bims-prodis Biomed News
on Proteomics in disease
Issue of 2019‒03‒17
thirty-six papers selected by
Nancy Gough
Bioserendipity
  1. Combined Tissue-Fluid Proteomics to Unravel Phenotypic Variability in Amyotrophic Lateral Sclerosis.
  2. Comprehensive lipidomic, metabolomic and proteomic profiling reveals the role of immune system in vitiligo.
  3. Analyzing Cerebrospinal Fluid Proteomes to Characterize Central Nervous System Disorders: A Highly Automated Mass Spectrometry-Based Pipeline for Biomarker Discovery.
  4. Circulating proteomic panels for diagnosis and risk stratification of acute-on-chronic liver failure in patients with viral hepatitis B.
  5. Integrated analysis of relapsed B-cell precursor Acute Lymphoblastic Leukemia identifies subtype-specific cytokine and metabolic signatures.
  6. LC‑MS/MS proteomic analysis revealed novel associations of 37 proteins with T2DM and notable upregulation of immunoglobulins.
  7. A multiplex immunoassay of serum biomarkers for the detection of uveal melanoma.
  8. Serological proteomic screening and evaluation of a recombinant egg antigen for the diagnosis of low-intensity Schistosoma mansoni infections in endemic area in Brazil.
  9. Discovering Protein Biomarkers from Clinical Peripheral Blood Mononuclear Cells Using Data-Independent Acquisition Mass Spectrometry.
  10. Fatigue in Sjögren's Syndrome: A Search for Biomarkers and Treatment Targets.
  11. Development of a Highly Multiplexed SRM Assay for Biomarker Discovery in Formalin-Fixed Paraffin-Embedded Tissues.
  12. Proteomic analysis of tear fluid reveals disease-specific patterns in patients with Parkinson's disease - A pilot study.
  13. Intact Protein Analysis by LC-MS for Characterizing Biomarkers in Cerebrospinal Fluid.
  14. Proteomic profiling of urine: implications for lupus nephritis.
  15. Proteomics profiling of plasma exosomes in epithelial ovarian cancer: A potential role in the coagulation cascade, diagnosis and prognosis.
  16. Protein Biomarker Discovery Using Human Blood Plasma Microparticles.
  17. Protein disulfide isomerase plasma levels in healthy humans reveal proteomic signatures involved in contrasting endothelial phenotypes.
  18. Fluorescence Proteomic Technology to Analyze Peripheral Blood Mononuclear Cells in Chronic Chagas Disease.
  19. Serum biomarker panels for the diagnosis of gastric cancer.
  20. Systems biology approaches to investigating the roles of extracellular vesicles in human diseases.
  21. Altered proteome of high-density lipoproteins from paediatric acute lymphoblastic leukemia survivors.
  22. Inflammatory effects of in vivo glycated albumin from cardiovascular patients.
  23. High-throughput epitope profiling of antibodies in the plasma of Alzheimer's disease patients using random peptide microarrays.
  24. Decorin as a prognostic biomarker in patients with malignant peripheral nerve sheath tumors.
  25. Development of parallel reaction monitoring assays for cerebrospinal fluid proteins associated with Alzheimer's disease.
  26. Mass spectrometry-based proteome profiling of extracellular vesicles and their roles in cancer biology.
  27. Identification of a novel biomarker-CCL5 using antibody microarray for colorectal cancer.
  28. Pre- and Post-analytical Factors in Biomarker Discovery.
  29. Functional proteomic atlas of HIV infection in primary human CD4+ T cells.
  30. Cytometric Bead Array (CBA) for Measuring Cytokine Levels in Chagas Disease Patients.
  31. A comparison between the abdominal and femoral adipose tissue proteome of overweight and obese women.
  32. Graphically encoded suspension array for multiplex immunoassay and quantification of autoimmune biomarkers in patient sera.
  33. Lys-C/Trypsin Tandem-Digestion Protocol for Gel-Free Proteomic Analysis of Colon Biopsies.
  34. Phase II study of stereotactic radiosurgery for the treatment of patients with oligoprogression on erlotinib.
  35. Development of treatment options for Chinese patients with advanced squamous cell lung cancer: focus on afatinib.
  36. Glucocorticoids promote breast cancer metastasis.
  1. Sci Rep. 2019 Mar 14. 9(1): 4478
    Combined Tissue-Fluid Proteomics to Unravel Phenotypic Variability in Amyotrophic Lateral Sclerosis.
    Emanuela Leoni, Michael Bremang, Vikram Mitra, Irene Zubiri, Stephan Jung, Ching-Hua Lu, Rocco Adiutori, Vittoria Lombardi, Claire Russell, Sasa Koncarevic, Malcolm Ward, Ian Pike, Andrea Malaspina.
      The lack of biomarkers for early diagnosis, clinical stratification and to monitor treatment response has hampered the development of new therapies for amyotrophic lateral sclerosis (ALS), a clinically heterogeneous neurodegenerative disorder with a variable site of disease initiation and rate of progression. To identify new biomarkers and therapeutic targets, two separate proteomic workflows were applied to study the immunological response and the plasma/brain proteome in phenotypic variants of ALS. Conventional multiplex (TMT) proteomic analysis of peripheral blood mononuclear cells (PBMCs) was performed alongside a recently introduced method to profile neuronal-derived proteins in plasma using brain tissue-enhanced isobaric tagging (TMTcalibrator). The combined proteomic analysis allowed the detection of regulated proteins linked to ALS pathogenesis (RNA-binding protein FUS, superoxide dismutase Cu-Zn and neurofilaments light polypeptide) alongside newly identified candidate biomarkers (myosin-9, fructose-bisphosphate aldolase and plectin). In line with the proteomic results, orthogonal immunodetection showed changes in neurofilaments and ApoE in bulbar versus limb onset fast progressing ALS. Functional analysis of significantly regulated features showed enrichment of pathways involved in regulation of the immune response, Rho family GTPases, semaphorin and integrin signalling. Our cross-phenotype investigation of PBMCs and plasma/brain proteins provides a more sensitive biomarker exploratory platform than conventional case-control studies in a single matrix. The reported regulated proteins may represent novel biomarker candidates and potentially druggable targets.
