bims-prodis 2019-05-12 papers

bims-prodis

Biomed News

on Proteomics in disease

Issue of 2019–05–12
eightteen papers selected by
Nancy Gough, Bioserendipity

Proteomic profiling of human prostate cancer-associated fibroblasts (CAF) reveals LOXL2-dependent regulation of the tumor microenvironment.
Quantitative proteomics of changes in succinylated proteins expression profiling in left appendages tissue from valvular heart disease patients with atrial fibrillation.
Comparative proteomic analysis of human serum before and after liver transplantation using quantitative proteomics.
Comparison of Proteome Composition of Serum Enriched in Extracellular Vesicles Isolated from Polycythemia Vera Patients and Healthy Controls.
Proteomic analysis of meningiomas reveals clinically-distinct molecular patterns.
Serum proteome changes and accelerated reduction of fat mass after laparoscopic gastric plication in morbidly obese patients.
Proteomic Profiling of Exosomes Derived from Plasma of HIV-Infected Alcohol Drinkers and Cigarette Smokers.
Comparative proteomic analysis of plasma of children with congenital heart disease.
Plasma fibronectin levels identified via quantitative proteomics profiling predicts hepatitis B surface antigen seroclearance in chronic hepatitis B.
[Proteomic analysis of the cerebrospinal fluid from patients with amyotrophic lateral sclerosis based on tandem mass spectrometry technique].
Melanoma proteomics suggests functional differences related to mutational status.
Forensic evidence of sulfur mustard exposure in real cases of human poisoning by detection of diverse albumin-derived protein adducts.
Uncovering the role of hnRNP K, an RNA-binding protein, in B-cell lymphomas.
Characterization of Glycolytic Enzymes and Pyruvate Kinase M2 in Type 1 and 2 Diabetic Nephropathy.
A longitudinal big data approach for precision health.
Serum Protein Biomarkers of Fibrosis Aid in Risk Stratification of Future Stricturing Complications in Pediatric Crohn's Disease.
Integrated omics-based pathway analyses uncover CYP epoxygenase-associated networks as theranostic targets for metastatic triple negative breast cancer.
Relative proteome quantification of alpha, beta, gamma and delta globin chains in early eluting peaks of Bio-Rad variant II® CE-HPLC of hemoglobin from healthy and beta-thalassemia subjects in Malaysia.

Mol Cell Proteomics. 2019 May 06. pii: mcp.RA119.001496. [Epub ahead of print]

Proteomic profiling of human prostate cancer-associated fibroblasts (CAF) reveals LOXL2-dependent regulation of the tumor microenvironment.

Elizabeth V Nguyen, Brooke A Pereira, Mitchell G Lawrence, Xiuquan Ma, Richard J Rebello, Howard Chan, Birunthi Niranjan, Yunjian Wu, Stuart Ellem, Xiaoqing Guan, Jianmin Wu, Joanna N Skhinas, Thomas R Cox, Gail P Risbridger, Renea A Taylor, Natalie L Lister, Roger Daly.

  In prostate cancer, cancer-associated fibroblasts (CAF) exhibit contrasting biological properties to non-malignant prostate fibroblasts (NPF) and promote tumorigenesis. Resolving intercellular signaling pathways between CAF and prostate tumor epithelium may offer novel opportunities for research translation. To this end, the proteome and phosphoproteome of four pairs of patient-matched CAF and NPF were characterized to identify discriminating proteomic signatures. Samples were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) with a hyper reaction monitoring data-independent acquisition (HRM-DIA) workflow. Proteins that exhibited a significant increase in CAF versus NPF were enriched for the functional categories 'cell adhesion' and the 'extracellular matrix'. The CAF phosphoproteome exhibited enhanced phosphorylation of proteins associated with the 'spliceosome' and 'actin binding'. STRING analysis of the CAF proteome revealed a prominent interaction hub associated with collagen synthesis, modification, and signaling. It contained multiple collagens, including the fibrillar types COL1A1/2 and COL5A1; the receptor tyrosine kinase discoidin domaincontaining receptor 2 (DDR2), a receptor for fibrillar collagens; and lysyl oxidase-like 2 (LOXL2), an enzyme which promotes collagen crosslinking. Increased activity and/or expression of LOXL2 and DDR2 in CAF were confirmed by enzymatic assays and Western blot analyses. Pharmacological inhibition of CAF-derived LOXL2 perturbed extracellular matrix (ECM) organization and decreased CAF migration in a wound healing assay. Furthermore, it significantly impaired the motility of co-cultured RWPE-2 prostate tumor epithelial cells. These results indicate that CAF-derived LOXL2 is an important mediator of intercellular communication within the prostate tumor microenvironment and is a potential therapeutic target.

Keywords:  Cancer biomarker(s); Cancer-associated fibroblasts; Fibroblasts; LOXL2; Non-malignant prostate fibroblasts; Phosphoproteome; Prostate cancer; Prostate cancer biomarkers; Tumor microenvironment*

DOI:  https://doi.org/10.1074/mcp.RA119.001496
Clin Chim Acta. 2019 May 03. pii: S0009-8981(19)31844-3. [Epub ahead of print]

Quantitative proteomics of changes in succinylated proteins expression profiling in left appendages tissue from valvular heart disease patients with atrial fibrillation.

