bims-prodis Biomed News
on Proteomics in disease
Issue of 2019–04–21
29 papers selected by
Nancy Gough, Bioserendipity



  1. EBioMedicine. 2019 Apr 11. pii: S2352-3964(19)30242-7. [Epub ahead of print]
       BACKGROUND: Pancreatic cancer is a heterogenous disease with a poor prognosis. This study aimed to discover and validate prognostic tissue biomarkers in pancreatic cancer using a mass spectrometry (MS) based proteomics approach.
    METHODS: Global protein sequencing of fresh frozen pancreatic cancer and healthy pancreas tissue samples was conducted by MS to discover potential protein biomarkers. Selected candidate proteins were further verified by targeted proteomics using parallel reaction monitoring (PRM). The expression of biomarker candidates was validated by immunohistochemistry in a large tissue microarray (TMA) cohort of 141 patients with resectable pancreatic cancer. Kaplan-Meier and Cox proportional hazard modelling was used to investigate the prognostic utility of candidate protein markers.
    FINDINGS: In the initial MS-discovery phase, 165 proteins were identified as potential biomarkers. In the subsequent MS-verification phase, a panel of 45 candidate proteins was verified by the development of a PRM assay. Brain acid soluble protein 1 (BASP1) was identified as a new biomarker candidate for pancreatic cancer possessing largely unknown biological and clinical functions and was selected for further analysis. Importantly, bioinformatic analysis indicated that BASP1 interacts with Wilms tumour protein (WT1) in pancreatic cancer. TMA-based immunohistochemistry analysis showed that BASP1 was an independent predictor of prolonged survival (HR 0.468, 95% CI 0.257-0.852, p = .013) and predicted favourable response to adjuvant chemotherapy, whereas WT1 indicated a worsened survival (HR 1.636, 95% CI 1.083-2.473, p = .019) and resistance to chemotherapy. Interaction analysis showed that patients with negative BASP1 and high WT1 expression had the poorest outcome (HR 3.536, 95% CI 1.336-9.362, p = .011).
    INTERPRETATION: We here describe an MS-based proteomics platform for developing biomarkers for pancreatic cancer. Bioinformatic analysis and clinical data from our study suggest that BASP1 and its putative interaction partner WT1 can be used as biomarkers for predicting outcomes in pancreatic cancer patients.
    Keywords:  BASP1; Biomarkers; Chemotherapy response; Mass spectrometry; Pancreatic cancer; Prognosis; WT1
    DOI:  https://doi.org/10.1016/j.ebiom.2019.04.008
  2. Clin Proteomics. 2019 ;16 14
       Background: Endometriosis is a common gynaecological disorder affecting 5-10% of women of reproductive age who often experience chronic pelvic pain and infertility. Definitive diagnosis is through laparoscopy, exposing patients to potentially serious complications, and is often delayed. Non-invasive biomarkers are urgently required to accelerate diagnosis and for triaging potential patients for surgery.
    Methods: This retrospective case control biomarker discovery and validation study used quantitative 2D-difference gel electrophoresis and tandem mass tagging-liquid chromatography-tandem mass spectrometry for protein expression profiling of eutopic and ectopic endometrial tissue samples collected from 28 cases of endometriosis and 18 control patients undergoing surgery for investigation of chronic pelvic pain without endometriosis or prophylactic surgery. Samples were further sub-grouped by menstrual cycle phase. Selected differentially expressed candidate markers (LUM, CPM, TNC, TPM2 and PAEP) were verified by ELISA in a set of 87 serum samples collected from the same and additional women. Previously reported biomarkers (CA125, sICAM1, FST, VEGF, MCP1, MIF and IL1R2) were also validated and diagnostic performance of markers and combinations established.
    Results: Cycle phase and endometriosis-associated proteomic changes were identified in eutopic tissue from over 1400 identified gene products, yielding potential biomarker candidates. Bioinformatics analysis revealed enrichment of adhesion/extracellular matrix proteins and progesterone signalling. The best single marker for discriminating endometriosis from controls remained CA125 (AUC = 0.63), with the best cross-validated multimarker models improving the AUC to 0.71-0.81, depending upon menstrual cycle phase and control group.
    Conclusions: We have identified menstrual cycle- and endometriosis-associated protein changes linked to various cellular processes that are potential biomarkers and that provide insight into the biology of endometriosis. Our data indicate that the markers tested, whilst not useful alone, have improved diagnostic accuracy when used in combination and demonstrate menstrual cycle specificity. Tissue heterogeneity and blood contamination is likely to have hindered biomarker discovery, whilst a small sample size precludes accurate determination of performance by cycle phase. Independent validation of these biomarker panels in a larger cohort is however warranted, and if successful, they may have clinical utility in triaging patients for surgery.
    Keywords:  Biomarkers; Ectopic tissue; Endometriosis; Eutopic tissue; Serum
    DOI:  https://doi.org/10.1186/s12014-019-9235-3
  3. J Food Drug Anal. 2019 Apr;pii: S1021-9498(18)30155-8. [Epub ahead of print]27(2): 387-403
      Prostate, bladder and kidney cancer are the three most common types of genitourinary cancer in the world. Of these, prostate and bladder cancers are within the top 10 most common cancers in men. Notably, kidney cancer causes no obvious symptoms in the early stages. To satisfy clinical-management requirements, researchers have developed numerous biomarkers by applying proteomic approaches using clinical serum, urine and tissue specimens, as well as cell and animal models. Through application of biomarker pipeline protocols, including discovery, verification and validation phases, and mass-spectrometric based proteomic platforms coupled with multiplexed quantification assays, these studies have led to recent rapid progress in this area. With improvements in mass-spectrometric based proteomic techniques, numerous promising biomarker candidates and marker panels for various clinical purposes have been proposed. Verification of novel protein biomarker candidates is very resource demanding (e.g. on the clinical and laboratory sides). With the support of national consortia, it is now possible to investigate the future clinical use of such biomarker strategies and assess their cost-effectiveness in personalized medicine.