    DOI:  https://doi.org/10.1038/s41598-019-40632-4
  2. Clin Exp Dermatol. 2019 Mar 12.
    Comprehensive lipidomic, metabolomic and proteomic profiling reveals the role of immune system in vitiligo.
    L Liang, Y Li, X Tian, J Zhou, L Zhong.
      PURPOSE: Vitiligo is a common depigmentation disorder resulting from destruction of melanocytes, with both genetic and environmental influences. Although genomic analyses have been performed to investigate the pathogenesis of vitiligo, the lipidomics, metabolomics and proteomics of serum have not been reported and the role of small molecules and serum proteins in vitiligo remains unknown.EXPERIMENTAL DESIGN: To study the metabolite and protein profiles in vitiligo and healthy controls, plasma samples from 60 participants (29 vitiligo and 31 healthy) were analyzed. Untargeted lipidomics, metabolomics and iTRAQ-based proteomics were performed using ultra-performance liquid chromatography-tandem mass spectrometry. In addition, to validate differentially expressed metabolites in vitiligo patients, plasma enzyme-linked immunosorbent assay was performed.
    RESULTS: We identified differential expression of several metabolites and proteins involved in the immune system. Among these metabolites and proteins, lysophosphatidylcholine (lysoPC, LPC), platelet-activating factor (PAF), sn-glycerol-3-phosphocholine (GPC), succinic acid, C-X-C motif chemokine 4 (CXCL4) and CXCL7 were significantly elevated in the plasma of vitiligo patients, while aspartate was down-regulated.
    CONCLUSIONS AND CLINICAL RELEVANCE: Our study characterizes several serum metabolites and proteins which could be potential candidate biomarkers in vitiligo and provides a comprehensive insight into the role of immune system and aspartate metabolism in vitiligo. This article is protected by copyright. All rights reserved.
    Keywords:  c; immune system; lipidomics; metabolomics; vitiligo
    DOI:  https://doi.org/10.1111/ced.13961
  3. Methods Mol Biol. 2019 ;1959 89-112
    Analyzing Cerebrospinal Fluid Proteomes to Characterize Central Nervous System Disorders: A Highly Automated Mass Spectrometry-Based Pipeline for Biomarker Discovery.
    Antonio Núñez Galindo, Charlotte Macron, Ornella Cominetti, Loïc Dayon.
      Over the past decade, liquid chromatography tandem mass spectrometry (LC MS/MS)-based workflows become standard for biomarker discovery in proteomics. These medium- to high-throughput (in terms of protein content) profiling approaches have been applied to clinical research. As a result, human proteomes have been characterized to a greater extent than ever before. However, proteomics in clinical research and biomarker discovery studies has generally been performed with small cohorts of subjects (or pooled samples from larger cohorts). This is problematic, as when aiming to identify novel biomarkers, small studies suffer from inherent and important limitations, as a result of the reduced biological diversity and representativity of human populations. Consequently, larger-scale proteomics will be key to delivering robust biomarker candidates and enabling translation to clinical practice.Cerebrospinal fluid (CSF) is a highly clinically relevant body fluid, and an important source of potential biomarkers for brain-associated damage, such as that induced by traumatic brain injury and stroke, and brain diseases, such as Alzheimer's disease and Parkinson's disease. We have developed a scalable automated proteomic pipeline (ASAP2) for biomarker discovery. This workflow is compatible with larger clinical research studies in terms of sample size, while still allowing several hundred proteins to be measured in CSF by MS. In this chapter, we describe the whole proteomic workflow to analyze human CSF. We further illustrate our protocol with some examples from an analysis of hundreds of human CSF samples, in the specific context of biomarker discovery to characterize central nervous system disorders.
    Keywords:  Alzheimer; Automation; Biomarker; Brain; CSF; Clinical research; Depletion; Human; Isobaric tagging; Large scale; Mass spectrometry
    DOI:  https://doi.org/10.1007/978-1-4939-9164-8_6
  4. Theranostics. 2019 ;9(4): 1200-1214
    Circulating proteomic panels for diagnosis and risk stratification of acute-on-chronic liver failure in patients with viral hepatitis B.
    Zeyu Sun, Xiaoli Liu, Daxian Wu, Hainv Gao, Jing Jiang, Ying Yang, Jie Wu, Qikang Gao, Jie Wang, Zhengyi Jiang, Youping Xu, Xiao Xu, Lanjuan Li.
