Comparative Proteomics Analysis of Exosomes Identifies Key Pathways and Protein Markers Related to Breast Cancer Metastasis.

Proteomics analysis of circulating exosomes derived from cancer cells represents a promising approach to the elucidation of cell-cell communication and the discovery of putative biomarker candidates for cancer diagnosis and treatment. Nonetheless, the proteome of exosomes derived from cell lines with different metastatic capabilities still warrants further investigation. Here, we present a comprehensive quantitative proteomics investigation of exosomes isolated from immortalized mammary epithelial cells and matched tumor lines with different metastatic potentials in an attempt to discover exosome markers specific to breast cancer (BC) metastasis. A total of 2135 unique proteins were quantified with a high confidence level from 20 isolated exosome samples, including 94 of the TOP 100 exosome markers archived by ExoCarta. Moreover, 348 altered proteins were observed, among which several metastasis-specific markers, including cathepsin W (CATW), magnesium transporter MRS2 (MRS2), syntenin-2 (SDCB2), reticulon-4 (RTN), and UV excision repair protein RAD23 homolog (RAD23B), were also identified. Notably, the abundance of these metastasis-specific markers corresponds well with the overall survival of BC patients in clinical settings. Together, these data provide a valuable dataset for BC exosome proteomics investigation and prominently facilitate the elucidation of the molecular mechanisms underlying primary tumor development and progression.

High-quality and robust protein quantification in large clinical/pharmaceutical cohorts with IonStar proteomics investigation.

Robust, reliable quantification of large sample cohorts is often essential for meaningful clinical or pharmaceutical proteomics investigations, but it is technically challenging. When analyzing very large numbers of samples, isotope labeling approaches may suffer from substantial batch effects, and even with label-free methods, it becomes evident that low-abundance proteins are not reliably measured owing to unsufficient reproducibility for quantification. The MS1-based quantitative proteomics pipeline IonStar was designed to address these challenges. IonStar is a label-free approach that takes advantage of the high sensitivity/selectivity attainable by ultrahigh-resolution (UHR)-MS1 acquisition (e.g., 120-240k full width at half maximum at m/z = 200) which is now widely available on ultrahigh-field Orbitrap instruments. By selectively and accurately procuring quantitative features of peptides within precisely defined, very narrow m/z windows corresponding to the UHR-MS1 resolution, the method minimizes co-eluted interferences and substantially enhances signal-to-noise ratio of low-abundance species by decreasing noise level. This feature results in high sensitivity, selectivity, accuracy and precision for quantification of low-abundance proteins, as well as fewer missing data and fewer false positives. This protocol also emphasizes the importance of well-controlled, robust experimental procedures to achieve high-quality quantification across a large cohort. It includes a surfactant cocktail-aided sample preparation procedure that achieves high/reproducible protein/peptide recoveries among many samples, and a trapping nano-liquid chromatography-mass spectrometry strategy for sensitive and reproducible acquisition of UHR-MS1 peptide signal robustly across a large cohort. Data processing and quality evaluation are illustrated using an example dataset ( http://proteomecentral.proteomexchange.org ), and example results from pharmaceutical project and one clinical project (patients with acute respiratory distress syndrome) are shown. The complete IonStar pipeline takes ~1-2 weeks for a sample cohort containing ~50-100 samples.

Identification of Potential Megalin/Cubilin Substrates Using Extensive Proteomics Quantification from Kidney Megalin-Knockdown Mice.

Megalin and cubilin, endocytic proteins present in the proximal tubule of the kidney, are responsible for reabsorbing filtered proteins from urine. Our hypothesis was that potential substrates of megalin/cubilin could be identified by examining urinary protein differences between control (WT) mice and kidney-specific megalin knockdown (KD) mice. Using the IonStar proteomics approach, 877 potential megalin/cubilin substrates were discovered, with 23 of these compounds representing known megalin/cubilin substrates. Some of the proteins with the largest fold changes in the urine between KD and WT included the known megalin substrates retinol-binding protein and vitamin D-binding protein. Of the total proteins identified as novel substrates, about three-quarters of compounds had molecular weights (MWs) below 69 kDa, the MW of albumin, and the remaining had higher MWs, with about 5% of the proteins having MWs greater than 150 kDa. Sex differences in the number of identified substrates occurred, but this may be due to differences in kidney megalin expression between both male and female megalin KD and WT animals, with the ratio of megalin between WT and KD being 2.76 and 2.14 for female and male mice, respectively. The top three ingenuity canonical pathways based on the urinary proteins in both female and male KD mice were acute phase response signaling, liver X receptor/retinoid X receptor activation, and intrinsic prothrombin activation pathways. In conclusion, analysis of urine samples from kidney-specific megalin KD and WT mice was found to be useful for the identification of potential endogenous substrates for megalin and cubilin.

