinary approach consisting of venom proteomics, biochemical and pharmacological analyses, and in vivo research to comparatively analyse N. naja venoms across a broad region (>6000 km; seven populations) covering India’s six distinct biogeographical zones.FindingsBy generating the most comprehensive pan-Indian proteomic and toxicity profiles to date, we unveil considerable differences in the composition, pharmacological effects and potencies of geographically-distinct venoms from this species and, through the use of immunological assays and preclinical experiments, demonstrate alarming repercussions on antivenom therapy. We find that commercially-available antivenom fails to effectively neutralise envenomations by the pan-Indian populations of N. naja, including a complete lack of neutralisation against the desert Naja population.ConclusionOur findings highlight the significant influence of ecology and environment on snake venom composition and potency, and stress the pressing need to innovate pan-India effective antivenoms to safeguard the lives, limbs and livelihoods of the country’s 200,000 annual snakebite victims.

Advances in high-throughput proteomic analysis

基于质谱的蛋白质组学技术已经日趋成熟,可以对细胞和组织中的成千上万种蛋白质进行全面的定性和定量分析,逐步实现“深度覆盖”。随着生物医学日益增长的大队列蛋白质组学分析需求,如何在保持较为理想的覆盖深度下实现短时间、快速的“高通量”蛋白质组学分析已成为当前亟需解决的关键问题之一。常规的蛋白质组学分析流程通常包括样品前处理、色谱分离、质谱检测和数据分析。该文从以上4个方面展开介绍近10年以来高通量蛋白质组学分析技术取得的一系列研究进展,主要包括:(1)基于高通量、自动化移液工作站的蛋白质组样品前处理方法;(2)基于微升流速液相色谱与质谱联用的高通量蛋白质组检测方法;(3)利用灵敏度高、扫描速度快的质谱仪实现短色谱梯度分离下蛋白质组深度覆盖的分析方法;(4)基于人工智能、深度神经网络、机器学习等的蛋白质组学大数据分析方法。此外,对高通量蛋白质组学面临的挑战及其发展进行展望。总而言之,预期在不久的将来高通量蛋白质组学技术将会逐步“落地转化”,成为大队列蛋白质组学分析的利器。

Quantifying Proteome and Protein Modifications in Activated T Cells by Multiplexed Isobaric Labeling Mass Spectrometry.

The dynamic regulation of protein function by altered protein expression and post-translational modifications (PTMs) is essential for T cell function, but it has remained difficult to systemically quantify such events. Mass spectrometry (MS)-based proteomics has become a mainstream tool for comprehensive profiling of proteome and PTMs, especially with the development of multiplexed isobaric labeling methods, such as tandem mass tag (TMT), coupled with high-resolution two-dimensional liquid chromatography and tandem mass spectrometry (LC/LC-MS/MS). Here, we introduce a deep proteomics profiling protocol with an optimized 11-plex TMT-LC/LC-MS/MS platform to quantitate whole proteome, phosphoproteome, acetylome, and methylome in activated T cells. The major steps include preparation of activated T cells, protein extraction and digestion, TMT labeling, basic pH reverse phase LC, modified peptide enrichment, acidic pH reverse phase LC-MS/MS, and computational data processing. Approximately 10,000 proteins, 30,000 phosphosites, 2,000 lysine acetylated sites, and 1,000 lysine methylated sites can be identified and quantified from 1mg of proteins per sample. Quality control steps are implemented in this protocol, and future development, such as nanoscale 16-plex TMT analysis, is discussed. This multiplexed and robust method provides a powerful tool for dissecting proteomic and PTM signatures in T cells at the systems level, and it is equally suitable for other biological samples, including effector T cell subsets.

Investigating the Adipose Tissue Secretome: A Protocol to Generate High-Quality Samples Appropriate for Comprehensive Proteomic Profiling.

In this chapter we describe in detail how to prepare a sample containing the complete set of secretion products from primary adipocytes, which are suitable for comprehensive and sensitive secretome analysis. The underlying protocol should be seen as starting point guiding through critical steps of the complex workflow of preperation of secretomes. For diverse research questions and in the context of different sample types used, the protocol has to be carefully adjusted in order to approximate to the real secretome.