    Keywords:  Biomarker; Bladder cancer; Kidney cancer; Prostate cancer; Proteomics
    DOI:  https://doi.org/10.1016/j.jfda.2018.09.005
  4. J Clin Med. 2019 Apr 16. pii: E517. [Epub ahead of print]8(4):
      Coarctation of the aorta is a form of left ventricular outflow tract obstruction in paediatric patients that can be presented with either bicuspid (BAV) or normal tricuspid (TAV) aortic valve. The congenital BAV is associated with hemodynamic changes and can therefore trigger different molecular remodelling in the coarctation area. This study investigated the proteomic and phosphoproteomic changes associated with BAV for the first time in neonatal coarctation patients. Aortic tissue was collected just proximal to the coarctation site from 23 neonates (BAV; n = 10, TAV; n = 13) that were matched for age (age range 4-22 days). Tissue from half of the patients was frozen and used for proteomic and phosphoproteomic analysis whilst the remaining tissue was formalin fixed and used for analysis of elastin content using Elastic Van-Gieson (EVG) staining. A total of 1796 protein and 75 phosphoprotein accession numbers were detected, of which 34 proteins and one phosphoprotein (SSH3) were differentially expressed in BAV patients compared to TAV patients. Ingenuity Pathway Analysis identified the formation of elastin fibres as a significantly enriched function (p = 1.12 × 10-4) due to the upregulation of EMILIN-1 and the downregulation of TNXB. Analysis of paraffin sections stained with EVG demonstrated increased elastin content in BAV patients. The proteomic/phosphoproteomic analysis also suggested changes in inositol signalling pathways and reduced expression of the antioxidant SOD3. This work demonstrates for the first time that coarcted aortic tissue in neonatal BAV patients has an altered proteome/phosphoproteome consistent with observed structural vascular changes when compared to TAV patients.
    Keywords:  aortic coarctation; bicuspid aortic valve; congenital heart disease
    DOI:  https://doi.org/10.3390/jcm8040517
  5. Curr Eye Res. 2019 Apr 17.
       PURPOSE: The protein composition of aqueous humour (AH) has held significant relevance and remains to be the prime sample in the discovery of biomarkers in glaucoma. The purpose of this study is to analyze the aqueous humour protein concentrations in primary open angle glaucoma (POAG) and primary angle closure glaucoma (PACG) and further examine the proteome changes compared to cataract control.
    METHODS: Aqueous humour was collected from 90 POAG, 72 PACG, 78 cataract (control) in this study. The total protein was quantified using Bradford's assay. Samples were subjected to trypsin digestion followed by LC-MS/MS for proteomic studies (n=3 per group). The extracellular matrix has a major influence on the aqueous humour outflow, the regulator proteins osteopontin (OPN), cathepsin D and cystatin C detected by mass spectrometry are validated in aqueous humour samples by western blot and turbidimetric immunoassay.
    RESULTS: We observed a significant increase in protein levels of POAG (p=0.0009). Interestingly, a similar increase in PACG compared to cataract (p<0.0001) and POAG (p=0.02). Proteomics analysis identified 184, 190 and 299 proteins in control, POAG and PACG. OPN was increased in POAG (p=0.0319) and PACG (p=0.0103) compared to control. The precursor form of cathepsin D was increased in POAG and decreased in PACG, though not significant compared to control. Cystatin C was also increased in both POAG (p=0.0310) and PACG (p=0.0125) compared to control.
    CONCLUSION: In this study, we report for the first time that PACG cohort had higher total protein compared to controls. A qualitative comparison of proteomes revealed increased numbers of proteins identified in PACG. We assume that elevated levels of OPN and cystatin C in POAG and PACG along with altered cathepsin levels may contribute to ECM aberration in glaucoma.
    Keywords:  Cathepsin D; Glaucoma; aqueous humour; cystatin C; osteopontin
    DOI:  https://doi.org/10.1080/02713683.2019.1608261
  6. Clin Proteomics. 2019 ;16 12
       Background: Esophageal cancer (EC) is one of the malignant tumors with a poor prognosis. The early stage of EC is asymptomatic, so identification of cancer biomarkers is important for early detection and clinical practice.
    Methods: In this study, we compared the protein expression profiles in esophageal squamous cell carcinoma (ESCC) tissues and adjacent normal esophageal tissues from five patients through high-resolution label-free mass spectrometry. Through bioinformatics analysis, we found the differentially expressed proteins of ESCC. To perform the rapid identification of biomarkers, we adopted a high-throughput protein identification technique of Quantitative Dot Blot (QDB). Meanwhile, the QDB results were verified by classical immunohistochemistry.
    Results: In total 2297 proteins were identified, out of which 308 proteins were differentially expressed between ESCC tissues and normal tissues. By bioinformatics analysis, the four up-regulated proteins (PTMA, PAK2, PPP1CA, HMGB2) and the five down-regulated proteins (Caveolin, Integrin beta-1, Collagen alpha-2(VI), Leiomodin-1 and Vinculin) were selected and validated in ESCC by Western Blot. Furthermore, we performed the QDB and IHC analysis in 64 patients and 117 patients, respectively. The PTMA expression was up-regulated gradually along the progression of ESCC, and the PTMA expression ratio between tumor and adjacent normal tissue was significantly increased along with the progression. Therefore, we suggest that PTMA might be a potential candidate biomarker for ESCC.
    Conclusion: In this study, label-free quantitative proteomics combined with QDB revealed that PTMA expression was up-regulated in ESCC tissues, and PTMA might be a potential candidate for ESCC. Since Western Blot cannot achieve rapid and high-throughput screening of mass spectrometry results, the emergence of QDB meets this demand and provides an effective method for the identification of biomarkers.