Proteomic Network Analysis of Bronchoalveolar Lavage Fluid in Ex-Smokers to Discover Implicated Protein Targets and Novel Drug Treatments for Chronic Obstructive Pulmonary Disease.

Bronchoalveolar lavage of the epithelial lining fluid (BALF) can sample the profound changes in the airway lumen milieu prevalent in chronic obstructive pulmonary disease (COPD). We compared the BALF proteome of ex-smokers with moderate COPD who are not in exacerbation status to non-smoking healthy control subjects and applied proteome-scale translational bioinformatics approaches to identify potential therapeutic protein targets and drugs that modulate these proteins for the treatment of COPD. Proteomic profiles of BALF were obtained from (1) never-smoker control subjects with normal lung function (n = 10) or (2) individuals with stable moderate (GOLD stage 2, FEV1 50−80% predicted, FEV1/FVC < 0.70) COPD who were ex-smokers for at least 1 year (n = 10). After identifying potential crucial hub proteins, drug−proteome interaction signatures were ranked by the computational analysis of novel drug opportunities (CANDO) platform for multiscale therapeutic discovery to identify potentially repurposable drugs. Subsequently, a literature-based knowledge graph was utilized to rank combinations of drugs that most likely ameliorate inflammatory processes. Proteomic network analysis demonstrated that 233 of the >1800 proteins identified in the BALF were significantly differentially expressed in COPD versus control. Functional annotation of the differentially expressed proteins was used to detail canonical pathways containing the differential expressed proteins. Topological network analysis demonstrated that four putative proteins act as central node proteins in COPD. The drugs with the most similar interaction signatures to approved COPD drugs were extracted with the CANDO platform. The drugs identified using CANDO were subsequently analyzed using a knowledge-based technique to determine an optimal two-drug combination that had the most appropriate effect on the central node proteins. Network analysis of the BALF proteome identified critical targets that have critical roles in modulating COPD pathogenesis, for which we identified several drugs that could be repurposed to treat COPD using a multiscale shotgun drug discovery approach.

A Presurgical-Window Intervention Trial of Isothiocyanate-Rich Broccoli Sprout Extract in Patients with Breast Cancer.

SCOPE: Dietary isothiocyanates (ITCs) from cruciferous vegetables have shown potent anti-breast cancer activities in preclinical models, but their anticancer effects in vivo in breast cancer patients remain elusive. A proof-of-principle, presurgical window of opportunity trial is conducted to assess the anticancer effects of dietary ITCs in breast cancer patients. METHODS AND RESULTS: Thirty postmenopausal breast cancer patients are randomly assigned to receive ITC-rich broccoli sprout extract (BSE) (200 µmol ITC per day) or a placebo for 2 weeks. Expression of biomarkers related to ITCs functions are measured in breast cancer tissue specimens at pre- and post-interventions using immunohistochemistry staining. First morning urine samples are collected at both timepoints for proteomic analysis. Overall, the study shows high compliance (100%) and low toxicity (no grade 4 adverse event). Trends of increase in cleaved caspase 3 and tumor-infiltrating lymphocytes (TILs) and trends of decrease in Ki-67 and nuclear to cytoplasm ratio of estrogen receptor (ER)-α are observed in the BSE arm only, consistent with the significantly altered signaling pathways identified in urinary proteomic analysis. CONCLUSIONS: Anticancer activities of ITCs are observed in breast cancer patients, supporting the potential beneficial roles of ITC-containing cruciferous vegetables in breast cancer prognosis.

Surface hydrophobics mediate functional dimerization of CYP121A1 of Mycobacterium tuberculosis.