      Chronic HBV infection (CHB) can lead to acute-on-chronic liver failure (HBV-ACLF) characterized by high mortality. This study aimed to reveal ACLF-related proteomic alterations, from which protein based diagnostic and prognostic scores for HBV-ACLF were developed. Methods: Ten healthy controls, 16 CHB, and 19 HBV-ACLF according to COSSH (Chinese group on the study of severe hepatitis B) criteria were enrolled to obtain the comprehensive proteomic portrait related to HBV-ACLF initiation and progression. Potential markers of HBV-ACLF were further selected based on organ specificity and functionality. An additional cohort included 77 healthy controls, 92 CHB and 71 HBV-ACLF was used to validate the proteomic signatures via targeted proteomic assays. Results: Significant losses of plasma proteins related to multiple functional clusters, including fatty acid metabolism/transport, immuno-response, complement and coagulation systems, were observed in ACLF patients. In the validation study, 28 proteins were confirmed able to separate ACLF, CHB patients. A diagnostic classifier P4 (APOC3, HRG, TF, KLKB1) was built to differentiate ACLF from CHB with high accuracy (auROC = 0.956). A prognostic model P8 (GC, HRG, HPR, SERPINA6, age, NEU, INR and total protein) was built to distinguish survivors from non-survivors in 28 and 90-days follow-up (auROC = 0.882, 0.871), and to stratify ACLF patients into risk subgroups showing significant difference in 28 and 90-days mortality (HR=7.77, 7.45, both P<0.0001). In addition, P8 score correlated with ACLF grades and numbers of extra-hepatic organ failures in ACLF patients, and was able to predict ACLF-associated coagulation and brain failure within 90 days (auROC = 0.815, 0.842). Conclusions: Proteomic signatures developed in this study reflected the deficiency of key hematological functions in HBV-ACLF patients, and show potential for HBV-ACLF diagnosis and risk prediction in complementary to current clinical based parameters.
    Keywords:  HBV-ACLF; biomarkers; mass spectrometry; proteomics
    DOI:  https://doi.org/10.7150/thno.31991
  5. Sci Rep. 2019 Mar 12. 9(1): 4188
    Integrated analysis of relapsed B-cell precursor Acute Lymphoblastic Leukemia identifies subtype-specific cytokine and metabolic signatures.
    Michael P Schroeder, Lorenz Bastian, Cornelia Eckert, Nicola Gökbuget, Alva Rani James, Jutta Ortiz Tanchez, Cornelia Schlee, Konstandina Isaakidis, Björn Häupl, Katharina Baum, Oscar Arturo Migueles Lozano, Khouloud Kouidri, Kuan-Ting Pan, Henning Urlaub, Stefan Schwartz, Thomas Burmeister, Arend von Stackelberg, Dieter Hoelzer, Heike Pfeiffer, Michael A Rieger, Stefanie Göllner, Thomas Oellerich, Martin Horstman, Martin Schrappe, Jana Wolf, Renate Kirschner-Schwabe, Monika Brüggemann, Carsten Müller-Tidow, Hubert Serve, Martin Neumann, Claudia D Baldus.
      Recent efforts reclassified B-Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL) into more refined subtypes. Nevertheless, outcomes of relapsed BCP-ALL remain unsatisfactory, particularly in adult patients where the molecular basis of relapse is still poorly understood. To elucidate the evolution of relapse in BCP-ALL, we established a comprehensive multi-omics dataset including DNA-sequencing, RNA-sequencing, DNA methylation array and proteome MASS-spec data from matched diagnosis and relapse samples of BCP-ALL patients (n = 50) including the subtypes DUX4, Ph-like and two aneuploid subtypes. Relapse-specific alterations were enriched for chromatin modifiers, nucleotide and steroid metabolism including the novel candidates FPGS, AGBL and ZNF483. The proteome expression analysis unraveled deregulation of metabolic pathways at relapse including the key proteins G6PD, TKT, GPI and PGD. Moreover, we identified a novel relapse-specific gene signature specific for DUX4 BCP-ALL patients highlighting chemotaxis and cytokine environment as a possible driver event at relapse. This study presents novel insights at distinct molecular levels of relapsed BCP-ALL based on a comprehensive multi-omics integrated data set including a valuable proteomics data set. The relapse specific aberrations reveal metabolic signatures on genomic and proteomic levels in BCP-ALL relapse. Furthermore, the chemokine expression signature in DUX4 relapse underscores the distinct status of DUX4-fusion BCP-ALL.
    DOI:  https://doi.org/10.1038/s41598-019-40786-1
  6. Int J Mol Med. 2019 Mar 08.
    LC‑MS/MS proteomic analysis revealed novel associations of 37 proteins with T2DM and notable upregulation of immunoglobulins.
    Rabab Asghar Abdulwahab, Ayodele Alaiya, Zakia Shinwari, Abdul Ameer A Allaith, Hayder A Giha.
      Type 2 diabetes mellitus (T2DM) is a disease associated with a number of metabolic disturbances, including protein metabolism. In the present study, blood samples were obtained from Bahraini subjects, including 6 patients with T2DM and 6 age‑ and sex‑matched, non‑diabetic, healthy controls. Depleted and non‑depleted sera were prepared from the collected blood, and the global protein expression changes were evaluated by liquid chromatography tandem mass spectrometry. Only significantly and markedly differentially‑expressed proteins (P<0.05, analysis of variance; maximum fold change ≥1.5) were considered as candidate proteins for informatics analysis. Accordingly, a total of 62 proteins were identified to be differentially expressed in T2DM, compared with control subjects, and they were grouped functionally into 16 classes of proteins. The largest class was that of the immune‑associated proteins. Additionally, ~25 of these proteins (40%) had previously been associated with DM; however, the association of the other 37 proteins with T2DM was a novel observation. The majority of the identified proteins were upregulated in T2DM. The identified proteins could be involved in the pathogenesis of the disease or serve as disease biomarkers. Further validation of the identified proteins in a large study cohort is required, in order to fully access their potential clinical usefulness.
    DOI:  https://doi.org/10.3892/ijmm.2019.4127
  7. Clin Proteomics. 2019 ;16 10
    A multiplex immunoassay of serum biomarkers for the detection of uveal melanoma.
    Jin Song, Shannath L Merbs, Lori J Sokoll, Daniel W Chan, Zhen Zhang.