Proteomic signatures of radioresistance: Alteration of inflammation, angiogenesis and metabolism-related factors in radioresistant oesophageal adenocarcinoma

The clinical management of locally advanced oesophageal adenocarcinoma (OAC) involves neoadjuvant chemoradiotherapy (CRT), but as radioresistance remains a major clinical challenge, complete pathological response to CRT only occurs in 20-30% of patients. In this study we used an established isogenic cell line model of radioresistant OAC to detect proteomic signatures of radioresistance to identify novel molecular and cellular targets of radioresistance in OAC. A total of 5785 proteins were identified of which 251 were significantly modulated in OE33R cells, when compared to OE33P. Gene ontology and pathway analysis of these significantly modulated proteins demonstrated altered metabolism in radioresistant cells accompanied by an inhibition of apoptosis. In addition, inflammatory and angiogenic pathways were positively regulated in radioresistant cells compared to the radiosensitive cells. In this study, we demonstrate, for the first time, a comprehensive proteomic profile of the established isogenic cell line model of radioresistant OAC. This analysis provides insights into the molecular and cellular pathways which regulate radioresistance in OAC. Furthermore, it identifies pathway specific signatures of radioresistance that will direct studies on the development of targeted therapies and personalised approaches to radiotherapy.

Comprehensive Profiling of Proteome and Ubiquitome Changes in Human Lens Epithelial Cell Line after Ultraviolet-B Irradiation.

Ultraviolet-B (UVB) is a recognized risk factor for age-related cataract (ARC) and can cause various changes, including ubiquitination, in lens epithelial cells (LECs). However, the relationship between ubiquitination and ARC is unclear. Herein, we used UVB-irradiated human lens epithelial cell line (SRA01/04) representing the cell model of ARC to investigate the profile changes in the proteome and ubiquitome. A total of 552 differentially expressed proteins (DEPs) and 871 differentially ubiquitinated proteins (DUPs) were identified, and 9 ubiquitination motifs were found. Bioinformatics analysis revealed diverse pathways and biological processes of differential proteins and several DNA damage repair proteins that were potentially mediated via ubiquitin-proteasome pathway. We validated the decreased protein expression of DNA-directed RNA polymerase II subunit RPB2 (POLR2B) in both human cataract capsule tissues and UVB-treated SRA01/04 cells and found that treatment with proteasome inhibitor (MG-132) could reverse the protein level of POLR2B in UVB-irradiated SRA01/04 cells. Our data provide novel information regarding protein expressions and ubiquitination modifications in UVB-induced oxidative damage model. This study might offer a cell-level reference to further investigate the pathogenesis of ARC.

Quantitative Proteomics in Drosophila with Holidic Stable-Isotope Labeling of Amino Acids in Fruit Flies (SILAF).

Protein-focused research has been challenging in Drosophila melanogaster due to few specific antibodies for Western blotting and the lack of effective labeling methods for quantitative proteomics. Herein, we describe the preparation of a holidic medium that allows stable-isotope labeling of amino acids in fruit flies (SILAF). Furthermore, in this chapter, we provide a protocol for mitochondrial enrichments from Drosophila larvae and flies together with a procedure to generate high-quality peptides for further analysis by mass spectrometry. Samples obtained following this protocol can be used for various functional studies such as comprehensive proteome profiling or quantitative analysis of posttranslational modifications upon enrichment. SILAF is based on standard fly routines in a basic wet lab environment and provides a flexible and cost-effective tool for quantitative protein expression analysis.

Comprehensive Proteomic Profiling of Urinary Exosomes and Identification of Potential Non-invasive Early Biomarkers of Alzheimer's Disease in 5XFAD Mouse Model.