Tuberculosis is caused by the pathogenic bacterium Mycobacterium tuberculosis (Mtb) and remains the leading cause of death by infection world-wide. The Mtb genome encodes a disproportionate number of twenty cytochrome P450 enzymes, of which the essential enzyme cytochrome P450 121A1 (CYP121A1) remains a target of drug design efforts. CYP121A1 mediates a phenol coupling reaction of the tyrosine dipeptide cyclo-L-Tyr-L-Tyr (cYY). In this work, a structure and function investigation of dimerization was performed as an overlooked feature of CYP121A1 function. This investigation showed that CYP121A1 dimers form via intermolecular contacts on the distal surface and are mediated by a network of solvent-exposed hydrophobic residues. Disruption of CYP121A1 dimers by site-directed mutagenesis leads to a partial loss of specificity for cYY, resulting in an approximate 75% decrease in catalysis. 19F labeling and nuclear magnetic resonance of the enzyme FG-loop was also combined with protein docking to develop a working model of a functional CYP121A1 dimer. The results obtained suggest that participation of a homodimer interface in substrate selectivity represents a novel paradigm of substrate binding in CYPs, while also providing important mechanistic insight regarding a relevant drug target in the development of novel anti-tuberculosis agents.

Improving retinal mitochondrial function as a treatment for age-related macular degeneration.

Age-related macular degeneration (AMD) is the leading cause of blindness among the elderly. Currently, there are no treatments for dry AMD, which is characterized by the death of retinal pigment epithelium (RPE) and photoreceptors. Reports from human donors with AMD suggest that RPE mitochondrial defects are a key event in AMD pathology. Thus, the most effective strategy for treating dry AMD is to identify compounds that enhance mitochondrial function and subsequently, preserve the RPE. In this study, primary cultures of RPE from human donors with (n = 20) or without (n = 8) AMD were used to evaluate compounds that are designed to protect mitochondria from oxidative damage (N-acetyl-l-cysteine; NAC), remove damaged mitochondria (Rapamycin), increase mitochondrial biogenesis (Pyrroloquinoline quinone; PQQ), and improve oxidative phosphorylation (Nicotinamide mononucleotide, NMN). Mitochondrial function measured after drug treatments showed an AMD-dependent response; only RPE from donors with AMD showed improvements. All four drugs caused a significant increase in maximal respiration (p < 0.05) compared to untreated controls. Treatment with Rapamycin, PQQ, or NMN significantly increased ATP production (p < 0.05). Only Rapamycin increased basal respiration (p < 0.05). Notably, robust responses were observed in only about 50% of AMD donors, with attenuated responses observed in the remaining AMD donors. Further, within the responders, individual donors exhibited a distinct reaction to each drug. Our results suggest drugs targeting pathways involved in maintaining healthy mitochondria can improve mitochondrial function in a select population of RPE from AMD donors. The unique response of individual donors to specific drugs supports the need for personalized medicine when treating AMD.

Evidence of Allosteric Coupling between Substrate Binding and Adx Recognition in the Vitamin D Carbon-24 Hydroxylase CYP24A1.

Metabolic inactivation of 1,25(OH)2D3 requires molecular recognition between the mitochondrial enzyme cytochrome P450 24A1 (CYP24A1) and its cognate redox partner adrenodoxin (Adx). Recent evidence supports a model of CYP24A1 function in which substrate binding and Adx recognition are structurally linked. However, the details of this allosteric connection are not clear. In this study, we utilize chemical cross-linking coupled to mass spectrometry, nuclear magnetic resonance (NMR) spectroscopy, and CYP24A1 functional assays to inform a working model of a CYP24A1-Adx complex. We report that differential cross-linking internal to CYP24A1 points toward an Adx-induced conformational change that perturbs the F and G helices, which are required for substrate binding. Moreover, the modeled complex suggests that a semiconserved nonpolar interaction at the interface may influence CYP24A1 regioselectivity. Taken together, these findings contribute to our understanding of Adx recognition in a critical vitamin D-inactivating enzyme and provide broader insight regarding the variability inherent in CYP-Adx interactions.

Regulator of G protein signaling 12 enhances osteoclastogenesis by suppressing Nrf2-dependent antioxidant proteins to promote the generation of reactive oxygen species.