Fan Bai, Tao Tu, Fen Qin, Yingxu Ma, Na Liu, Yaozhong Liu, Xiaobo Liao, Shenghua Zhou, Qiming Liu.

   BACKGROUND: Previous studies have suggested that proteomic modifications are closely associated with cardiovascular diseases. The aim of this study was to identify potential mechanisms by profiling the changes in succinylated protein expression in left appendage tissues from patients with valvular heart disease and atrial fibrillation (AF).
METHODS: Using dimethyl labeling for relative and absolute quantification-coupled high-performance liquid chromatography-tandem mass spectrometry, we analyzed the proteomics profiles and succinylation events in 18 left atrial appendage tissue samples from patients who underwent cardiac valvular surgery, including nine patients with permanent AF and nine patients with sinus rhythm (SR).
RESULTS: In total, after setting the quantification ratio > 1.3 and < 1:1.3 representing the up- and downregulated cutoff values, respectively, 132 proteins were classified as targets of upregulation and 117 proteins as targets of downregulation. Within these proteins, 246 sites exhibited upregulated succinylation and 45 sites exhibited downregulated succinylation. Protein-protein interaction networks showed that the proteins exhibiting lysine succinylation and AF status were highly enriched in energy metabolism, extracellular matrix-related, and cellular structure-related proteins. These results were confirmed by western blot.
CONCLUSIONS: The differences in succinylation level of energy metabolism-related proteins indicates the possible involvement of these proteins in AF of valvular heart disease patients, and provide insight for further analysis of their biological functions.

Keywords:  Atrial fibrillation; Isobaric tag for relative and absolute quantification; Proteomics; Succinylation

DOI:  https://doi.org/10.1016/j.cca.2019.05.002
Oncotarget. 2019 Apr 02. 10(26): 2508-2514

Comparative proteomic analysis of human serum before and after liver transplantation using quantitative proteomics.

Ruohan Zhang, Minglin Ou, Yue Zhang, Qiang Yan, Huaizhou Chen, Liusheng Lai, Ying Li, Feilong Xu, Donge Tang, Xuyong Sun, Jianhui Dong, Yong Dai, Weiguo Sui.

  Liver cancer is the second leading cause of cancer mortality worldwide. Safer and more effective diagnostic methods for liver cancer are desirable, and biomarkers represent a potentially alternative method for diagnosis. The present study was designed to identify liver cancer biomarkers. We quantified the changes in serum protein levels between liver transplantation and healthy (control) females using isobaric tags for relative and absolute quantitation (iTRAQ) as well as proteomic analysis. A total of 1399 proteins were identified; of these, three proteins showed significantly different concentrations between the before transplantation group and the control group. These proteins may thus be relevant to liver cancer and constitute potential liver cancer biomarkers.

Keywords:  iTRAQ; liver transplantation; pathway; protein markers; proteomics

DOI:  https://doi.org/10.18632/oncotarget.26761
Proteomes. 2019 May 06. pii: E20. [Epub ahead of print]7(2):

Comparison of Proteome Composition of Serum Enriched in Extracellular Vesicles Isolated from Polycythemia Vera Patients and Healthy Controls.

Anna Fel, Aleksandra E Lewandowska, Petro E Petrides, Jacek R Wiśniewski.

  Extracellular vesicles (EVs), e.g., exosomes and microvesicles, are one of the main networks of intercellular communication. In myeloproliferative neoplasms, such as polycythemia vera (PV), excess of EVs originating from overabundant blood cells can directly contribute to thrombosis through their procoagulant activity. However, the proteomic composition of these vesicles in PV patients has not been investigated before. In this work, we examined the proteomic composition of serum EVs of PV patients in comparison to healthy controls. We processed EV-enriched serum samples using the Multiple Enzyme Filter Aided Sample Preparation approach (MED-FASP), conducted LC-MS/MS measurements on a Q-Exactive HF-X mass spectrometer, and quantitatively analyzed the absolute concentrations of identified proteins by the Total Protein Approach (TPA). Thirty-eight proteins were present at statistically significant different concentrations between PV patients' study group and healthy controls' group. The main protein components deregulated in PV were primarily related to excessive amounts of cells, increased platelet activation, elevated immune and inflammatory response, and high concentrations of procoagulant and angiogenic agents. Our study provides the first quantitative analysis of the serum EVs' proteome in PV patients. This new knowledge may contribute to a better understanding of the secondary systemic effects of PV disease and further development of diagnostic or therapeutic procedures.

Keywords:  extracellular vesicles; multi-enzyme digestion filter aided sample preparation (MED-FASP); polycythemia vera; proteomics; total protein approach

DOI:  https://doi.org/10.3390/proteomes7020020
Neuro Oncol. 2019 May 11. pii: noz084. [Epub ahead of print]

Proteomic analysis of meningiomas reveals clinically-distinct molecular patterns.

Michail-Dimitrios Papaioannou, Ugliesa Djuric, Jennifer Kao, Shirin Karimi, Gelareh Zadeh, Kenneth Aldape, Phedias Diamandis.