BackgroundAlzheimer’s disease (AD) is an incurable neurodegenerative disease characterized by irreversible progressive cognitive deficits. Identification of candidate biomarkers, before amyloid-β-plaque deposition occurs, is therefore of great importance for early intervention of AD.ObjectiveTo investigate the potential non-invasive early biomarkers of AD in 5XFAD mouse model, we investigate the proteome of urinary exosomes present in 1-month-old (before amyloid-β accumulation) 5XFAD mouse models and their littermate controls. Another two groups of 2 and 6 months-old urinary samples were collected for monitoring the dynamic change of target proteins during AD progression.MethodsProteomic, bioinformatics analysis, multiple reaction monitoring (MRM), western blotting (WB) or ELISA were performed for analyzing these urinary exosomes.ResultsA total of 316 proteins including 44 brain cell markers were identified using liquid chromatography tandem mass spectrometry. Importantly, 18 proteins were unique to the 5XFAD group. Eighty-eight proteins including 11 brain cell markers were differentially expressed. Twenty-two proteins were selected to be verified by WB. Furthermore, based on an independent set of 12 urinary exosomes samples, five in these proteins were further confirmed significant difference. Notably, Annexin 2 and Clusterin displayed significant decreased in AD model during the course detected by ELISA. AOAH, Clusterin, and Ly86 are also brain cell markers that were first reported differential expression in urinary exosomes of AD model.ConclusionOur data demonstrated that some urinary exosome proteins, especially Annexin 2 and Clusterin, as nanometer-sized particles, enable detection of differences before amyloid-β-plaque deposition in 5XFAD mouse model, which may present an ideal non-invasive source of biomarkers for prevention of AD.

Proteomic evaluation of the fallopian tube: Insight into the pathogenesis of serous ovarian cancer

Objective: Serous ovarian cancer was thought to originate from ovarian surface epithelium, but contemporary opinion suggests precancerous lesions in the fallopian tube may be the origin for this disease. We sought to conduct a comprehensive proteomic profiling of serous ovarian cancer and adjacent fallopian tubes to improve our understanding of disease pathogenesis.

Comprehensive plasma proteomic profiling reveals biomarkers for active tuberculosis.

BACKGROUNDTuberculosis (TB) kills more people than any other infection, and new diagnostic tests to identify active cases are required. We aimed to discover and verify novel markers for TB in nondepleted plasma.METHODSWe applied an optimized quantitative proteomics discovery methodology based on multidimensional and orthogonal liquid chromatographic separation combined with high-resolution mass spectrometry to study nondepleted plasma of 11 patients with active TB compared with 10 healthy controls. Prioritized candidates were verified in independent UK (n = 118) and South African cohorts (n = 203).RESULTSWe generated the most comprehensive TB plasma proteome to date, profiling 5022 proteins spanning 11 orders-of-magnitude concentration range with diverse biochemical and molecular properties. We analyzed the predominantly low–molecular weight subproteome, identifying 46 proteins with significantly increased and 90 with decreased abundance (peptide FDR ≤ 1%, q ≤ 0.05). Verification was performed for novel candidate biomarkers (CFHR5, ILF2) in 2 independent cohorts. Receiver operating characteristics analyses using a 5-protein panel (CFHR5, LRG1, CRP, LBP, and SAA1) exhibited discriminatory power in distinguishing TB from other respiratory diseases (AUC = 0.81).CONCLUSIONWe report the most comprehensive TB plasma proteome to date, identifying novel markers with verification in 2 independent cohorts, leading to a 5-protein biosignature with potential to improve TB diagnosis. With further development, these biomarkers have potential as a diagnostic triage test.FUNDINGColciencias, Medical Research Council, Innovate UK, NIHR, Academy of Medical Sciences, Program for Advanced Research Capacities for AIDS, Wellcome Centre for Infectious Diseases Research.

Diabetes Induced Alterations in Murine Vitreous Proteome Are Mitigated by IL-6 Trans-Signaling Inhibition.