Regulators of G-protein Signaling are a conserved family of proteins required in various biological processes including cell differentiation. We previously demonstrated that Rgs12 is essential for osteoclast differentiation and its deletion in vivo protected mice against pathological bone loss. To characterize its mechanism in osteoclastogenesis, we selectively deleted Rgs12 in C57BL/6J mice targeting osteoclast precursors using LyzM-driven Cre mice or overexpressed Rgs12 in RAW264.7 cells. Rgs12 deletion in vivo led to an osteopetrotic phenotype evidenced by increased trabecular bone, decreased osteoclast number and activity but no change in osteoblast number and bone formation. Rgs12 overexpression increased osteoclast number and size, and bone resorption activity. Proteomics analysis of Rgs12-depleted osteoclasts identified an upregulation of antioxidant enzymes under the transcriptional regulation of Nrf2, the master regulator of oxidative stress. We confirmed an increase of Nrf2 activity and impaired reactive oxygen species production in Rgs12-deficient cells. Conversely, Rgs12 overexpression suppressed Nrf2 through a mechanism dependent on the 26S proteasome, and promoted RANKL-induced phosphorylation of ERK1/2 and NFκB, which was abrogated by antioxidant treatment. Our study therefore identified a novel role of Rgs12 in regulating Nrf2, thereby controlling cellular redox state and osteoclast differentiation.

Characterization and Proteomic-Transcriptomic Investigation of Monocarboxylate Transporter 6 Knockout Mice: Evidence of a Potential Role in Glucose and Lipid Metabolism.

Monocarboxylate transporter 6 [(MCT6), SLC16A5] is an orphan transporter with no known endogenous substrates or physiological role. Previous in vitro and in vivo experiments investigated MCT6 substrate/inhibitor specificity in Xenopus laevis oocytes; however, these data remain limited. Transcriptomic changes in the livers of mice undergoing different dieting schemes have suggested that Mct6 plays a role in glucose and lipid metabolism. The objectives of this study were 1) to develop a novel knockout (KO) mouse model (Mct6-/-) using CRISPR/Cas9 technology, 2) to characterize the KO animal model by examining physiological and biochemical parameters, and 3) to understand the physiological role of MCT6 in vivo through global proteomic and liver transcriptomic profiling. mRNA tissue analysis demonstrated knockout of Mct6, which showed greater than 90% knockdown of Mct6 (Slc16a5) gene expression in all major tissues analyzed when normalized to Mct6+/+ mice. Proteomic analyses identified greater than 4000 unique proteins in kidney, liver, and colon tissues, among which 51, 38, and 241 proteins were significantly altered, respectively (for each tissue), between Mct6+/+ and Mct6-/- mice. Additionally, Mct6-/- mice demonstrated significant changes in 199 genes in the liver compared with Mct6+/+ mice. In silico biological pathway analyses revealed significant changes in proteins and genes involved in glucose and lipid metabolism-associated pathways. This study is the first to provide evidence for an association of Mct6 in the regulation of glucose and lipid metabolism. SIGNIFICANCE STATEMENT: This paper focuses on elucidating the innate biological role of an orphan transporter in vivo, which has not been investigated thus far. Using efficient and high-throughput technologies, such as CRISPR/Cas9 gene editing, liquid chromatography-tandem mass spectrometry-based proteomic and RNA-sequencing transcriptomic analyses, our laboratory provides the first existence and characterization of a Mct6 knockout mouse model. The evidence gathered in this paper, as well as other laboratories, support the importance of MCT6 in regulating a variety of glucose and lipid metabolic pathways, which may indicate its significance in metabolic diseases.

Trypanosoma brucei PRMT1 Is a Nucleic Acid Binding Protein with a Role in Energy Metabolism and the Starvation Stress Response.