    Keywords:  Esophageal squamous cell carcinoma (ESCC); Label-free quantitative proteomics; Prothymosin alpha (PTMA); Quantitative Dot Blot (QDB)
    DOI:  https://doi.org/10.1186/s12014-019-9232-6
  7. Biomed Res Int. 2019 ;2019 2735038
      Seminal plasma is a complex mixture of secretions from various glands in the male genital tract. Compared to sperm cells, it contains important proteins that are both directly and indirectly associated with sperm motility. Here, we constructed quantitative proteomes of human seminal plasma from three normozoospermic and asthenozoospermic individuals. A total of 524 proteins were identified, and 366 of them were found to be quantified in all six samples. We first investigated the absolute expression features of these proteins and found that the variations of protein identification among different samples and other published datasets were mainly due to some lowly expressed proteins. By integration of various proteomic datasets and bioinformatics databases, we comprehensively annotated the biological functions, physiological originations, and disease associations of these proteins. We found that our dataset could benefit the studies of both male infertility and other male diseases. Finally, based on the relative expression values determined by chemical labeling, we identified a total of 29 differentially expressed proteins, which could be used as candidate targets for studying the molecular bases of sperm motility or developing precise diagnostic biomarkers of asthenozoospermia. We further successfully verified the expression trends of four representative proteins by Western blotting. Compared to a previous dataset based on label-free quantification, our results showed that most of the important proteins could be identified in the sample collected only once for each individual, providing the bases for personalized examination of seminal plasma proteins in clinic.
    DOI:  https://doi.org/10.1155/2019/2735038
  8. Nat Biomed Eng. 2019 Apr 15.
      Acute myelogenous leukaemia (AML) is associated with risk factors that are largely unknown and with a heterogeneous response to treatment. Here, we provide a comprehensive quantitative understanding of AML proteomic heterogeneities and hallmarks by using the AML Proteome Atlas, a proteomics database that we have newly derived from MetaGalaxy analyses, for the proteomic profiling of 205 patients with AML and 111 leukaemia cell lines. The analysis of the dataset revealed 154 functional patterns based on common molecular pathways, 11 constellations of correlated functional patterns and 13 signatures that stratify the outcomes of patients. We find limited overlap between proteomics data and both cytogenetics and genetic mutations. Moreover, leukaemia cell lines show limited proteomic similarities with cells from patients with AML, suggesting that a deeper focus on patient-derived samples is needed to gain disease-relevant insights. The AML Proteome Atlas provides a knowledge base for proteomic patterns in AML, a guide to leukaemia cell line selection, and a broadly applicable computational approach for quantifying the heterogeneities of protein expression and proteomic hallmarks in AML.
    DOI:  https://doi.org/10.1038/s41551-019-0387-2
  9. Cell Syst. 2019 Apr 03. pii: S2405-4712(19)30081-X. [Epub ahead of print]
      High-grade serous ovarian carcinoma (HGSC) is the most common and lethal subtype of gynecologic malignancy in women. The current standard of treatment combines cytoreductive surgery and chemotherapy. Despite the efficacy of initial treatment, most patients develop cancer recurrence, and 70% of patients die within 5 years of initial diagnosis. CA125 is the current FDA-approved biomarker used in the clinic to monitor response to treatment and recurrence, but its impact on patient survival is limited. New strategies for the discovery of HGSC biomarkers are urgently needed. Here, we describe a proteomics strategy to detect tumor-associated proteins in serum of HGSC patient-derived xenograft models. We demonstrate proof-of-concept applicability using two independent, longitudinal serum cohorts from HGSC patients.
    Keywords:  N-glycosylation; biomarker; ovarian cancer; patient-derived xenograft; serum proteomics; targeted proteomics
    DOI:  https://doi.org/10.1016/j.cels.2019.03.011
  10. PeerJ. 2019 ;7 e6699
      Mass spectrometry-based proteomics facilitate disease understanding by providing protein abundance information about disease progression. For the same type of disease studies, multiple mass spectrometry datasets may be generated. Integrating multiple mass spectrometry datasets can provide valuable information that a single dataset analysis cannot provide. In this article, we introduce a meta-analysis software, MetaMSD (Meta Analysis for Mass Spectrometry Data) that is specifically designed for mass spectrometry data. Using Stouffer's or Pearson's test, MetaMSD detects significantly more differential proteins than the analysis based on the single best experiment. We demonstrate the performance of MetaMSD using simulated data, urinary proteomic data of kidney transplant patients, and breast cancer proteomic data. Noting the common practice of performing a pilot study prior to a main study, this software will help proteomics researchers fully utilize the benefit of multiple studies (or datasets), thus optimizing biomarker discovery. MetaMSD is a command line tool that automatically outputs various graphs and differential proteins with confidence scores. It is implemented in R and is freely available for public use at https://github.com/soyoungryu/MetaMSD. The user manual and data are available at the site. The user manual is written in such a way that scientists who are not familiar with R software can use MetaMSD.
    Keywords:  Differential Proteins; Mass Spectrometry; Meta-Analysis; Proteomics
    DOI:  https://doi.org/10.7717/peerj.6699
  11. J Food Drug Anal. 2019 Apr;pii: S1021-9498(18)30157-1. [Epub ahead of print]27(2): 475-482
      Immunoglobulins (Igs) are major serum proteins which play important roles in immunity. Both untargeted and targeted proteomic workflows can be applied to investigate antigen-binding sites and the glycosylation profiles of Igs. For a more-comprehensive picture of IgG from human serum, we developed an IgG purification process and coupled the standardized method to untargeted and targeted proteomic workflows for IgG investigations. Parameters such as the type of purification beads, volume of the bead slurry, incubation conditions, and binding capacities were evaluated in this study. Only 2 μL of human serum was required for each sample. The performance of coupling the purification process to untargeted proteomics in the IgG analysis was evaluated by comparing normalized abundances of IgG subclass-specific peptides with quantification results from an ELISA. Pearson's correlation values were all >0.82. Targeted proteomic workflow was applied to serum samples from patients with autoimmune pancreatitis and from healthy controls, and the results corresponded to clinical findings that IgG4-related peptides/glycopeptides showed higher abundances in the diseased group. The developed IgG purification process is simple and requires small sample volume, and it can be coupled to targeted and untargeted proteomic workflows for clinical investigations in the future.