PurposeDiabetic retinopathy (DR) is a microvascular complication caused by prolonged hyperglycemia and characterized by leaky retinal vasculature and ischemia-induced angiogenesis. Vitreous humor is a gel-like biofluid in the posterior segment of the eye between the lens and the retina. Disease-related changes are observed in the biochemical constituents of the vitreous, including proteins and macromolecules. Recently, we found that IL-6 trans-signaling plays a significant role in the vascular leakage and retinal pathology associated with DR. Therefore, in this study, comprehensive proteomic profiling of the murine vitreous was performed to identify diabetes-induced alterations and to determine effects of IL-6 trans-signaling inhibition on these changes.MethodsVitreous samples from mice were collected by evisceration, and proteomic analyses were performed using liquid chromatography–tandem mass spectrometry (LC-MS/MS).ResultsA total of 154 proteins were identified with high confidence in control mice and were considered to be characteristic of healthy murine vitreous fluid. The levels of 72 vitreous proteins were significantly altered in diabetic mice, including several members of heat shock proteins, 14-3-3 proteins, and tubulins. Alterations in 52 out of 72 proteins in diabetic mice were mitigated by IL-6 trans-signaling inhibition.ConclusionsProteomic analysis of murine vitreous fluid performed in this study provides important information about the changes caused by diabetes in the ocular microenvironment. These diabetes-induced alterations in the murine vitreous proteome were mitigated by IL-6 trans-signaling inhibition. These findings further support that IL-6 trans-signaling may be an important therapeutic target for the treatment of DR.

Stromal expression of hemopexinis associated with lymph-node metastasisin pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma is one of the most aggressive types of cancer. Certain proteins in the tumor microenvironment have attracted considerable attention owing to their association with tumor invasion and metastasis. Here, we used proteomics to identify proteins associated with lymph-node metastasis, which is one of the prognostic factors. We selected lymph node metastasis-positive and -negative patients (n = 5 each) who underwent pancreatectomy between 2005 and 2015 and subjected to comprehensive proteomic profiling of tumor stroma. A total of 490 proteins were detected by mass spectrometry. Software analysis revealed that nine of these proteins were differentially expressed between the two patient groups. We focused on hemopexin and ferritin light chain based on immunohistochemistry results. We assessed the clinicopathological data of 163 patients and found that hemopexin expression was associated with UICC N2 (p = 0.0399), lymph node ratio (p = 0.0252), venous invasion (p = 0.0096), and lymphatic invasion (p = 0.0232). Notably, in vitro assays showed that hemopexin promotes invasion of the pancreatic cancer cells. Our findings suggest that hemopexin is a lymph node metastasis-associated protein that could potentially serve as a useful therapeutic target or biomarker of pancreatic ductal adenocarcinoma.

Proximal Biotinylation-Based Combinatory Approach for Isolating Integral Plasma Membrane Proteins.

Comprehensive profiling of the cell-surface proteome has been challenging due to the lack of tools for an effective and reproducible way to isolate plasma membrane proteins from mammalian cells. Here we employ a proximity-dependent biotinylation approach to label and isolate plasma membrane proteins without an extra in vitro labeling step, which we call Plasma Membrane-BioID. The lipid-modified BirA* enzyme (MyrPalm BirA*) was targeted to the inner leaflet of the plasma membrane, where it effectively biotinylated plasma membrane proteins. Biotinylated proteins were then affinity-purified and analyzed by mass spectrometry. Our analysis demonstrates that combining conventional sucrose density gradient centrifugation and Plasma Membrane-BioID is ideal to overcome the inherent limitations of the identification of integral membrane proteins, and it yields highly pure plasma components for downstream proteomic analysis.

Integrated transcriptomic and proteomic analysis of the acetic acid stress in Issatchenkia orientalis.

Issatchenkia orientalis known as a multi-tolerant non-Saccharomyces yeast, which tolerant environmental stresses, exhibits potential in wine making and bioethanol production. It is essential for the growth of I.orientalis to tolerant acetic acid in the mixed cultures with Saccharomyces cerevisiae. In this work, RNA-sequence and TMT (Tandem Mass Tag) were used to examine the comprehensive transcriptomic and proteomic profiles of I.orientalis in response to acetic acid. The results showed that 876 genes were identified differentially transcribed in I.orientalis genome and 399 proteins expressed in proteome after 4hr acetic acid (90mM, pH 4.5). The comprehensive analysis showed a series of determinants of acetic acid tolerance: Glycolysis and TCA cycle provide enough nicotinamide adenine dinucleotide to effectively convert acetic acid. Genes associated with potassium, iron, zinc, and glutathione synthesis were upregulated. The same changes of differentially expressed genes and proteins were mainly concentrated in chaperones, coenzyme, energy production, and transformation. PRACTICAL APPLICATIONS: In addition to the main fermentation products, wine yeast also produces metabolite acetic acid in the fermentation process, and yeast cells are exposed to acetic acid stress, which restrains cell proliferation. Issatchenkia orientalis exhibits great potential in winemaking and bioethanol production. The yeast is known as a multi-tolerant non-Saccharomyces yeast that can tolerate a variety of environmental stresses. In this study, RNA-Seq and TMT were conducted to investigate the changes in transcriptional and proteomic profile of I.orientalis under acetic acid stress. The knowledge of the transcription and expression changes of the I.orientalis is expected to understand the tolerance mechanisms in I.orientalis and to guide traditional fermentation processes by Saccharomyces cerevisiae improving its high resistance to acetic acid stress.