In Trypanosoma brucei and related kinetoplastid parasites, transcription of protein coding genes is largely unregulated. Rather, mRNA binding proteins, which impact processes such as transcript stability and translation efficiency, are the predominant regulators of gene expression. Arginine methylation is a posttranslational modification that preferentially targets RNA binding proteins and is, therefore, likely to have a substantial impact on T. brucei biology. The data presented here demonstrate that cells depleted of T. brucei PRMT1 (TbPRMT1), a major type I protein arginine methyltransferase, exhibit decreased virulence in an animal model. To understand the basis of this phenotype, quantitative global proteomics was employed to measure protein steady-state levels in cells lacking TbPRMT1. The approach revealed striking changes in proteins involved in energy metabolism. Most prominent were a decrease in glycolytic enzyme abundance and an increase in proline degradation pathway components, changes that resemble the metabolic remodeling that occurs during T. brucei life cycle progression. The work describes several RNA binding proteins whose association with mRNA was altered in TbPRMT1-depleted cells, and a large number of TbPRMT1-interacting proteins, thereby highlighting potential TbPRMT1 substrates. Many proteins involved in the T. brucei starvation stress response were found to interact with TbPRMT1, prompting analysis of the response of TbPRMT1-depleted cells to nutrient deprivation. Indeed, depletion of TbPRMT1 strongly hinders the ability of T. brucei to form cytoplasmic mRNA granules under starvation conditions. Finally, this work shows that TbPRMT1 itself binds nucleic acids in vitro and in vivo, a feature completely novel to protein arginine methyltransferases.IMPORTANCETrypanosoma brucei infection causes human African trypanosomiasis, also known as sleeping sickness, a disease with a nearly 100% fatality rate when untreated. Current drugs are expensive, toxic, and highly impractical to administer, prompting the community to explore various unique aspects of T. brucei biology in search of better treatments. In this study, we identified the protein arginine methyltransferase (PRMT), TbPRMT1, as a factor that modulates numerous aspects of T. brucei biology. These include glycolysis and life cycle progression signaling, both of which are being intensely researched toward identification of potential drug targets. Our data will aid research in those fields. Furthermore, we demonstrate for the first time a direct association of a PRMT with nucleic acids, a finding we believe could translate to other organisms, including humans, thereby impacting research in fields as distant as human cancer biology and immune response modulation.

Comparative Characterization of Osteoclasts Derived From Murine Bone Marrow Macrophages and RAW 264.7 Cells Using Quantitative Proteomics.

Osteoclasts are bone-resorbing cells differentiated from macrophage/monocyte precursors in response to macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL). In vitro models are principally based on primary bone marrow macrophages (BMMs), but RAW 264.7 cells are frequently used because they are widely available, easy to culture, and more amenable to genetic manipulation than primary cells. Increasing evidence, however, has shown that the vastly different origins of these two cell types may have important effects on cell behavior. In particular, M-CSF is a prerequisite for the differentiation of BMMs, by promoting survival and proliferation and priming the cells for RANKL induction. RAW 264.7 cells readily form osteoclasts in the presence of RANKL, but M-CSF is not required. Based on these key differences, we sought to understand their functional implications and how it might affect osteoclast differentiation and related signaling pathways. Using a robust and high-throughput proteomics strategy, we quantified the global protein changes in osteoclasts derived from BMMs and RAW 264.7 cells at 1, 3, and 5 days of differentiation. Data are available via ProteomeXchange with the identifier PXD009610. Correlation analysis of the proteomes demonstrated low concordance between the two cell types (R2 ≈ 0.13). Bioinformatics analysis indicate that RANKL-dependent signaling was intact in RAW 264.7 cells, but biological processes known to be dependent on M-CSF were significantly different, including cell cycle control, cytoskeletal organization, and apoptosis. RAW 264.7 cells exhibited constitutive activation of Erk and Akt that was dependent on the activity of Abelson tyrosine kinase, and the timing of Erk and Akt activation was significantly different between BMMs and RAW 264.7 cells. Our findings provide the first evidence for major discrepancies between BMMs and RAW 264.7 cells, indicating that careful consideration is needed when using the RAW 264.7 cell line for studying M-CSF-dependent signaling and functions. © 2018 American Society for Bone and Mineral Research. © 2018 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Surfactant Cocktail-Aided Extraction/Precipitation/On-Pellet Digestion Strategy Enables Efficient and Reproducible Sample Preparation for Large-Scale Quantitative Proteomics.