   BACKGROUND: Meningiomas represent one of the most common brain tumors and exhibit a clinically heterogenous behaviour, sometimes difficult to predict with classic histopathologic features. While emerging molecular profiling efforts have linked specific genomic drivers to distinct clinical patterns, the proteomic landscape of meningiomas remains largely unexplored.
METHODS: We utilize liquid chromatography tandem mass spectrometry using an Orbitrap mass analyzer to quantify global protein abundances of a clinically well-annotated formalin-fixed paraffin embedded (FFPE) cohort (n=61) of meningiomas spanning all World Health Organization (WHO) grades and various degrees of clinical aggressiveness.
RESULTS: In total, we quantify 3042 unique proteins comparing patterns across different clinical parameters. Unsupervised clustering analysis highlighted distinct proteomic (n=106 proteins, Welch's t-test, p<0.01) and pathway-level (e.g. Notch and PI3K/AKT/mTOR) differences between convexity and skull base meningiomas. Supervised comparative analyses of different pathological grades revealed distinct patterns between benign (Grade I) and atypical/malignant (Grade II/III) meningiomas with specific oncogenes enriched in higher grade lesions. Independent of WHO grade, clinically aggressive meningiomas, that rapidly recurred (<3 years), had distinctive protein patterns converging on mRNA processing and impaired activation of the matrisome complex. Larger sized meningiomas (>3cm maximum tumor diameter) and those with previous radiation exposure, revealed perturbed pro-proliferative (e.g. EGFR) and metabolic as well as inflammatory response pathways (mitochondrial activity, interferon) respectively.
CONCLUSIONS: Our proteomic study demonstrates that meningiomas of different grades and clinical parameters present distinct proteomic profiles. These proteomic variations offer potential future utility in helping better predict patient outcome and in nominating novel therapeutic targets for personalized care.

Keywords:  Proteome-wide analysis; mass spectrometry; meningiomas; neuro-oncology; stratification

DOI:  https://doi.org/10.1093/neuonc/noz084
J Proteomics. 2019 May 02. pii: S1874-3919(19)30136-8. [Epub ahead of print]

Serum proteome changes and accelerated reduction of fat mass after laparoscopic gastric plication in morbidly obese patients.

Parisa Savedoroudi, Tue Bjerg Bennike, Kenneth Kastaniegaard, Mohammad Talebpour, Alireza Ghassempour, Allan Stensballe.

  Laparoscopic Gastric Plication (LGP) is a relatively new bariatric surgical procedure which no part of the stomach is removed. It is not clearly understood how LGP leads to fatty tissue reduction. We aimed to investigate the impact of LGP on serum proteome and understand molecular mechanisms of LGP-induced weight loss post-surgery. A Prospective observational study of 16 obese individuals who underwent LGP was performed. A Label-free quantitative shotgun proteomics approach was used to compare serum proteome of subjects before surgery with serum of the same individuals 1 to 2 months post-surgery (T1) and 4 to 5 months post-surgery (T2). The proteome analysis revealed that 48 proteins were differentially regulated between pre-surgery and T1, and seven proteins between pre-surgery and T2 of which six proteins were shared between the two timepoints. Among differentially regulated proteins, four proteins (SRGN, FETUB, LCP1 and CFP) have not previously been described in the context of BMI/weight loss. Despite few differences following LGP, most regulated serum proteins are in accordance with alternative weight loss procedures. Pathway analysis revealed changes to lipid- and inflammatory pathways, including PPARα/RXRα, LXR/RXR and FXR/RXR activation, especially at T1. At T2, the pathways related to inflammation and immune system are most affected. SIGNIFICANCE: Among the available clinical therapies for morbid obesity, bariatric surgery is considered as the most effective approach to achieve long-term weight loss, alongside a significant improvement in metabolic syndrome. However, very little is known about the underlying mechanism associated with significant weight loss post-surgery. Understanding such mechanisms could lead to development of safer non-surgical weight loss approaches. We here present the first analysis of the impact of LGP on the serum proteome, to bring new insights into the underlying molecular mechanism. Our findings indicate that LGP has a comprehensive systemic effect based on the blood serum proteome profile which might account for accelerated reduction of fat mass after surgery, thus, food restriction is not the only reason for weight loss following this unique surgical approach. As secretory regions of the stomach are preserved in LGP and it is associated with minimal physiological and anatomical changes, the findings are of high importance in the field of bariatric surgery and weight loss.

Keywords:  Bariatric surgery; Label free quantitation; Laparoscopic gastric plication; Obesity; Proteomics

DOI:  https://doi.org/10.1016/j.jprot.2019.05.001
J Neuroimmune Pharmacol. 2019 May 08.

Proteomic Profiling of Exosomes Derived from Plasma of HIV-Infected Alcohol Drinkers and Cigarette Smokers.

Sunitha Kodidela, Yujie Wang, Benjamin J Patters, Yuqing Gong, Namita Sinha, Sabina Ranjit, Kelli Gerth, Sanjana Haque, Theodore Cory, Carole McArthur, Anil Kumar, Jim Y Wan, Santosh Kumar.