    Keywords:  Human serum; IgG purification; Liquid chromatography-mass spectrometry; Proteomics
    DOI:  https://doi.org/10.1016/j.jfda.2018.10.001
  12. Sci Rep. 2019 Apr 15. 9(1): 6059
      A hypercoagulable state leading to a high risk of a thrombotic event is one of the most common complications observed in β-thalassemia/HbE disease, particularly in patients who have undergone a splenectomy. However, the hypercoagulable state, as well as the molecular mechanism of this aspect of the pathogenesis of β-thalassemia/HbE, remains poorly understood. To address this issue, fifteen non-splenectomized β-thalassemia/HbE patients, 8 splenectomized β-thalassemia/HbE patients and 20 healthy volunteers were recruited to this study. Platelet activation and hypercoagulable parameters including levels of CD62P and prothrombin fragment 1 + 2 were analyzed by flow cytometry and ELISA, respectively. A proteomic analysis was conducted to compare the platelet proteome between patients and normal subjects, and the results were validated by western blot analysis. The β-thalassemia/HbE patients showed significantly higher levels of CD62P and prothrombin fragment 1 + 2 than normal subjects. The levels of platelet activation and hypercoagulation found in patients were strongly associated with splenectomy status. The platelet proteome analysis revealed 19 differential spots which were identified to be 19 platelet proteins, which included 10 cytoskeleton proteins, thrombin generation related proteins, and antioxidant enzymes. Our findings highlight markers of coagulation activation and molecular pathways known to be associated with the pathogenesis of platelet activation, the hypercoagulable state, and consequently with the thrombosis observed in β-thalassemia/HbE patients.
    DOI:  https://doi.org/10.1038/s41598-019-42432-2
  13. J Proteomics. 2019 Apr 15. pii: S1874-3919(19)30118-6. [Epub ahead of print]
      Mild olfactory dysfunction has been observed in frontotemporal dementias (FTD). However, the underlying molecular mechanisms associated to this deficit are poorly understood. We applied quantitative proteomics to analyze pathological effects on the olfactory bulb (OB) from progressive supranuclear palsy (PSP) and frontotemporal lobar degeneration (FTLD-TDP43) subjects respect to elderly non-FTD group. Our data revealed: i) a mitochondrial and calcium homeostasis impairment in PSP and ii) a disruption of protein synthesis and vesicle trafficking in FTLD-TDP43. Although differential OB proteomes clearly differ between both FTD phenotypes, functional analyses pointed out an imbalance in survival signaling in both pathologies. A common alteration of olfactory mitogen-activated protein kinases (MAPKs), calcium/calmodulin dependent protein kinase II (CAMKII), and protein kinase C (PKC) signaling pathways was observed in PSP and FTLD subjects. In contrast, a specific shut off in mitogen-activated protein kinase kinase 4 (SEK1/MKK4)/stress-activated protein kinase (SAPK) axis was exclusively observed in PSP, whereas a specific phosphoinositide-dependent protein kinase 1 (PDK1) inactivation was observed in FTLD-TDP43. In summary, our data contribute to a better understanding of the molecular mechanisms that are modulated in PSP and FTLD-TDP43 at olfactory level, highlighting cross-disease similarities and differences in the regulation of survival pathways across FTD spectrum. SIGNIFICANCE: This work reflects differential olfactory molecular disarrangements in PSP and FTLD-TDP43, two clinically similar FTD disorders, but with different neuropathological signature. Besides FTDs present mild olfactory dysfunction, our data provide basic information for understanding the implication of the OB in the pathophysiology of FTDs.
    DOI:  https://doi.org/10.1016/j.jprot.2019.04.011
  14. Biomed Pharmacother. 2019 Apr 10. pii: S0753-3322(19)31031-5. [Epub ahead of print]114 108856
      Patient survival time generally reflects the tumor progression and represents a key clinical parameter. In this study, we aimed to comprehensively characterize the prognosis-associated molecular alterations in hepatocellular carcinoma (HCC). In this study, copy-number changes, gene mutations, mRNA expression, and reverse phase protein arrays data in HCC samples profiled by The Cancer Genome Atlas (TCGA) were obtained. Tumors were then stratified into two groups based on the clinical outcome and identified genomic, transcriptomic, and proteomic traits associated to HCC prognosis. We found that several copy number amplifications and deletions can discriminate HCC patients with poor prognosis from those with better prognosis. Mutated DNAH8 showed a worse prognosis-specific pattern and correlated with a reduced disease-free survival in HCC. By integrating RNA sequencing data, we found that HCC samples with poor prognosis are consistently associated with the up-regulation of cell cycle process, such as chromosome separation, DNA replication, cytokinesis, and etc. At the proteomic level, seven proteins were significantly enriched in samples with poor prognosis, including acetylated α-Tubulin, p62-LCK-ligand, ARID1 A, MSH6, B-Raf, Cyclin B1, and PEA15. Acetylated α-Tubulin was frequently expressed in HCC tissues and acted as a promising prognostic factor for HCC. These alterations lay a foundation for developing relevant therapeutic strategies and improve our knowledge of the pathogenesis of HCC.
    Keywords:  Biomarker; Copy number variation; Hepatocellular carcinoma; Prognosis; Proteomics
    DOI:  https://doi.org/10.1016/j.biopha.2019.108856
  15. Exp Parasitol. 2019 Apr 13. pii: S0014-4894(19)30051-7. [Epub ahead of print]
      Adult Brugia malayi proteins with high potential as epidemiological markers, diagnostic and therapeutic targets, and/or vaccine candidates were revealed by using microfilaremic human sera and an immunoproteomic approach. They were HSP70, cytoplasmic intermediate filament protein, independent phosphoglycerate mutase, and enolase. Brugia malayi microfilaria-specific proteins that formed circulating immune complexes (ICs) were investigated. The IC-forming proteins were orthologues of hypothetical protein Bm1_12480, Pao retrotransposon peptidase family protein, uncoordinated protein 44, NAD-binding domain containing protein of the UDP-glucose/GDP-mannose dehydrogenase family which contained ankyrin repeat region, ZU5 domain with C-terminal death domain, C2 domain containing protein, and FLJ90013 protein of the eukaryotic membrane protein family. Antibodies to these proteins were not free in the microfilaremic sera, raising the possible role of the IC-forming proteins in an immune evasion mechanism of the circulating microfilariae to avoid antibody-mediated-host immunity. Moreover, detection of these ICs should be able to replace the inconvenient night blood sampling for microfilaria in an evaluation of efficacy of anti-microfilarial agents.