      Background: Approximately 50% of uveal melanoma (UM) patients develop metastases preferentially in the liver leading to death within 15 months. Currently, there is no effective treatment for metastatic UM, in part because the tumor burden is typically high when liver metastases are detected through abnormal liver function tests (LFTs) or imaging studies. The use of LFTs results followed by diagnostic tests has high specificity and predictive values but low sensitivity, and better tests are needed for early diagnosis of the primary tumor as well as its metastatic spread. To evaluate serum biomarkers for the early detection of UM, multiplex immunoassays were developed.Methods: Magnetic bead-based multiplex immunoassays were developed for the selected serum biomarkers using a Bio-Plex 200 system. The dynamic ranges, lower limits of detection and quantification, cross-reactivity, and intra- and inter-assay precision were assessed. All proteins were analyzed in sera of 48 patients diagnosed with UM (14 metastatic, 9 disease-free (DF) ≥ 5 years, 25 unknown) and 36 healthy controls. The performance of the biomarkers was evaluated individually and in combination for their ability to detect UM.
    Results: A 7-plex immunoassay of OPN, MIA, CEACAM-1, MIC-1, SPON1, POSTN and HSP27 was developed with negligible cross-reactivity, recovery of 84-105%, and intra-assay and inter-assay precision of 2.3-7.5% or 2.8-20.8%, respectively. Logistic regression identified a two-marker panel of HSP27 and OPN that significantly improved the individual biomarker performance in discriminating UM from healthy controls. The improved discrimination of a two-marker panel of MIA and MIC-1 was also observed between metastatic UM and DF, however not statistically significant due to the small sample size.
    Conclusions: The multiplex immunoassay provides sufficient analytical performance to evaluate serum biomarkers that complement each other in detection of UM, and warrants further validation with a larger number of patient samples.
    Keywords:  Biomarker; Immunoassay; Multiplex; Serum; Uveal melanoma
    DOI:  https://doi.org/10.1186/s12014-019-9230-8
  8. PLoS Negl Trop Dis. 2019 Mar 14. 13(3): e0006974
    Serological proteomic screening and evaluation of a recombinant egg antigen for the diagnosis of low-intensity Schistosoma mansoni infections in endemic area in Brazil.
    Vanessa Silva-Moraes, Lisa Marie Shollenberger, William Castro-Borges, Ana Lucia Teles Rabello, Donald A Harn, Lia Carolina Soares Medeiros, Wander de Jesus Jeremias, Liliane Maria Vidal Siqueira, Caroline Stephane Salviano Pereira, Maria Luysa Camargos Pedrosa, Nathalie Bonatti Franco Almeida, Aureo Almeida, Jose Roberto Lambertucci, Nídia Francisca de Figueiredo Carneiro, Paulo Marcos Zech Coelho, Rafaella Fortini Queiroz Grenfell.
      BACKGROUND: Despite decades of use of control programs, schistosomiasis remains a global public health problem. To further reduce prevalence and intensity of infection, or to achieve the goal of elimination in low-endemic areas, there needs to be better diagnostic tools to detect low-intensity infections in low-endemic areas in Brazil. The rationale for development of new diagnostic tools is that the current standard test Kato-Katz (KK) is not sensitive enough to detect low-intensity infections in low-endemic areas. In order to develop new diagnostic tools, we employed a proteomics approach to identify biomarkers associated with schistosome-specific immune responses in hopes of developing sensitive and specific new methods for immunodiagnosis.METHODS AND FINDINGS: Immunoproteomic analyses were performed on egg extracts of Schistosoma mansoni using pooled sera from infected or non-infected individuals from a low-endemic area of Brazil. Cross reactivity with other soil-transmitted helminths (STH) was determined using pooled sera from individuals uniquely infected with different helminths. Using this approach, we identified 23 targets recognized by schistosome acute and chronic sera samples. To identify immunoreactive targets that were likely glycan epitopes, we compared these targets to the immunoreactivity of spots treated with sodium metaperiodate oxidation of egg extract. This treatment yielded 12/23 spots maintaining immunoreactivity, suggesting that they were protein epitopes. From these 12 spots, 11 spots cross-reacted with sera from individuals infected with other STH and 10 spots cross-reacted with the negative control group. Spot number 5 was exclusively immunoreactive with sera from S. mansoni-infected groups in native and deglycosylated conditions and corresponds to Major Egg Antigen (MEA). We expressed MEA as a recombinant protein and showed a similar recognition pattern to that of the native protein via western blot. IgG-ELISA gave a sensitivity of 87.10% and specificity of 89.09% represented by area under the ROC curve of 0.95. IgG-ELISA performed better than the conventional KK (2 slides), identifying 56/64 cases harboring 1-10 eggs per gram of feces that were undiagnosed by KK parasitological technique.
    CONCLUSIONS: The serological proteome approach was able to identify a new diagnostic candidate. The recombinant egg antigen provided good performance in IgG-ELISA to detect individuals with extreme low-intensity infections (1 egg per gram of feces). Therefore, the IgG-ELISA using this newly identified recombinant MEA can be a useful tool combined with other techniques in low-endemic areas to determine the true prevalence of schistosome infection that is underestimated by the KK method. Further, to overcome the complexity of ELISA in the field, a second generation of antibody-based rapid diagnostic tests (RDT) can be developed.
    DOI:  https://doi.org/10.1371/journal.pntd.0006974
  9. Methods Mol Biol. 2019 ;1959 151-161
    Discovering Protein Biomarkers from Clinical Peripheral Blood Mononuclear Cells Using Data-Independent Acquisition Mass Spectrometry.
    Xin Ku, Wei Yan.