Comprehensive Proteomic Profiling of Pressure Ulcers in Patients with Spinal Cord Injury Identifies a Specific Protein Pattern of Pathology.

Objective: Severe pressure ulcers (PUs) do not respond to conservative wound therapy and need surgical repair. To better understand the pathogenesis and to advance on new therapeutic options, we focused on the proteomic analysis of PU, which offers substantial opportunities to identify significant changes in protein abundance during the course of PU formation in an unbiased manner.Approach: To better define the protein pattern of this pathology, we performed a proteomic approach in which we compare severe PU tissue from spinal cord injury (SCI) patients with control tissue from the same patients.Results: We found 76 proteins with difference in abundance. Of these, 10 proteins were verified as proteins that define the pathology: antithrombin-III, alpha-1-antitrypsin, kininogen-1, alpha-2-macroglobulin, fibronectin, apolipoprotein A-I, collagen alpha-1 (XII) chain, haptoglobin, apolipoprotein B-100, and complement factor B.Innovation: This is the first study to analyze differential abundance protein of PU tissue from SCI patients using high-throughput protein identification and quantification by tandem mass tags followed by liquid chromatography tandem mass spectrometry.Conclusion: Differential abundance proteins are mainly involved in tissue regeneration. These proteins might be considered as future therapeutic options to enhance the physiological response and permit cellular repair of damaged tissue.

Comparative pharmacoproteomics reveals potential targets for berberine, a promising therapy for colorectal cancer

Berberine (BBR), a natural isoquinoline alkaloid, has been shown to be a promising therapeutic agent for colorectal cancer (CRC), but the molecular mechanism remains unclear. Here, we used mass spectrometry-based label-free proteomics to explore the potential targets of BBR in CRC cells. Comprehensive proteomic profiles demonstrated that of 8051 identified proteins, 503 and 277 differentially expressed proteins (DEPs) were screened out of CACO2 and LOVO cells, respectively. 83 DEPs were overlapped and most of these were down-regulated. A pathway enrichment analysis pinpointed mitochondrial translation, respiratory electron transport and the citric acid (TCA) cycle as biological effectors. The data of proteomics was subsequently confirmed by citrate synthase (CS), Tu translation elongation factor (TUFM), pentatricopeptide repeat domain 3 (PTCD3) and mitochondrial ribosomal protein L48 (MRPL 48) protein measurement. CS protein expression in CRC cells and tissues was higher than it was in normal specimens. Additionally, forcible downregulation of CS led to remarkable cell proliferation inhibition. Taken together, we concluded that the anticancer effects of BBR are attributable to mitochondrial protein synthesis, TCA and respiratory electron transport inhibition and that CS might be a useful therapeutic target in CRC treatment.

Abstract TS1-1: Advanced enabling technologies for cancer proteomics

Abstract Mass spectrometry-based proteomics is a promising technology in characterization of human proteome at a genome scale, facilitating proteogenomic characterization of cancer specimens. Recent advances in mass spectrometry instrumentation, sample handling platforms, and proteomic analysis strategies allow for not only large-scale, comprehensive proteome profiling, but also focused systems biology studies, biomarker verification, as well as analysis of protein expression and phosphorylation in very small populations of cells. This will lead to significantly improved understanding of aberrant regulatory and signal transduction networks underlying cancer for translational applications. Citation Format: T Liu. Advanced enabling technologies for cancer proteomics [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr TS1-1.