  Abuse of alcohol and tobacco could exacerbate HIV pathogenesis by transferring materials through exosomes (small nanovesicles). Exosomes present a stable and accessible source of information concerning the health and/or disease status of patients, which can provide diagnostic and prognostic biomarkers for myriad conditions. Therefore, we aimed to study the specific exosomal proteins that are altered in both HIV-infected subjects and alcohol/tobacco users. Exosomes were isolated from plasma of the following subjects: a) HIV-negative subjects (healthy), b) HIV-positive subjects (HIV), c) HIV-negative alcohol drinkers (drinkers), d) HIV-negative tobacco smokers (smokers), e) HIV-positive drinkers (HIV + drinkers), and f) HIV-positive smokers (HIV + smokers). Quantitative proteomic profiling was then performed from these exosomes. Sixteen proteins were significantly altered in the HIV group, ten in drinkers, four in HIV + drinkers, and fifteen in smokers compared to healthy subjects. Only one protein, fibulin-1 (FBLN1), was significantly altered in HIV + smokers. Interestingly, hemopexin was not significantly altered in drinkers or HIV patients but was significantly altered in HIV + drinkers. Further, our study is the first to show properdin expression in plasma exosomes, which was decreased in HIV + smokers and HIV + drinkers compared to HIV patients. The present findings suggest that hemopexin and properdin show potential as markers for physiological effects that may arise in HIV-infected individuals who abuse alcohol and tobacco. Graphical abstract This study presents a proteomic analysis of plasma-derived exosomes from HIV-infected alcohol drinkers and smokers. Among the proteins altered due to drug-abuse, hemopexin and properdin were of highest significance. These proteins can be potential biomarkers for co-morbid conditions associated with drug abuse in HIV-patients.

Keywords:  Alcohol; HIV; Plasma exosome; Proteomics; Smoking

DOI:  https://doi.org/10.1007/s11481-019-09853-2
Electrophoresis. 2019 May 09.

Comparative proteomic analysis of plasma of children with congenital heart disease.

Qiong Huang, Zhi Geng, Tao Chen, Xiangjun Cheng, Haitao Gu, Qingguo Li, Dianfu Li, Rui Liu.

  Congenital heart disease (CHD) is one of the largest class of birth defects. Eight subjects with ventricular septal defect (VSD, a kind of CHD) and 11 health children were enrolled in tandem mass tags label-based quantitative proteomic analysis to compare plasma proteins differentially abundance. A total of 66 proteins were significantly upregulated or downregulated in VSD patients compared with healthy children. These proteins were involved in pathways linked to platelet activation, fructose and mannose metabolism, complement and coagulation cascades, glycolysis/gluconeogenesis, regulation of actin cytoskeleton, and carbon metabolism. The amount of ten proteins changed significantly (p < 0.05) in newly recruited 30 VSD compared with 15 control children, which were validated by enzyme-linked immunosorbent assay. The areas under the receiver operating characteristic curve values of fructose-bisphosphate aldolase B (ALDOB) and thymosin beta-4 (Tβ4) were higher than those of other candidate proteins. ALDOB and Tβ4 might be potential biomarkers applied for identifying VSD in the further works. This article is protected by copyright. All rights reserved.

Keywords:  Biomarkers; Congenital heart diseases; Plasma; Proteomic analysis; Ventricular septal defect

DOI:  https://doi.org/10.1002/elps.201900098
J Infect Dis. 2019 May 06. pii: jiz223. [Epub ahead of print]

Plasma fibronectin levels identified via quantitative proteomics profiling predicts hepatitis B surface antigen seroclearance in chronic hepatitis B.

Fen Liu, Wai-Kay Seto, Danny Ka-Ho Wong, Fung-Yu Huang, Ka-Shing Cheung, Lung-Yi Mak, Rakesh Sharma, Saisai Zhang, James Fung, Ching-Lung Lai, Man-Fung Yuen.

   BACKGROUND: Seroclearance of hepatitis B surface antigen (HBsAg) is a potentially achievable target of chronic hepatitis B (CHB). Plasma proteins relevant to HBsAg seroclearance remain undetermined.
METHODS: We prospectively recruited treatment-naïve CHB patients with spontaneous HBsAg seroclearance and matched HBsAg-positive controls. Plasma protein profiling was performed using isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomics, with the expression of candidate proteins validated in a separate cohort. The predictive value of fibronectin was assessed at 3 years, 1 year (Year -1) before and at the time (Year 0) of HBsAg seroclearance.
RESULTS: 487 plasma proteins were identified via proteomics, with 97 proteins showing altered expression. In verification cohort (n=90), median plasma fibronectin levels in patients with HBsAg seroclearance was higher than in controls (p=0.009). In the longitudinal cohort (n=164), patients with HBsAg seroclearance, when compared to controls, had a higher median fibronectin levels at Year -1 (413.26 vs. 227.95 µg/ml), and Year 0 (349.45 vs. 208.72 µg/ml) (both p<0.001). In patients with an annual HBsAg log reduction >0.5, Year -1 fibronectin level achieved an AUROC of 0.884 in predicting HBsAg seroclearance.
CONCLUSIONS: Using proteomics-based technology, plasma fibronectin may be associated with HBsAg seroclearance, and a potential predictor of "functional cure".

Keywords:  HBV; HBsAg; biomarker; functional cure; iTRAQ

DOI:  https://doi.org/10.1093/infdis/jiz223
Nan Fang Yi Ke Da Xue Xue Bao. 2019 Apr 30. 39(4): 428-436

[Proteomic analysis of the cerebrospinal fluid from patients with amyotrophic lateral sclerosis based on tandem mass spectrometry technique].

Dandan Su, Yong Zhang, Fangfang Bi, Bo Xiao.