      Global proteomics analyses are traditionally performed in data-dependent acquisition (DDA) mode, which results in inadequate reproducibility across large sample cohorts due to the under-sampling inherent to shotgun proteomics. Recently, data-independent acquisition (DIA) strategies were introduced to allow reproducible detection and quantification of thousands of proteins with consistent sensitivity across samples. Here, we present an approach to analyze changes to the protein network in human peripheral blood mononuclear cells (PBMCs) from clinical blood samples, using DIA as a unique platform for biomarker discovery. We describe how to generate spectral PBMC proteome libraries by applying peptide fractionation followed by DDA analysis, and then how to apply DIA methods to PBMC samples from individual patients using a high-resolution Orbitrap Fusion mass spectrometer.
    Keywords:  Biomarker; Data-independent acquisition; Label-free quantification; Mass spectrometry; Peripheral blood mononuclear cell (PBMC)
    DOI:  https://doi.org/10.1007/978-1-4939-9164-8_10
  10. Front Immunol. 2019 ;10 312
    Fatigue in Sjögren's Syndrome: A Search for Biomarkers and Treatment Targets.
    Iris L A Bodewes, Peter J van der Spek, Leticia G Leon, Annemarie J M Wijkhuijs, Cornelia G van Helden-Meeuwsen, Liselotte Tas, Marco W J Schreurs, Paul L A van Daele, Peter D Katsikis, Marjan A Versnel.
      Background: Primary Sjögren's syndrome (pSS) is a systemic autoimmune disease, where patients often suffer from fatigue. Biological pathways underlying fatigue are unknown. In this study aptamer-based SOMAscan technology is used to identify potential biomarkers and treatment targets for fatigue in pSS. Methods: SOMAscan® Assay 1.3k was performed on serum samples of healthy controls (HCs) and pSS patients characterized for interferon upregulation and fatigue. Differentially expressed proteins (DEPs) between pSS patients and HC or fatigued and non-fatigued pSS patients were validated and discriminatory capacity of markers was tested using independent technology. Results: Serum concentrations of over 1,300 proteins were compared between 63 pSS patients and 20 HCs resulting in 58 upregulated and 46 downregulated proteins. Additionally, serum concentrations of 30 interferon positive (IFNpos) and 30 interferon negative (IFNneg) pSS patients were compared resulting in 25 upregulated and 13 downregulated proteins. ELISAs were performed for several DEPs between pSS patients and HCs or IFNpos and IFNneg all showing a good correlation between protein levels measured by ELISA and relative fluorescence units (RFU) measured by the SOMAscan. Comparing 22 fatigued and 23 non-fatigued pSS patients, 16 serum proteins were differentially expressed, of which 14 were upregulated and 2 were downregulated. Top upregulated DEPs included neuroactive synaptosomal-associated protein 25 (SNAP-25), alpha-enolase (ENO1) and ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1). Furthermore, the proinflammatory mediator IL36a and several complement factors were upregulated in fatigued compared to non-fatigued pSS patients. ROC analysis indicated that DEPs showed good capacity to discriminate fatigued and non-fatigued pSS patients. Conclusion: In this study we validated the use of aptamer-based proteomics and identified a novel set of proteins which were able to distinguish fatigued from non-fatigued pSS patients and identified a so-called "fatigue signature."
    Keywords:  SOMAscan; Sjögren's syndrome; fatigue; interferon; proteomics
    DOI:  https://doi.org/10.3389/fimmu.2019.00312
  11. Methods Mol Biol. 2019 ;1959 185-203
    Development of a Highly Multiplexed SRM Assay for Biomarker Discovery in Formalin-Fixed Paraffin-Embedded Tissues.
    Carine Steiner, Pierre Lescuyer, Jean-Christophe Tille, Paul Cutler, Axel Ducret.
      The search for novel and clinically relevant biomarkers still represents a major clinical challenge and mass-spectrometry-based technologies are essential tools to help in this process. In this application, we demonstrate how selected reaction monitoring (SRM) can be applied in a highly multiplexed way to analyze formalin-fixed paraffin-embedded (FFPE) tissues. Such an assay can be used to analyze numerous samples for narrowing down a list of potential biomarkers to the most relevant candidates. The use of FFPE tissues is of high relevance in this context as large sample collections linked with valuable clinical information are available in hospitals around the world. Here we describe in detail how we proceeded to develop such an assay for 200 proteins in breast tumor FFPE tissues. We cover the selection of suitable peptides, which are different in FFPE compared to fresh frozen tissues and show how we deliberately biased our assay toward proteins with a high probability of being measurable in human clinical samples.
    Keywords:  Biomarker discovery; Formalin-fixed paraffin-embedded (FFPE) tissues; Multiplexed protein assay; Selected reaction monitoring; Targeted mass spectrometry
    DOI:  https://doi.org/10.1007/978-1-4939-9164-8_13
  12. Parkinsonism Relat Disord. 2019 Mar 06. pii: S1353-8020(19)30094-X. [Epub ahead of print]
    Proteomic analysis of tear fluid reveals disease-specific patterns in patients with Parkinson's disease - A pilot study.
    Matthias Boerger, Sebastian Funke, Andreas Leha, Anna-Elisa Roser, Ann-Katrin Wuestemann, Fabian Maass, Mathias Bähr, Franz Grus, Paul Lingor.
      BACKGROUND: The diagnosis of Parkinson's disease (PD) is still challenging and biomarkers could contribute to an improved diagnostic accuracy. Tear fluid (TF) is an easily accessible body fluid reflecting pathophysiological changes in systemic and ocular diseases and is already used as a biomarker source for several ophthalmological disorders. Here, we analyzed the TF of patients with PD and controls (CTR) to describe disease-related changes in TF and identify putative biomarkers for the diagnosis of PD.METHODS: Unstimulated TF samples of a pilot cohort with 36 PD patients and 18 CTR were collected via Schirmer tear test strips and then analyzed via a Bottom-up liquid chromatography electrospray ionization tandem mass spectrometry (BULCMS) workflow, followed by functional analysis encompassing protein-protein interaction as well as cellular component and pathway analysis.