    Keywords:  Brugia malayi; Circulating immune complexes; Immunoproteomics
    DOI:  https://doi.org/10.1016/j.exppara.2019.04.005
  16. PLoS Med. 2019 Apr;16(4): e1002781
    ACS and GC6–74 cohort study groups
       BACKGROUND: A nonsputum blood test capable of predicting progression of healthy individuals to active tuberculosis (TB) before clinical symptoms manifest would allow targeted treatment to curb transmission. We aimed to develop a proteomic biomarker of risk of TB progression for ultimate translation into a point-of-care diagnostic.
    METHODS AND FINDINGS: Proteomic TB risk signatures were discovered in a longitudinal cohort of 6,363 Mycobacterium tuberculosis-infected, HIV-negative South African adolescents aged 12-18 years (68% female) who participated in the Adolescent Cohort Study (ACS) between July 6, 2005 and April 23, 2007, through either active (every 6 months) or passive follow-up over 2 years. Forty-six individuals developed microbiologically confirmed TB disease within 2 years of follow-up and were selected as progressors; 106 nonprogressors, who remained healthy, were matched to progressors. Over 3,000 human proteins were quantified in plasma with a highly multiplexed proteomic assay (SOMAscan). Three hundred sixty-one proteins of differential abundance between progressors and nonprogressors were identified. A 5-protein signature, TB Risk Model 5 (TRM5), was discovered in the ACS training set and verified by blind prediction in the ACS test set. Poor performance on samples 13-24 months before TB diagnosis motivated discovery of a second 3-protein signature, 3-protein pair-ratio (3PR) developed using an orthogonal strategy on the full ACS subcohort. Prognostic performance of both signatures was validated in an independent cohort of 1,948 HIV-negative household TB contacts from The Gambia (aged 15-60 years, 66% female), longitudinally followed up for 2 years between March 5, 2007 and October 21, 2010, sampled at baseline, month 6, and month 18. Amongst these contacts, 34 individuals progressed to microbiologically confirmed TB disease and were included as progressors, and 115 nonprogressors were included as controls. Prognostic performance of the TRM5 signature in the ACS training set was excellent within 6 months of TB diagnosis (area under the receiver operating characteristic curve [AUC] 0.96 [95% confidence interval, 0.93-0.99]) and 6-12 months (AUC 0.76 [0.65-0.87]) before TB diagnosis. TRM5 validated with an AUC of 0.66 (0.56-0.75) within 1 year of TB diagnosis in the Gambian validation cohort. The 3PR signature yielded an AUC of 0.89 (0.84-0.95) within 6 months of TB diagnosis and 0.72 (0.64-0.81) 7-12 months before TB diagnosis in the entire South African discovery cohort and validated with an AUC of 0.65 (0.55-0.75) within 1 year of TB diagnosis in the Gambian validation cohort. Signature validation may have been limited by a systematic shift in signal magnitudes generated by differences between the validation assay when compared to the discovery assay. Further validation, especially in cohorts from non-African countries, is necessary to determine how generalizable signature performance is.
    CONCLUSIONS: Both proteomic TB risk signatures predicted progression to incident TB within a year of diagnosis. To our knowledge, these are the first validated prognostic proteomic signatures. Neither meet the minimum criteria as defined in the WHO Target Product Profile for a progression test. More work is required to develop such a test for practical identification of individuals for investigation of incipient, subclinical, or active TB disease for appropriate treatment and care.
    DOI:  https://doi.org/10.1371/journal.pmed.1002781
  17. J Clin Periodontol. 2019 Apr 15.
       AIM: The aim of this study was to explore which peri-implant crevicular fluid (PICF) protein pattern is associated with the active peri-implantitis process.
    MATERIALS AND METHODS: PICF from 25 peri-implantitis sites were subjected to proteomic analysis using liquid chromatography-tandem mass spectrometry before and at 3, 6 and 12 months after treatment, to identify associations between PICF protein pattern and implant loss, bleeding on probing, pocket depth, and enamel matrix derivative (EMD) treatment.
    RESULTS: Clustering of subjects based on their 3 to 12 months PICF proteomic profiles by principal component analysis defined two major clusters. Cluster 2 differentiated from cluster 3 by 52 proteins (R2 =90%, Q2 =80%) and belonging to cluster 2 was associated with implant loss (p=0.009) and bleeding on probing (p=0.001). Cluster 3 was associated with implant survival and EMD treatment (p=0.044).
    CONCLUSION: Here we demonstrate that a specific PICF proteomic profile associates with active peri-implantitis process and implant loss. This article is protected by copyright. All rights reserved.
    Keywords:  enamel matrix derivative; implant; peri-implantitis; proteome
    DOI:  https://doi.org/10.1111/jcpe.13108
  18. Cell. 2019 Apr 10. pii: S0092-8674(19)30267-3. [Epub ahead of print]
      Breast cancer is a heterogeneous disease. Tumor cells and associated healthy cells form ecosystems that determine disease progression and response to therapy. To characterize features of breast cancer ecosystems and their associations with clinical data, we analyzed 144 human breast tumor and 50 non-tumor tissue samples using mass cytometry. The expression of 73 proteins in 26 million cells was evaluated using tumor and immune cell-centric antibody panels. Tumors displayed individuality in tumor cell composition, including phenotypic abnormalities and phenotype dominance. Relationship analyses between tumor and immune cells revealed characteristics of ecosystems related to immunosuppression and poor prognosis. High frequencies of PD-L1+ tumor-associated macrophages and exhausted T cells were found in high-grade ER+ and ER- tumors. This large-scale, single-cell atlas deepens our understanding of breast tumor ecosystems and suggests that ecosystem-based patient classification will facilitate identification of individuals for precision medicine approaches targeting the tumor and its immunoenvironment.