   OBJECTIVE: To investigate the differentially expressed proteins in the cerebrospinal fluid (CSF) of patients with amyotrophic lateral sclerosis (ALS) at the proteomics level using tandem mass spectrometry label (TMT) technique and explore the pathogenic mechanism and related pathways of ALS.
METHODS: Between November, 2017 and April, 2018, 5 patients with medulla oblongata onset ALS and 5 patients with limb onset ALS were selected from the Departments of Neurology of 928 Hospital of Army Joint Logistics Support Force of PLA and Xiangya Hospital of Central South University, with 5 patients with migraine and low intracranial pressure headache serving as the healthy controls.CSF samples were obtained from all the participants, and the differentially expressed proteins in the CSF were identified using tandem mass spectrometry (TMT) technique with bioinformatics analysis.
RESULTS: A total of 1530 proteins were identified and quantified in the CSF samples.The expression of 48 proteins was up-regulated and 6 proteins were down-regulated in medulla oblongata onset ALS patients; 16 proteins were up-regulated and 19 were down-regulated in limb onset ALS patients.GO analysis showed that these proteins, which were distributed both within and outside the cells, were involved in cell physiological process, single organ process and biological regulation and had binding function, catalytic activity, and receptor activity.KEGG pathway analysis showed that the up-regulated proteins in the CSF from patients with medulla oblongata onset ALS participated in 3 pathways involving the lysosomes, metabolism, and measles.The down-regulated proteins in the CSF from patients with limb onset ALS participated in 7 pathways involving the complement and coagulation cascade, Staphylococcus aureus infection and herpes simplex infection, and all the pathways contained complement components.
CONCLUSIONS: The CSF samples of ALS patients with medullary onset and limb onset have differentially expressed proteins.The lysosomal pathway is involved in the occurrence and progression of ALS with medullary onset, and the immune responses are involved in the occurrence and progression of ALS with limb onset.

Keywords:  amyotrophic lateral sclerosis; cerebrospinal fluid; proteomics; tandem mass tag

DOI:  https://doi.org/10.12122/j.issn.1673-4254.2019.04.08
Sci Rep. 2019 May 10. 9(1): 7217

Melanoma proteomics suggests functional differences related to mutational status.

Lucía Trilla-Fuertes, Angelo Gámez-Pozo, Guillermo Prado-Vázquez, Andrea Zapater-Moros, Mariana Díaz-Almirón, Claudia Fortes, María Ferrer-Gómez, Rocío López-Vacas, Verónica Parra Blanco, Iván Márquez-Rodas, Ainara Soria, Juan Ángel Fresno Vara, Enrique Espinosa.

Melanoma is the most lethal cutaneous cancer. New drugs have recently appeared; however, not all patients obtain a benefit of these new drugs. For this reason, it is still necessary to characterize melanoma at molecular level. The aim of this study was to explore the molecular differences between melanoma tumor subtypes, based on BRAF and NRAS mutational status. Fourteen formalin-fixed, paraffin-embedded melanoma samples were analyzed using a high-throughput proteomics approach, combined with probabilistic graphical models and Flux Balance Analysis, to characterize these differences. Proteomics analyses showed differences in expression of proteins related with fatty acid metabolism, melanogenesis and extracellular space between BRAF mutated and BRAF non-mutated melanoma tumors. Additionally, probabilistic graphical models showed differences between melanoma subgroups at biological processes such as melanogenesis or metabolism. On the other hand, Flux Balance Analysis predicts a higher tumor growth rate in BRAF mutated melanoma samples. In conclusion, differential biological processes between melanomas showing a specific mutational status can be detected using combined proteomics and computational approaches.

DOI: https://doi.org/10.1038/s41598-019-43512-z
Arch Toxicol. 2019 May 08.

Forensic evidence of sulfur mustard exposure in real cases of human poisoning by detection of diverse albumin-derived protein adducts.

Harald John, Marianne Koller, Franz Worek, Horst Thiermann, Markus Siegert.

  We present the forensic analyses of plasma samples of human victims exposed to sulfur mustard (SM) in a crisis region in the Middle East in 2015. A few hours after exposure, poisoned persons showed typical signs and symptoms of percutaneous SM exposure including erythema and later on blisters and hardly healing skin wounds. Blood samples were collected 15 days after poisoning to be analyzed for the presence of long-lived protein-adduct biomarkers to verify SM poisoning. We applied a novel bioanalytical toolbox targeting four human serum albumin-derived biomarkers that were made accessible after plasma proteolysis. These adducts contained the SM-specific hydroxyethylthioethyl moiety either bound to the thiol group of a cysteine residue (C34*) or to the side-chain carboxylic group of a glutamic acid residue (E230*). Peptide biomarkers were produced from plasma of the victims using proteinase K (C34*PF), pronase (C34*P) and pepsin (AE230*VSKL and LQQC34*PFEDHVKL) for enzymatic protein cleavage. Separation and detection were carried out by selective micro-liquid chromatography-electrospray ionization high-resolution tandem mass spectrometry (µLC-ESI MS/HR MS). In addition to this site-specific adduct detection, a general approach after alkaline hydrolysis of the plasma protein fraction was applied. Liberated thiodiglycol (TDG) was derivatized with heptafluorobutyric anhydride and detected by gas chromatography-electron ionization mass spectrometry (GC-EI MS). The different bioanalytical methods yielded congruent results confirming SM poisoning for all patients who showed clinical signs and symptoms. This is the first time that real cases of SM poisoning were confirmed and presented by such a broad compilation of protein-derived biomarkers.