For quantitative proteomics, efficient, robust, and reproducible sample preparation with high throughput is critical yet challenging, especially when large cohorts are involved, as is often required by clinical/pharmaceutical studies. We describe a rapid and straightforward surfactant cocktail-aided extraction/precipitation/on-pellet digestion (SEPOD) strategy to address this need. Prior to organic solvent precipitation and on-pellet digestion, SEPOD treats samples with a surfactant cocktail (SC) containing multiple nonionic/anionic surfactants, which achieves (i) exhaustive/reproducible protein extraction, including membrane-bound proteins; (ii) effective removal of detrimental nonprotein matrix components (e.g., >94% of phospholipids); (iii) rapid/efficient proteolytic digestion owing to dual (surfactants + precipitation) denaturation. The optimal SC composition and concentrations were determined by Orthogonal-Array-Design investigation of their collective/individuals effects on protein extraction/denaturation. Key parameters for cleanup and digestion were experimentally identified as well. The optimized SEPOD procedures allowed a rapid 6 h digestion providing a clean digest with high peptide yields and excellent quantitative reproducibility (especially low-abundance proteins). Compared with filter-assisted sample preparation (FASP) and in-solution digestion, SEPOD showed superior performance by recovering substantially more peptide/proteins (including integral membrane proteins), yielding significantly higher peptide intensities and improving quantification for peptides with extreme physicochemical properties. SEPOD was further applied in a large-cohort temporal investigation of 44 IAV-infected mouse lungs, providing efficient and reproducible peptide yields (77.9 ± 4.6%) across all samples. With the IonStar pipeline, >6 400 unique protein groups were quantified (≥2 peptide/protein, peptide-FDR < 0.05%), ∼99% without missing data in any sample with <7% technical median-intragroup CV. Altered proteome patterns revealed interesting novel insights into pathophysiological changes by IAV infection. In summary, SEPOD offers a feasible solution for rapid, efficient, and reproducible preparation of biological samples, facilitating high-quality proteomic quantification of large sample cohorts.

Proteomic Analysis of Charcoal-Stripped Fetal Bovine Serum Reveals Changes in the Insulin-like Growth Factor Signaling Pathway.

Charcoal-stripped fetal bovine serum (CS-FBS) is commonly used to study androgen responsiveness and androgen metabolism in cultured prostate cancer (CaP) cells. Switching CaP cells from FBS to CS-FBS may reduce the activity of androgen receptor (AR), inhibit cell proliferation, or modulate intracellular androgen metabolism. The removal of proteins by charcoal stripping may cause changes in biological functions and has not yet been investigated. Here we profiled proteins in FBS and CS-FBS using an ion-current-based quantitative platform consisting of reproducible surfactant-aided precipitation/on-pellet digestion, long-column nanoliquid chromatography separation, and ion-current-based analysis. A total of 143 proteins were identified in FBS, among which 14 proteins including insulin-like growth factor 2 (IGF-2) and IGF binding protein (IGFBP)-2 and -6 were reduced in CS-FBS. IGF-1 receptor (IGF1R) and insulin receptor were sensitized to IGFs in CS-FBS. IGF-1 and IGF-2 stimulation fully compensated for the loss of AR activity to maintain cell growth in CS-FBS. Endogenous production of IGF and IGFBPs was verified in CaP cells and clinical CaP specimens. This study provided the most comprehensive protein profiles of FBS and CS-FBS and offered an opportunity to identify new protein regulators and signaling pathways that regulate AR activity, androgen metabolism, and proliferation of CaP cells.

Mapping dynamic histone modification patterns during arsenic-induced malignant transformation of human bladder cells.

Arsenic is a known potent risk factor for bladder cancer. Increasing evidence suggests that epigenetic alterations, e.g., DNA methylation and histones posttranslational modifications (PTMs), contribute to arsenic carcinogenesis. Our previous studies have demonstrated that exposure of human urothelial cells (UROtsa cells) to monomethylarsonous acid (MMAIII), one of arsenic active metabolites, changes the histone acetylation marks across the genome that are correlated with MMAIII-induced UROtsa cell malignant transformation. In the current study, we employed a high-resolution and high-throughput liquid chromatography tandem mass spectrometry (LC-MS/MS) to identify and quantitatively measure various PTM patterns during the MMAIII-induced malignant transformation. Our data showed that MMAIII exposure caused a time-dependent increase in histone H3 acetylation on lysine K4, K9, K14, K18, K23, and K27, but a decrease in acetylation on lysine K5, K8, K12, and K16 of histone H4. Consistent with this observation, H3K18ac was increased while H4K8ac was decreased in the leukocytes collected from people exposed to high concentrations of arsenic compared to those exposed to low concentrations. MMAIII was also able to alter histone methylation patterns: MMAIII transformed cells experienced a loss of H3K4me1, and an increase in H3K9me1 and H3K27me1. Collectively, our data shows that arsenic exposure causes dynamic changes in histone acetylation and methylation patterns during arsenic-induced cancer development. Exploring the genomic location of the altered histone marks and the resulting aberrant expression of genes will be of importance in deciphering the mechanism of arsenic-induced carcinogenesis.