    RESULTS: BULCMS analysis lead to the identification of 571 tear proteins (false discovery rate, FDR < 1%), whereby 31 proteins were exclusively detected in the PD group and 7 only in the CTR group. Whereas 21 proteins were significantly increased in the PD versus CTR groups, 19 proteins were significantly decreased. Core networks of proteins involved in immune response, lipid metabolism and oxidative stress were distinctly altered in PD patients.
    CONCLUSIONS: To our best knowledge, this is the first description of TF proteome in PD patients. Tear protein level alterations suggest the contribution of different disease-related mechanisms in ocular pathology in PD and propose candidate proteins to be validated as potential biomarkers in larger cohorts.
    Keywords:  Biomarker; Dry eye syndrome; Parkinson's disease; Proteomics; Tear fluid
    DOI:  https://doi.org/10.1016/j.parkreldis.2019.03.001
  13. Methods Mol Biol. 2019 ;1959 163-172
    Intact Protein Analysis by LC-MS for Characterizing Biomarkers in Cerebrospinal Fluid.
    Jérôme Vialaret, Sylvain Lehmann, Christophe Hirtz.
      In the field of proteomics, the emerging "top-down" MS-based proteomics approach can be used to obtain a bird's eye view of all intact proteoforms present in a sample. This alternative to the "bottom-up" approach based on tryptic protein digestion processes has some unique advantages for assessing PTMs and sequence variations. However, it requires some dedicated tools for sample preparation and LC-MS analysis, which makes it more complex to handle than the bottom-up approach. In this study, a simple methodology is presented for characterizing intact proteins in biological fluid. This method yields quantitative information using an MS1 profiling approach and makes it possible to identify the proteins regulated under various clinical conditions.
    Keywords:  Biomarker; Cerebrospinal fluid; LC-MS; Protein precipitation; SPE; Top-down
    DOI:  https://doi.org/10.1007/978-1-4939-9164-8_11
  14. Expert Rev Proteomics. 2019 Mar 11.
    Proteomic profiling of urine: implications for lupus nephritis.
    Najla Aljaberi, Michael Bennett, Hermine I Brunner, Prasad Devarajan.
      INTRODUCTION: Lupus nephritis (LN) is a common and significant manifestation, affecting 60% of adults and 80% of children with systemic lupus erythematosus, with up to 30% of patients progressing to end stage renal disease. There remains an unmet need for non-invasive markers of disease activity, damage, and response to therapy. In addition, non-invasive biomarkers that predict therapeutic efficacy are needed to enable cost-effective clinical trials of novel agents. Areas covered: This review examines the methodological aspects of urinary proteomics, the role of proteome profiling in identifying promising urinary biomarkers in LN, and the translation of research findings into clinically useful tools in the management of LN. Expert opinion: Targeted and unbiased proteomics have identified several promising urinary biomarkers that predict LN activity, damage (chronicity), and response to therapy. In particular, a combination of biologically plausible urinary biomarkers termed as RAIL (Renal Activity Index for Lupus) has emerged as an excellent predictor of LN activity as well as response to therapy, being able to predict efficacy within 3 months of therapy. If validated in additional large prospective studies, the RAIL biomarkers will transform the care of patients with LN, allowing for a personalized and predictive approach and improved outcomes.
    Keywords:  biomarkers; lupus nephritis; systemic lupus erythematosus; urine
    DOI:  https://doi.org/10.1080/14789450.2019.1592681
  15. Int J Oncol. 2019 Mar 07.
    Proteomics profiling of plasma exosomes in epithelial ovarian cancer: A potential role in the coagulation cascade, diagnosis and prognosis.
    Wei Zhang, Xiaoxuan Ou, Xiaohua Wu.
      Ovarian cancer remains the most lethal type of cancer among all gynecological malignancies. The majority of patients are diagnosed with ovarian cancer at the late stages of the disease. Therefore, there exists an imperative need for the development of early ovarian cancer diagnostic techniques. Exosomes, secreted by various cell types, play pivotal roles in intercellular communication, which emerge as promising diagnostic and prognostic biomarkers for ovarian cancer. In this study, we present for the first time, at least to the best of our knowledge, the proteomics profiling of exosomes derived from the plasma of patients with ovarian cancer via liquid chromatography tandem mass spectrometry (LC‑MS/MS) with tandem mass tagging (TMT). The exosomes enriched from patient plasma samples were characterized by nanoparticle tracking analysis (NTA), dynamic light scattering (DLS), transmission electron microscopy (TEM) and western blot analysis. The size of the plasma exosomes fell into the range of 30 to 100 nm in diameter. The exosomal marker proteins, CD81 and TSG101, were clearly stained in the exosome samples; however, there was no staining for the endoplasmic reticulum protein, calnexin. A total of 294 proteins were identified with all exosome samples. Among these, 225 proteins were detected in both the cancerous and non‑cancerous samples. Apart from universal exosomal proteins, exosomes derived from ovarian cancer patient plasma also contained tumor‑specific proteins relevant to tumorigenesis and metastasis, particularly in epithelial ovarian carcinoma (EOC). Patients with EOC often suffer from coagulation dysfunction. The function of exosomes in coagulation was also examined. Several genes relevant to the coagulation cascade were screened out as promising diagnostic and prognostic factors that may play important roles in ovarian cancer progression and metastasis. On the whole, in this study, we successfully isolated and purified exosomes from plasma of patients with EOC, and identified a potential role of these exosomes in the coagulation cascade, as well as in the diagnosis and prognosis of patients.
    DOI:  https://doi.org/10.3892/ijo.2019.4742
  16. Methods Mol Biol. 2019 ;1959 51-64
    Protein Biomarker Discovery Using Human Blood Plasma Microparticles.