Keywords:  Biomarker; Hydroxyethylthioethyl moiety; LC–ESI MS/HRMS; Protein adducts; Vesicant

DOI:  https://doi.org/10.1007/s00204-019-02461-2
J Natl Cancer Inst. 2019 May 11. pii: djz078. [Epub ahead of print]

Uncovering the role of hnRNP K, an RNA-binding protein, in B-cell lymphomas.

Miguel Gallardo, Prerna Malaney, Marisa J L Aitken, Xiaorui Zhang, Todd M Link, Vrutant Shah, Sanzhar Alybayev, Meng-Han Wu, Laura R Pageon, Huaxian Ma, Rodrigo Jacamo, Li Yu, Zijun Y Xu-Monette, Haley Steinman, Hun Ju Lee, Dos Sarbassov, Inmaculada Rapado, Michelle C Barton, Joaquin Martinez-Lopez, Carlos Bueso-Ramos, Ken H Young, Sean M Post.

   BACKGROUND: hnRNP K is an RNA-binding protein that is aberrantly expressed in cancers. We and others have previously shown that reduced hnRNP K expression downmodulates tumor suppressive programs. However, overexpression of hnRNP K is the more commonly observed clinical phenomenon, yet its functional consequences and clinical significance remain unknown.
METHODS: Clinical implications of hnRNP K overexpression were examined through immunohistochemistry on samples from patients with diffuse large B-cell lymphoma (DLBCL) who did not harbor MYC alterations (n = 75). A novel transgenic mouse model that overexpresses hnRNP K specifically in B-cells was generated to directly examine the role of hnRNP K overexpression in mice (three transgenic lines). Molecular consequences of hnRNP K overexpression were determined through proteomics, formaldehyde-RNA-immunoprecipitation sequencing, and biochemical assays. Therapeutic response to BET-bromodomain inhibition in the context of hnRNP K overexpression was evaluated in vitro and in vivo (n = 3 per group). All statistical tests were two-sided.
RESULTS: hnRNP K is overexpressed in DLBCL patients without MYC genomic alterations. This overexpression is associated with dismal OS and PFS (p < 0.001). Overexpression of hnRNP K in transgenic mice resulted in the development of lymphomas and reduced survival (p < 0.001 for all transgenic lines; Line 171 (n = 30): HR = 64.23, 95% CI = 26.1-158.0; Line 173 (n = 31): HR = 25.27, 95% CI = 10.3-62.1; Line 177 (n = 25): HR = 119.5, 95% CI = 42.7-334.2, compared to wild-type mice). Clinical samples, mouse models, global screening assays, and biochemical studies revealed that hnRNP K's oncogenic potential stems from its ability to post-transcriptionally and translationally regulate MYC. Consequently, Hnrnpk overexpression renders cells sensitive to BET-bromodomain-inhibition in in vitro and transplantation models, which represents a strategy for mitigating hnRNP K-mediated c-Myc activation in patients.
CONCLUSION: Our findings indicate that hnRNP K is a bona fide oncogene when overexpressed and represents a novel mechanism for c-Myc activation in the absence of MYC lesions.

Keywords:  BET-bromodomain inhibitor; DLBCL; Mouse model; RNA-binding protein; c-Myc; diffuse-large B-cell lymphoma; hnRNP K; oncogene

DOI:  https://doi.org/10.1093/jnci/djz078
Diabetes Care. 2019 May 10. pii: dc182585. [Epub ahead of print]

Characterization of Glycolytic Enzymes and Pyruvate Kinase M2 in Type 1 and 2 Diabetic Nephropathy.

Daniel Gordin, Hetal Shah, Takanori Shinjo, Ronald St-Louis, Weier Qi, Kyoungmin Park, Samantha M Paniagua, David M Pober, I-Hsien Wu, Vanessa Bahnam, Megan J Brissett, Liane J Tinsley, Jonathan M Dreyfuss, Hui Pan, Yutong Dong, Monika A Niewczas, Peter Amenta, Thorsten Sadowski, Aimo Kannt, Hillary A Keenan, George L King.