    Keywords:  T cell; breast cancer; immunosuppression; macrophage; mass cytometry; single-cell analysis; tumor ecosystem; tumor heterogeneity
    DOI:  https://doi.org/10.1016/j.cell.2019.03.005
  19. J Food Drug Anal. 2019 Apr;pii: S1021-9498(18)30182-0. [Epub ahead of print]27(2): 483-493
      Oral cancer with high incidence rates is occurring in many countries including in India, Pakistan, Bangladesh, Sri Lanka and Taiwan. Smoking, alcoholism, and betel nut chewing are considered to be the main risk factors for oral cancer. Further, deaths from oral cancer have increased year by year. Although several oral cancer-associated biomarkers have been reported, very few useful biomarkers have been applied for early diagnosis. Therefore, the investigation of oral cancer-specific biomarkers is urgently needed. We previously investigated N-glycomes of oral cancer cells and patient plasma. We found that both mRNA levels of FUT8 and core-fucosylated glycoproteins increase in cases of oral cancer relative to normal cases. In this study we aim to discover novel core-fucosylated glycoprotein biomarkers for oral cancer diagnosis with glycoproteomic approaches. First, forty plasma samples obtained from the Human Bioinformation Bank of NCKUH were subjected to AAL (Aleuria aurantia lectin) affinity chromatography. Core-fucosylated proteins were collected and applied for LC-MS/MS followed by electrophoresis. Fourteen proteins were identified, and expression levels of proteins in plasma were verified by western blot. Expression levels of some glycoproteins were elevated in the oral cancer group, including ceruloplasmin, haptoglobin, and leucin-rich alpha-2-glycoprotein 1 (LRG1). However, levels of some glycoproteins decreased in the cancer group, including apolipoprotein A-I (apo A-I) and apolipoprotein A-IV (apo A-IV). Via ELISA analysis, we found that apo A-IV and apo A-IV/total protein ratios were decreased in plasma accompanied with cancer stages. The LRG1/total protein ratio was found to increase while plasma levels of LRG1 were not found to differ between the oral cancer plasma and normal groups. An ROC curve analysis reveals strong diagnosis performance when combining apo A-IV levels and LRG1/total protein ratios. Taken together, apo A-IV and LRG1, given their strong performance in detecting oral cancer, can serve as useful biomarkers and may be used as a useful tool for oral cancer screening and early diagnosis.
    Keywords:  Apo A-IV; Core fucose; LRG1; Oral cancer
    DOI:  https://doi.org/10.1016/j.jfda.2018.12.008
  20. FEBS Open Bio. 2019 Apr;9(4): 736-742
      We previously reported that exclusively breastfed infants born to mothers with pregestational obesity gain less weight during the first month after birth than those born to mothers of normal pregestational weight. This issue is potentially important since lower weight gain in breastfed infants of obese mothers might increase the risk of developing later obesity. Breast milk quality and quantity, together with breastfeeding practice, possibly influence infants' feeding behavior, appetite control, and regulation of growth later in life. The issue of whether breast milk protein patterns from obese mothers differ in composition from those of non-obese mothers remains largely unexplored. Here, we established a breast milk proteomic pattern that discriminates obese mothers and infants with delayed weight gain at 1 month after birth from normal-weight mothers with infants of the same age and with normal weight gain. Obese mothers were matched to normal-weight mothers (n = 26; body mass index 33.5 ± 3.2 vs 21.5 ± 1.5 kg·m-2). The mean weight gain of infants in the obese group at 1 month after birth was 430.8 g lower than that of the infants in the control group. Analysis of the breast milk delipidized fraction by surface-enhanced laser desorption/ionization on CM10 and Q10 arrays was followed by MS-assisted purification and LC-MS/MS microsequencing of a selected biomarker. We identified 15 candidate protein biomarkers, seven of which were overexpressed in the obese group and eight in the normal-weight group. One of the most significant candidate biomarkers, overexpressed in the obese group, was identified as a fragment of the sixth extracellular domain of the polymeric immunoglobulin receptor. Further structural identification of these candidate biomarkers and their validation in clinical assays may facilitate the development of a predictive immunoassay.
    Keywords:  SELDI biomarker; breast milk; infant weight gain; maternal obesity; pIgR
    DOI:  https://doi.org/10.1002/2211-5463.12610
  21. Contemp Clin Trials Commun. 2019 Jun;14 100345
    PREDICTS study team
       Purpose: The etiology of Inflammatory Bowel Disease (IBD) remains currently unknown but evidence would suggest that it results from a complex interplay between genetic susceptibility genes, the intestinal microbiome and the environment, resulting in an increased response towards microbial and self-antigens, followed by the development of pre-clinical intestinal inflammation as a precursor to overt clinical disease. Efforts are needed to provide insights into the characterization of the disease, the possible prediction of complications, and the detection of a pre-clinical disease state where, through early screening and intervention, disease course can be reversed, attenuated or even prevented. A consortium of academic, industry and governmental organization investigators initiated this study to enable an assessment of pre-disease biomarkers in patients newly diagnosed with Crohn's disease (CD) and ulcerative colitis (UC).
    Participants: A retrospective cohort of 1000 UC and 1000 CD cases with 500 matched controls was drawn from an active duty US military personnel population with relevant inclusion criteria with three associated pre-disease and a single disease-associated archived serum samples.
    Findings to date: The PREDICTS study has been established as a biorepository platform study to perform novel discovery and analysis efforts in the field of IBD and proteomic systems biology.
    Future plans: This study is poised to enable the assessment of novel biomarkers within the serum compartment to be analyzed with the goal of identifying pre-disease signals that ultimately predict disease risk, and further elucidate disease pathogenesis in the early stages of the disease process, and identify novel exposures that increase disease risk.