    Raghda Saad Zaghloul Taleb, Pacint Moez, Doreen Younan, Martin Eisenacher, Matthias Tenbusch, Barbara Sitek, Thilo Bracht.
      Cells shed into the extracellular space a population of membranous vesicles of plasma membrane origin called microparticles (MP). Given the fact that MP are abundantly present in body fluids including plasma, rich in cell-type or disease-specific proteins and formed in conditions of stress and injury, they have been extensively investigated as biomarkers in various diseases. With the advancement in the mass spectrometry-based proteome analysis, the knowledge of the protein composition of plasma MP (PMP) has been intensively expanded, which aids the discovery of novel diagnostic target proteins. However, the lack of standardized and accurate protocols for PMP isolation limits the implementation of PMP as biomarkers in clinical settings. Here, we describe in detail a robust protocol for PMP isolation from human blood plasma via ultracentrifugation followed by label-free quantitative proteome analysis of PMP.
    Keywords:  Blood-based biomarker; Label-free proteome quantification; Plasma microparticle proteome; Plasma microparticles; Ultracentrifugation
    DOI:  https://doi.org/10.1007/978-1-4939-9164-8_4
  17. Redox Biol. 2019 Feb 19. pii: S2213-2317(19)30021-7. [Epub ahead of print]22 101142
    Protein disulfide isomerase plasma levels in healthy humans reveal proteomic signatures involved in contrasting endothelial phenotypes.
    Percíllia Victória Santos de Oliveira, Sheila Garcia-Rosa, Ana Teresa Azevedo Sachetto, Ana Iochabel Soares Moretti, Victor Debbas, Tiphany Coralie De Bessa, Nathalia Tenguan Silva, Alexandre da Costa Pereira, Daniel Martins-de-Souza, Marcelo Larami Santoro, Francisco Rafael Martins Laurindo.
      Redox-related plasma proteins are candidate reporters of protein signatures associated with endothelial structure/function. Thiol-proteins from protein disulfide isomerase (PDI) family are unexplored in this context. Here, we investigate the occurrence and physiological significance of a circulating pool of PDI in healthy humans. We validated an assay for detecting PDI in plasma of healthy individuals. Our results indicate high inter-individual (median = 330 pg/mL) but low intra-individual variability over time and repeated measurements. Remarkably, plasma PDI levels could discriminate between distinct plasma proteome signatures, with PDI-rich (>median) plasma differentially expressing proteins related to cell differentiation, protein processing, housekeeping functions and others, while PDI-poor plasma differentially displayed proteins associated with coagulation, inflammatory responses and immunoactivation. Platelet function was similar among individuals with PDI-rich vs. PDI-poor plasma. Remarkably, such protein signatures closely correlated with endothelial function and phenotype, since cultured endothelial cells incubated with PDI-poor or PDI-rich plasma recapitulated gene expression and secretome patterns in line with their corresponding plasma signatures. Furthermore, such signatures translated into functional responses, with PDI-poor plasma promoting impairment of endothelial adhesion to fibronectin and a disturbed pattern of wound-associated migration and recovery area. Patients with cardiovascular events had lower PDI levels vs. healthy individuals. This is the first study describing PDI levels as reporters of specific plasma proteome signatures directly promoting contrasting endothelial phenotypes and functional responses.
    Keywords:  Endothelial cells; Plasma protein signatures; Plasma proteome; Protein disulfide isomerase; Thiol proteins
    DOI:  https://doi.org/10.1016/j.redox.2019.101142
  18. Methods Mol Biol. 2019 ;1955 363-380
    Fluorescence Proteomic Technology to Analyze Peripheral Blood Mononuclear Cells in Chronic Chagas Disease.
    John E Wiktorowicz, M Paola Zago, Nisha J Garg.
      The thiol moieties of cysteinyl residues in proteins undergo a number of modifications including nitrosylation, oxidation, persulfidation, sulfenylation, and others. These protein modifications may influence gain as well as loss of function in biological and disease conditions. Herein, we describe a quantitative approach that combines accurate, sensitive fluorescence modification of cysteinyl-S-nitrosyl (SNOFlo) groups that leaves electrophoretic mobility unaffected and offers the measurement of changes in S-nitrosylation (SNO) status relative to protein abundance. This approach has been useful in evaluating the global protein abundance and SNO profile of Chagas seropositive individuals that were categorized in clinically asymptomatic (C/A) and clinically symptomatic (C/S) subgroups and compared to normal healthy (N/H) controls. Through analyzing the proteome datasets with different bioinformatics and statistics tools, potential pathologic mechanisms in disease progression are identified. We also propose a panel of protein biomarkers that have a potential to identify the infected individuals at risk of developing clinical Chagas disease.
    Keywords:  2DE; Chagas cardiomyopathy; Mass spectrometry; Peripheral blood mononuclear cells; S-nitrosylation; Trypanosoma cruzi
    DOI:  https://doi.org/10.1007/978-1-4939-9148-8_27
  19. Cancer Med. 2019 Mar 14.
    Serum biomarker panels for the diagnosis of gastric cancer.
    Dan Wu, Pinglu Zhang, Ji Ma, Jinbo Xu, Li Yang, Weidan Xu, Haifeng Que, Meifen Chen, Hongtao Xu.