OBJECTIVE: Elevated glycolytic enzymes in renal glomeruli correlated with preservation of renal function in the Medalist Study, individuals with ≥50 years of type 1 diabetes. Specifically, pyruvate kinase M2 (PKM2) activation protected insulin-deficient diabetic mice from hyperglycemia-induced glomerular pathology. This study aims to extend these findings in a separate cohort of type 1 and type 2 diabetes and discover new circulatory biomarkers for renal protection through proteomics and metabolomics of Medalists' plasma. We hypothesize that increased glycolytic flux and improved mitochondrial biogenesis will halt the progression of diabetic nephropathy.
RESEARCH DESIGN AND METHODS: Immunoblots analyzed selected glycolytic and mitochondrial enzymes in postmortem glomeruli of non-Medalists with type 1 diabetes (n = 15), type 2 diabetes (n = 19), and no diabetes (n = 5). Plasma proteomic (SOMAscan) (n = 180) and metabolomic screens (n = 214) of Medalists with and without stage 3b chronic kidney disease (CKD) were conducted and significant markers validated by ELISA.
RESULTS: Glycolytic (PKM1, PKM2, and ENO1) and mitochondrial (MTCO2) enzymes were significantly elevated in glomeruli of CKD- versus CKD+ individuals with type 2 diabetes. Medalists' plasma PKM2 correlated with estimated glomerular filtration rate (r 2 = 0.077; P = 0.0002). Several glucose and mitochondrial enzymes in circulation were upregulated with corresponding downregulation of toxic metabolites in CKD-protected Medalists. Amyloid precursor protein (APP) was also significantly upregulated, tumor necrosis factor receptors downregulated, and both confirmed by ELISA.
CONCLUSIONS: Elevation of enzymes involved in the metabolism of intracellular free glucose and its metabolites in renal glomeruli are connected to preserving kidney function in both type 1 and type 2 diabetes. The renal profile of elevated glycolytic enzymes and reduced toxic glucose metabolites is reflected in the circulation, supporting their use as biomarkers for endogenous renal protective factors in people with diabetes.

DOI: https://doi.org/10.2337/dc18-2585
Nat Med. 2019 May;25(5): 792-804

A longitudinal big data approach for precision health.

Sophia Miryam Schüssler-Fiorenza Rose, Kévin Contrepois, Kegan J Moneghetti, Wenyu Zhou, Tejaswini Mishra, Samson Mataraso, Orit Dagan-Rosenfeld, Ariel B Ganz, Jessilyn Dunn, Daniel Hornburg, Shannon Rego, Dalia Perelman, Sara Ahadi, M Reza Sailani, Yanjiao Zhou, Shana R Leopold, Jieming Chen, Melanie Ashland, Jeffrey W Christle, Monika Avina, Patricia Limcaoco, Camilo Ruiz, Marilyn Tan, Atul J Butte, George M Weinstock, George M Slavich, Erica Sodergren, Tracey L McLaughlin, Francois Haddad, Michael P Snyder.

Precision health relies on the ability to assess disease risk at an individual level, detect early preclinical conditions and initiate preventive strategies. Recent technological advances in omics and wearable monitoring enable deep molecular and physiological profiling and may provide important tools for precision health. We explored the ability of deep longitudinal profiling to make health-related discoveries, identify clinically relevant molecular pathways and affect behavior in a prospective longitudinal cohort (n = 109) enriched for risk of type 2 diabetes mellitus. The cohort underwent integrative personalized omics profiling from samples collected quarterly for up to 8 years (median, 2.8 years) using clinical measures and emerging technologies including genome, immunome, transcriptome, proteome, metabolome, microbiome and wearable monitoring. We discovered more than 67 clinically actionable health discoveries and identified multiple molecular pathways associated with metabolic, cardiovascular and oncologic pathophysiology. We developed prediction models for insulin resistance by using omics measurements, illustrating their potential to replace burdensome tests. Finally, study participation led the majority of participants to implement diet and exercise changes. Altogether, we conclude that deep longitudinal profiling can lead to actionable health discoveries and provide relevant information for precision health.

DOI: https://doi.org/10.1038/s41591-019-0414-6
Am J Gastroenterol. 2019 May;114(5): 777-785

Serum Protein Biomarkers of Fibrosis Aid in Risk Stratification of Future Stricturing Complications in Pediatric Crohn's Disease.

Jing Wu, David M Lubman, Subra Kugathasan, Lee A Denson, Jeffrey S Hyams, Marla C Dubinsky, Anne M Griffiths, Robert N Baldassano, Joshua D Noe, Shervin Rabizadeh, Ajay S Gulati, Joel R Rosh, Wallace V Crandall, Peter D R Higgins, Ryan W Stidham.

OBJECTIVES: Avoiding fibrostenotic complications is of paramount concern in the management of Crohn's disease (CD). We sought to investigate the association of candidate biomarkers of fibrosis collected at diagnosis with the future development of fibrostenotic CD.
METHODS: Using the Risk Stratification and Identification of Immunogenetic and Microbial Markers of Rapid Disease Progression in Children with Crohn's Disease cohort, a multicenter prospective observational pediatric inception cohort, subjects with an inflammatory phenotype (B1) at diagnosis who later converted to a stricturing phenotype (B2) within 3 years were compared with those who remained B1. Serum collected at diagnosis underwent both parallel reaction monitoring-targeted proteomic analysis and conventional enzyme-linked immunosorbent assay for 10 candidate biomarkers of intestinal fibrosis. Cox proportional hazard regression was used for multivariable analysis of time-dependent outcomes.
RESULTS: In 116 subjects 58 subjects with verified B1 phenotype at diagnosis who later converted to B2 disease were compared with 58 subjects who remained B1 over 3 years of follow-up. Extracellular matrix protein 1 (ECM1) levels in the upper quartile (hazard ratio [HR] 3.43, 95% confidence limit [CL] 1.33, 8.42) were associated with future fibrostenotic disease. ASCA IgA (HR 4.99, 95% CL 1.50, 16.68) and CBir levels (HR 5.19, 95% CL 1.83, 14.74) were also associated with future intestinal fibrostenosis, although ECM1 continued to demonstrate independent association with conversion to B2 even with adjustment for serologies in multivariable analysis (HR 5.33, 95% CL 1.29, 22.13).
CONCLUSIONS: ECM1 and other biomarkers of fibrosis may aid in determining the risk of uncomplicated inflammatory disease converting to B2 stricturing phenotypes in children with CD. Prospective validation studies to verify test performance and optimize clinical utilization are needed before clinical implementation.