    DOI:  https://doi.org/10.1016/j.conctc.2019.100345
  22. Expert Rev Proteomics. 2019 Apr 18.
       INTRODUCTION: Plasma proteomics has been utilised extensively for studies that investigate various disease settings (e.g. cardiovascular disease), as well as to monitor the effect of pharmaceuticals on the plasma proteome (e.g. chemotherapy). However, plasma proteomic studies focusing on children represent a very small proportion of the plasma proteomics studies completed to date. Early disease detection and prevention is critical in paediatrics, as children must live with the disease outcomes for many years and often carry negative outcomes into adulthood. Paediatrics represents an area of plasma proteomics that is about to undergo a significant expansion. Areas covered: This review is based on a PubMed search focusing on 5 keywords, plasma, biomarkers, paediatric, proteomics and children. It is a comprehensive summary of plasma proteomic studies specific to the paediatric patient and discusses aspects such as the clinical setting, sample size, methodological approaches and outlines the significance of the findings. Expert commentary: Plasma proteomics is expanding significantly as a result of major advancements in proteomic technology. This is in synergy with the growing focus on true early disease detection, and prevention in early life. We are about to see a new era of advanced medical science built from paediatric proteomics.
    Keywords:  biomarkers; children; paediatric; plasma; proteomics
    DOI:  https://doi.org/10.1080/14789450.2019.1608186
  23. BMC Cancer. 2019 Apr 18. 19(1): 365
       BACKGROUND: Worldwide, breast cancer is the main cause of cancer mortality in women. Most cases originate in mammary ductal cells that produce the nipple aspirate fluid (NAF). In cancer patients, this secretome contains proteins associated with the tumor microenvironment. NAF studies are challenging because of inter-individual variability. We introduced a paired-proteomic shotgun strategy that relies on NAF analysis from both breasts of patients with unilateral breast cancer and extended PatternLab for Proteomics software to take advantage of this setup.
    METHODS: The software is based on a peptide-centric approach and uses the binomial distribution to attribute a probability for each peptide as being linked to the disease; these probabilities are propagated to a final protein p-value according to the Stouffer's Z-score method.
    RESULTS: A total of 1227 proteins were identified and quantified, of which 87 were differentially abundant, being mainly involved in glycolysis (Warburg effect) and immune system activation (activated stroma). Additionally, in the estrogen receptor-positive subgroup, proteins related to the regulation of insulin-like growth factor transport and platelet degranulation displayed higher abundance, confirming the presence of a proliferative microenvironment.
    CONCLUSIONS: We debuted a differential bioinformatics workflow for the proteomic analysis of NAF, validating this secretome as a treasure-trove for studying a paired-organ cancer type.
    Keywords:  Breast cancer; Breast secretion; Liquid biopsy; Nipple aspirate fluid; Paired analysis; Proteome
    DOI:  https://doi.org/10.1186/s12885-019-5547-y
  24. Urolithiasis. 2019 Apr 16.
      Urine proteins are thought to control calcium oxalate stone formation, but over 1000 proteins have been reported in stone matrix obscuring their relative importance. Proteins critical to stone formation should be present at increased relative abundance in stone matrix compared to urine, so quantitative protein distribution data were obtained for stone matrix compared to prior urine proteome data. Matrix proteins were isolated from eight stones (> 90% calcium oxalate content) by crystal dissolution and further purified by ultradiafiltration (> 10 kDa membrane). Proteomic analyses were performed using label-free spectral counting tandem mass spectrometry, followed by stringent filtering. The average matrix proteome was compared to the average urine proteome observed in random urine samples from 25 calcium oxalate stone formers reported previously. Five proteins were prominently enriched in matrix, accounting for a mass fraction of > 30% of matrix protein, but only 3% of urine protein. Many highly abundant urinary proteins, like albumin and uromodulin, were present in matrix at reduced relative abundance compared to urine, likely indicating non-selective inclusion in matrix. Furthermore, grouping proteins by isoelectric point demonstrated that the stone matrix proteome was highly enriched in both strongly anionic (i.e., osteopontin) and strongly cationic (i.e., histone) proteins, most of which are normally found in intracellular or nuclear compartments. The fact that highly anionic and highly cationic proteins aggregate at low concentrations and these aggregates can induce crystal aggregation suggests that protein aggregation may facilitate calcium oxalate stone formation, while cell injury processes are implicated by the presence of many intracellular proteins.
    Keywords:  Calcium oxalate; Kidney calculi; Nephrolithiasis; Urine proteome
    DOI:  https://doi.org/10.1007/s00240-019-01131-3
  25. Front Cell Infect Microbiol. 2019 ;9 65
      Tuberculosis (TB) is still a serious threat to human health which is caused by mycobacterium tuberculosis (Mtb). The main reason for failure to eliminate TB is lack of clearly understanding the molecular mechanism of Mtb pathogenesis. Determining human Mtb-interacting proteins enables us to characterize the mechanism and identify potential molecular targets for TB diagnosis and treatment. However, experimentally systematic Mtb interactors are not readily available. In this study, we performed an unbiased, comprehensive two-way proteome microarray based approach to systematically screen global human Mtb interactors and determine the binding partners of Mtb effectors. Our results, for the first time, screened 84 potential human Mtb interactors. Bioinformatic analysis further highlighted these protein candidates might engage in a wide range of cellular functions such as activation of DNA endogenous promoters, transcription of DNA/RNA and necrosis, as well as immune-related signaling pathways. Then, using Mtb proteome microarray followed His tagged pull-down assay and Co-IP, we identified one interacting partner (Rv0577) for the protein candidate NRF1 and three binding partners (Rv0577, Rv2117, Rv2423) for SMAD2, respectively. This study gives new insights into the profile of global Mtb interactors potentially involved in Mtb pathogenesis and demonstrates a powerful strategy in the discovery of Mtb effectors.
    Keywords:  Mtb proteome microarray; NRF1; SMAD2; host-pathogen interaction; human proteome microarray
    DOI:  https://doi.org/10.3389/fcimb.2019.00065
  26. Parkinsonism Relat Disord. 2019 Apr 11. pii: S1353-8020(19)30205-6. [Epub ahead of print]
       INTRODUCTION: As current clinical diagnostic protocols for Parkinson's disease (PD) may be prone to inaccuracies there is a need to identify and validate molecular biomarkers, such as circulating microRNAs, which will complement current practices and increase diagnostic accuracy. This study identifies, verifies and validates combinatory serum microRNA signatures as diagnostic classifiers of PD across different patient cohorts.