IonStar enables high-precision, low-missing-data proteomics quantification in large biological cohorts.

Reproducible quantification of large biological cohorts is critical for clinical/pharmaceutical proteomics yet remains challenging because most prevalent methods suffer from drastically declined commonly quantified proteins and substantially deteriorated quantitative quality as cohort size expands. MS2-based data-independent acquisition approaches represent tremendous advancements in reproducible protein measurement, but often with limited depth. We developed IonStar, an MS1-based quantitative approach enabling in-depth, high-quality quantification of large cohorts by combining efficient/reproducible experimental procedures with unique data-processing components, such as efficient 3D chromatographic alignment, sensitive and selective direct ion current extraction, and stringent postfeature generation quality control. Compared with several popular label-free methods, IonStar exhibited far lower missing data (0.1%), superior quantitative accuracy/precision [∼5% intragroup coefficient of variation (CV)], the widest protein abundance range, and the highest sensitivity/specificity for identifying protein changes (<5% false altered-protein discovery) in a benchmark sample set (n = 20). We demonstrated the usage of IonStar by a large-scale investigation of traumatic injuries and pharmacological treatments in rat brains (n = 100), quantifying >7,000 unique protein groups (>99.8% without missing data across the 100 samples) with a low false discovery rate (FDR), two or more unique peptides per protein, and high quantitative precision. IonStar represents a reliable and robust solution for precise and reproducible protein measurement in large cohorts.

Dynamics of Zebrafish Heart Regeneration Using an HPLC-ESI-MS/MS Approach.

Failure to properly repair damaged due to myocardial infarction is a major cause of heart failure. In contrast with adult mammals, zebrafish hearts show remarkable regenerative capabilities after substantial damage. To characterize protein dynamics during heart regeneration, we employed an HPLC-ESI-MS/MS (mass spectrometry) approach. Myocardium tissues were taken from sham-operated fish and ventricle-resected sample at three different time points (2, 7, and 14 days); dynamics of protein expression were analyzed by an ion-current-based quantitative platform. More than 2000 protein groups were quantified in all 16 experiments. Two hundred and nine heart-regeneration-related protein groups were quantified and clustered into six time-course patterns. Functional analysis indicated that multiple molecular function and metabolic pathways were involved in heart regeneration. Interestingly, Ingenuity Pathway Analysis revealed that P53 signaling was inhibited during the heart regeneration, which was further verified by real-time quantitative polymerase chain reaction (Q-PCR). In summary, we applied systematic proteomics analysis on regenerating zebrafish heart, uncovered the dynamics of regenerative genes expression and regulatory pathways, and provided invaluable insight into design regenerative-based strategies in human hearts.

Intrinsically Disordered Proteins as Important Players during Desiccation Stress of Soybean Radicles.

Intrinsically disordered proteins (IDPs) play a variety of important physiological roles in all living organisms. However, there is no comprehensive analysis of the abundance of IDPs associated with environmental stress in plants. Here, we show that a set of heat-stable proteins (i.e., proteins that do not denature after boiling at 100 °C for 10 min) was present in R0mm and R15mm radicles (i.e., before radicle emergence and 15 mm long radicles) of soybean (Glycine max) seeds. This set of 795 iTRAQ-quantified heat-stable proteins contained a high proportion of wholly or highly disordered proteins (15%), which was significantly higher than that estimated for the whole soybean proteome containing 55,787 proteins (9%). The heat-stable proteome of soybean radicles that contain many IDPs could protect lactate dehydrogenase (LDH) during freeze-thaw cycles. Comparison of the 795 heat-stable proteins in the R0mm and R15mm soybean radicles revealed that many of these proteins changed abundance during seedling growth with 170 and 89 proteins being more abundant in R0mm and R15mm, respectively. KEGG analysis identified 18 proteins from the cysteine and methionine metabolism pathways and nine proteins from the phenylpropanoid biosynthesis pathway. As an important type of IDP related to stress, 30 late embryogenesis abundant proteins were also found. Ten selected proteins with high levels of predicted intrinsic disorder were able to efficiently protect LDH from the freeze-thaw-induced inactivation, but the protective ability was not correlated with the disorder content of these proteins. These observations suggest that protection of the enzymes and other proteins in a stressed cell can be one of the biological functions of plant IDPs.