DOI: https://doi.org/10.14309/ajg.0000000000000237
J Exp Clin Cancer Res. 2019 May 09. 38(1): 187

Integrated omics-based pathway analyses uncover CYP epoxygenase-associated networks as theranostic targets for metastatic triple negative breast cancer.

Maria Karmella Apaya, Jeng-Yuan Shiau, Guo-Shiou Liao, Yu-Jen Liang, Chia-Wei Chen, Hsin-Chou Yang, Chi-Hong Chu, Jyh-Cherng Yu, Lie-Fen Shyur.

   BACKGROUND: Current prognostic tools and targeted therapeutic approaches have limited value for metastatic triple negative breast cancer (TNBC). Building upon current knowledge, we hypothesized that epoxyeicosatrienoic acids (EETs) and related CYP450 epoxygenases may have differential roles in breast cancer signaling, and better understanding of which may uncover potential directions for molecular stratification and personalized therapy for TNBC patients.
METHODS: We analyzed the oxylipin metabolome of paired tumors and adjacent normal mammary tissues from patients with pathologically confirmed breast cancer (N = 62). We used multivariate statistical analysis to identify important metabolite contributors and to determine the predictive power of tumor tissue metabolite clustering. In vitro functional assays using a panel of breast cancer cell lines were carried out to further confirm the crucial roles of endogenous and exogenous EETs in the metastasis transformation of TNBC cells. Deregulation of associated downstream signaling networks associated with EETs/CYPs was established using transcriptomics datasets from The Cancer Genome Atlas (TCGA) and Molecular Taxonomy of Breast Cancer International Consortium (METABRIC). Comparative TNBC proteomics using the same tissue specimens subjected to oxylipin metabolomics analysis was used as validation set.
RESULTS: Metabolite-by-metabolite comparison, tumor immunoreactivity, and gene expression analyses showed that CYP epoxygenases and arachidonic acid-epoxygenation products, EET metabolites, are strongly associated with TNBC metastasis. Notably, all the 4 EET isomers (5,6-, 8,9-, 11,12-, and 14,15-EET) was observed to profoundly drive the metastasis transformation of mesenchymal-like TNBC cells among the TNBC (basal- and mesenchymal-like), HER2-overexpressing and luminal breast cancer cell lines examined. Our pathway analysis revealed that, in hormone-positive breast cancer subtype, CYP epoxygenase overexpression is more related to immune cell-associated signaling, while EET-mediated Myc, Ras, MAPK, EGFR, HIF-1α, and NOD1/2 signaling are the molecular vulnerabilities of metastatic CYP epoxygenase-overexpressing TNBC tumors.
CONCLUSIONS: This study suggests that categorizing breast tumors according to their EET metabolite ratio classifiers and CYP epoxygenase profiles may be useful for prognostic and therapeutic assessment. Modulation of CYP epoxygenase and EET-mediated signaling networks may offer an effective approach for personalized treatment of breast cancer, and may be an effective intervention option for metastatic TNBC patients.

Keywords:  CYP450 epoxygenase; Epoxyeicosatrienoic acid; Metastasis; Oxylipin metabolome; Triple negative breast cancer

DOI:  https://doi.org/10.1186/s13046-019-1187-y
Biochem Biophys Rep. 2019 Jul;18 100635

Relative proteome quantification of alpha, beta, gamma and delta globin chains in early eluting peaks of Bio-Rad variant II® CE-HPLC of hemoglobin from healthy and beta-thalassemia subjects in Malaysia.

Uday Younis Hussein Abdullah, Hishamshah M Ibrahim, Haitham Muhammed Jassim, Mohamad Zaki Salleh, Teh Lay Kek, Mohd Nur Fakhruzzaman Bin Noorizhab, Bin Alwi Zilfalil, Prapin Wilairat, Suthat Fucharoen.

  This is the first report of QQQ-mass spectrometric identification and quantification of the Hb subunits, alpha, beta, delta and gamma globin peptides, derived from enzymatic-digestion of proteins in the early unknown peaks of the Bio-Rad cation-exchange chromatography of haemoglobin. The objectives were to assess the relationship of the quantity of the free alpha, beta, delta and gamma globin chains with the phenotypic diversity of beta-thalassaemias (β-thal). The results demonstrate that the pools of free globin chains in red blood cells were correlating with the severity of the disease in patients with different phenotypes of β-thal. The mechanism and the regulation of synthesis of free globin chains pool in a normal individual and in patients with different β-thal phenotypes could arise from several mechanisms which will require further investigation. The role of the free globin pool in patients with β-thal for development of novel therapeutic approaches based on these potential targets requires further investigation. Pertinent biomarkers improves the diagnosis of the β-thal, especially in low-income countries where they are most common and allows more effective therapeutic intervention leading to more successful therapeutic outcome.

Keywords:  Anaemia; Globin chain; HPLC; Mass spectrometry; Quantification; Thalassemia; Unknown peaks

DOI:  https://doi.org/10.1016/j.bbrep.2019.100635