    METHODS: 370 PD (drug naïve) and control serum samples from the Norwegian ParkWest study were used for identification and verification of differential microRNA levels in PD which were validated in a blind study using 64 NY Parkinsonism in UMeå (NYPUM) study serum samples and tested for specificity in 48 Dementia Study of Western Norway (DemWest) study Alzheimer's disease (AD) serum samples using miRNA-microarrays, and quantitative (q) RT-PCR. Proteomic approaches identified potential molecular targets for these microRNAs.
    RESULTS: Using Affymetrix GeneChip® miRNA 4.0 arrays and qRT-PCR we comprehensively analyzed serum microRNA levels and found that the microRNA (PARKmiR)-combinations, hsa-miR-335-5p/hsa-miR-3613-3p (95% CI, 0.87-0.94), hsa-miR-335-5p/hsa-miR-6865-3p (95% CI, 0.87-0.93), and miR-335-5p/miR-3613-3p/miR-6865-3p (95% CI, 0.87-0.94) show a high degree of discriminatory accuracy (AUC 0.9-1.0). The PARKmiR signatures were validated in an independent PD cohort (AUC ≤ 0.71) and analysis in AD serum samples showed PARKmiR signature specificity to PD. Proteomic analyses showed that the PARKmiRs regulate key PD-associated proteins, including alpha-synuclein and Leucine Rich Repeat Kinase 2.
    CONCLUSIONS: Our study has identified and validated unique miRNA serum signatures that represent PD classifiers, which may complement and increase the accuracy of current diagnostic protocols.
    Keywords:  Alzheimer's disease; Biomarker; Parkinson's disease; microRNA
    DOI:  https://doi.org/10.1016/j.parkreldis.2019.04.010
  27. Proc Natl Acad Sci U S A. 2019 Apr 19. pii: 201818347. [Epub ahead of print]
      Dysregulation of signaling pathways in multiple sclerosis (MS) can be analyzed by phosphoproteomics in peripheral blood mononuclear cells (PBMCs). We performed in vitro kinetic assays on PBMCs in 195 MS patients and 60 matched controls and quantified the phosphorylation of 17 kinases using xMAP assays. Phosphoprotein levels were tested for association with genetic susceptibility by typing 112 single-nucleotide polymorphisms (SNPs) associated with MS susceptibility. We found increased phosphorylation of MP2K1 in MS patients relative to the controls. Moreover, we identified one SNP located in the PHDGH gene and another on IRF8 gene that were associated with MP2K1 phosphorylation levels, providing a first clue on how this MS risk gene may act. The analyses in patients treated with disease-modifying drugs identified the phosphorylation of each receptor's downstream kinases. Finally, using flow cytometry, we detected in MS patients increased STAT1, STAT3, TF65, and HSPB1 phosphorylation in CD19+ cells. These findings indicate the activation of cell survival and proliferation (MAPK), and proinflammatory (STAT) pathways in the immune cells of MS patients, primarily in B cells. The changes in the activation of these kinases suggest that these pathways may represent therapeutic targets for modulation by kinase inhibitors.
    Keywords:  B cells; autoimmunity; multiple sclerosis; phosphoproteomics; signaling pathways
    DOI:  https://doi.org/10.1073/pnas.1818347116
  28. Sci Transl Med. 2019 Apr 17. pii: eaav0936. [Epub ahead of print]11(488):
      Eradicating triple-negative breast cancer (TNBC) resistant to neoadjuvant chemotherapy (NACT) is a critical unmet clinical need. In this study, patient-derived xenograft (PDX) models of treatment-naïve TNBC and serial biopsies from TNBC patients undergoing NACT were used to elucidate mechanisms of chemoresistance in the neoadjuvant setting. Barcode-mediated clonal tracking and genomic sequencing of PDX tumors revealed that residual tumors remaining after treatment with standard frontline chemotherapies, doxorubicin (Adriamycin) combined with cyclophosphamide (AC), maintained the subclonal architecture of untreated tumors, yet their transcriptomes, proteomes, and histologic features were distinct from those of untreated tumors. Once treatment was halted, residual tumors gave rise to AC-sensitive tumors with similar transcriptomes, proteomes, and histological features to those of untreated tumors. Together, these results demonstrated that tumors can adopt a reversible drug-tolerant state that does not involve clonal selection as an AC resistance mechanism. Serial biopsies obtained from patients with TNBC undergoing NACT revealed similar histologic changes and maintenance of stable subclonal architecture, demonstrating that AC-treated PDXs capture molecular features characteristic of human TNBC chemoresistance. Last, pharmacologic inhibition of oxidative phosphorylation using an inhibitor currently in phase 1 clinical development delayed residual tumor regrowth. Thus, AC resistance in treatment-naïve TNBC can be mediated by nonselective mechanisms that confer a reversible chemotherapy-tolerant state with targetable vulnerabilities.
    DOI:  https://doi.org/10.1126/scitranslmed.aav0936
  29. Thyroid. 2019 Apr 16.
       BACKGROUND: Energetic metabolism is described to be deregulated in cancer and the Warburg effect is presented as a major hallmark. Recently, cellular heterogeneity in tumors and tumor microenvironment have been recognized to play an important role in several metabolic pathways in cancer. However, their contribution to papillary thyroid cancer (PTC) development and metabolism is still poorly described.
    METHODS: We performed a proteomic analysis of 5 PTC and investigated the cellular distribution of several upregulated metabolic proteins in the cancer and in the stromal cells of PTC.
    RESULTS: MS/MS analysis revealed the upregulation of many metabolism-related proteins, among which pyruvate carboxylase. Pyruvate carboxylase knockdown in thyroid cell lines alters their proliferative and motility capacities, and measurements of oxygen consumption rates showed that this enzyme is involved in the replenishment of the TCA cycle. Immunostainings of several upregulated metabolic proteins showed that thyroid cancer cells have an increased mitochondrial oxidative metabolism compared to stromal cells.
    CONCLUSION: PTC have a very active TCA cycle, continuously replenished by a pyruvate carboxylase mediated anaplerosis. This is specifically observed in the tumor cells.
    DOI:  https://doi.org/10.1089/thy.2018.0435