An IonStar Experimental Strategy for MS1 Ion Current-Based Quantification Using Ultrahigh-Field Orbitrap: Reproducible, In-Depth, and Accurate Protein Measurement in Large Cohorts.

In-depth and reproducible protein measurement in many biological samples is often critical for pharmaceutical/biomedical proteomics but remains challenging. MS1-based quantification using quadrupole/ultrahigh-field Orbitrap (Q/UHF-Orbitrap) holds great promise, but the critically important experimental approaches enabling reliable large-cohort analysis have long been overlooked. Here we described an IonStar experimental strategy achieving excellent quantitative quality of MS1 quantification. Key features include: (i) an optimized, surfactant-aided sample preparation approach provides highly efficient (>75% recovery) and reproducible (<15% CV) peptide recovery across large cell/tissue cohorts; (ii) a long column with modest gradient length (2.5 h) yields the optimal balance of depth/throughput on a Q/UHF-Orbitrap; (iii) a large-ID trap not only enables highly reproducible gradient delivery as for the first time observed via real-time conductivity monitoring, but also increases quantitative loading capacity by >8-fold and quantified >25% more proteins; (iv) an optimized HCD-OT markedly outperforms HCD-IT when analyzing large cohorts with high loading amounts; (v) selective removal of hydrophobic/hydrophilic matrix components using a novel selective trapping/delivery approach enables reproducible, robust LC-MS analysis of >100 biological samples in a single set, eliminating batch effect; (vi) MS1 acquired at higher resolution (fwhm = 120 k) provides enhanced S/N and quantitative accuracy/precision for low-abundance species. We examined this pipeline by analyzing a 5 group, 20 samples biological benchmark sample set, and quantified 6273 unique proteins (≥2 peptides/protein) under stringent cutoffs without fractionation, 6234 (>99.4%) without missing data in any of the 20 samples. The strategy achieved high quantitative accuracy (3-6% media error), low intragroup variation (6-9% media intragroup CV) and low false-positive biomarker discovery rates (3-8%) across the five groups, with quantified protein abundances spanning >6.5 orders of magnitude. Finally, this strategy is straightforward, robust, and broadly applicable in pharmaceutical/biomedical investigations.

Quantitative proteomic profiling of paired cancerous and normal colon epithelial cells isolated freshly from colorectal cancer patients.

PURPOSE: The heterogeneous structure in tumor tissues from colorectal cancer (CRC) patients excludes an informative comparison between tumors and adjacent normal tissues. Here, we develop and apply a strategy to compare paired cancerous (CEC) versus normal (NEC) epithelial cells enriched from patients and discover potential biomarkers and therapeutic targets for CRC. EXPERIMENTAL DESIGN: CEC and NEC cells are respectively isolated from five different tumor and normal locations in the resected colon tissue from each patient (N = 12 patients) using an optimized epithelial cell adhesion molecule (EpCAM)-based enrichment approach. An ion current-based quantitative method is employed to perform comparative proteomic analysis for each patient. RESULTS: A total of 458 altered proteins that are common among >75% of patients are observed and selected for further investigation. Besides known findings such as deregulation of mitochondrial function, tricarboxylic acid cycle, and RNA post-transcriptional modification, functional analysis further revealed RAN signaling pathway, small nucleolar ribonucleoproteins (snoRNPs), and infection by RNA viruses are altered in CEC cells. A selection of the altered proteins of interest is validated by immunohistochemistry analyses. CONCLUSION AND CLINICAL RELEVANCE: The informative comparison between matched CEC and NEC enhances our understanding of molecular mechanisms of CRC development and provides biomarker candidates and new pathways for therapeutic intervention.