      Gastric cancer is a leading cause of mortality due to neoplastic disease. Although early detection of gastric cancers can decrease the mortality rate, it remains a diagnostic challenge because of the lack of effective biomarkers. In this study, fifteen gastric cancer patients and ten healthy subjects were recruited to assess novel serum biomarkers for gastric cancer using antibody microarray technology. ELISA was utilized to validate the antibody array results. As a result, compared to the controls, eleven cytokines were found to be significantly increased in gastric cancer, including interferon gamma receptor 1 (IFNGR1), neurogenic locus notch homolog protein 3 (Notch-3), tumor necrosis factor receptor superfamily member 19L (TNFRSF19L), growth hormone receptor (GHR), signaling lymphocytic activation molecule family 8 (SLAMF8), folate receptor beta (FR-beta), integrin alpha 5, galectin-8, erythropoietin-producing hepatocellular A1 (EphA1), epiregulin, and fibroblast growth factor 12 (FGF-12) with P < 0.05. ELISA validation supported the results of the antibody array. More importantly, most of these eleven cytokines, including IFNGR1, TNFRSF19L, GHR, SLAMF8, FR-beta, and integrin alpha 5 were discovered to be elevated in gastric cancer serum samples for the first time in this study, suggesting that these proteins may serve as novel biomarkers for the early diagnosis and prognosis determination of gastric cancer.
    Keywords:  antibody array; biomarkers; gastric cancer; serum
    DOI:  https://doi.org/10.1002/cam4.2055
  20. Exp Mol Med. 2019 Mar 15. 51(3): 33
    Systems biology approaches to investigating the roles of extracellular vesicles in human diseases.
    András Gézsi, Árpád Kovács, Tamás Visnovitz, Edit I Buzás.
      Extracellular vesicles (EVs) are membrane-enclosed structures secreted by cells. In the past decade, EVs have attracted substantial attention as carriers of complex intercellular information. They have been implicated in a wide variety of biological processes in health and disease. They are also considered to hold promise for future diagnostics and therapy. EVs are characterized by a previously underappreciated heterogeneity. The heterogeneity and molecular complexity of EVs necessitates high-throughput analytical platforms for detailed analysis. Recently, mass spectrometry, next-generation sequencing and bioinformatics tools have enabled detailed proteomic, transcriptomic, glycomic, lipidomic, metabolomic, and genomic analyses of EVs. Here, we provide an overview of systems biology experiments performed in the field of EVs. Furthermore, we provide examples of how in silico systems biology approaches can be used to identify correlations between genes involved in EV biogenesis and human diseases. Using a knowledge fusion system, we investigated whether certain groups of proteins implicated in the biogenesis/release of EVs were associated with diseases and phenotypes. Furthermore, we investigated whether these proteins were enriched in publicly available transcriptomic datasets using gene set enrichment analysis methods. We found associations between key EV biogenesis proteins and numerous diseases, which further emphasizes the key role of EVs in human health and disease.
    DOI:  https://doi.org/10.1038/s12276-019-0226-2
  21. Sci Rep. 2019 Mar 12. 9(1): 4268
    Altered proteome of high-density lipoproteins from paediatric acute lymphoblastic leukemia survivors.
    Maryse Fournier, Eric Bonneil, Carole Garofalo, Guy Grimard, Caroline Laverdière, Maja Krajinovic, Simon Drouin, Daniel Sinnett, Valérie Marcil, Emile Levy.
      Acute lymphoblastic leukemia (ALL) is the most frequent malignancy in children. With the use of more modern, efficient treatments, 5-year survival has reached more than 90% in this population. However, this achievement comes with many secondary and long-term effects since more than 65% of the survivors experience at least one severe complication, including the metabolic syndrome and cardiovascular diseases. The main objective of the present work was to characterize the composition of HDL particles isolated from pediatric ALL survivors. HDLs from 8 metabolically healthy ALL survivors, 8 metabolically unhealthy ALL survivors and 8 age- and gender-matched controls were analyzed. The HDL fraction from the survivors contained less cholesterol than the controls. In addition, proteomic analyses revealed an enrichment of pro-thrombotic (e.g., fibrinogen) and pro-inflammatory (e.g., amyloid A) proteins in the HDLs deriving from metabolically unhealthy survivors. These results indicate an alteration in the composition of lipid and protein content of HDL from childhood ALL survivors with metabolic disorders. Although more work is needed to validate the functionality of these HDLs, the data seem relevant for survivor health given the detection of potential biomarkers related to HDL metabolism and functionality in cancer.
    DOI:  https://doi.org/10.1038/s41598-019-40906-x
  22. Biomed Pharmacother. 2019 Mar 12. pii: S0753-3322(18)35003-0. [Epub ahead of print]113 108763
    Inflammatory effects of in vivo glycated albumin from cardiovascular patients.
    Beatriz Paradela-Dobarro, Susana B Bravo, Adriana Rozados-Luís, Mercedes González-Peteiro, Alfonso Varela-Román, José Ramón González-Juanatey, Javier García-Seara, Ezequiel Alvarez.
      OBJECTIVES: Characterization of the type of glycation found in circulating proteins from cardiovascular patients in comparison with healthy control subjects and to explore the pathophysiological molecular effects of these glycomodified proteins on human umbilical vein endothelial cells (HUVEC) in culture.METHODS: Human serum albumin pools from 10 subjects each, of patients with heart failure (HF) presenting high or low glycation levels, and from healthy subjects were isolated and purified. The glycation levels of these pools were characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and compared between them. Analysis of endothelial dysfunction after the treatment of HUVEC with the pools was made by mRNA expression of adhesion molecules and by functional adhesion of mononuclear cells to HUVEC monolayers.
    RESULTS: Specific characterization of post-transductional modifications (advanced glycation end products) in high and low glycated albumins from patients was made in comparison with healthy subjects. Albumins from patients were able, at very low concentrations (12.5 μg/mL), to significantly up-regulate (˜0.2 - 2 fold) the gene expression of adhesion molecules in HUVEC. At the functional level, the albumin from patients with high glycation levels (at 12.5 and 25 μg/mL) significantly enhanced (˜10%) the adhesion of mononuclear cells to HUVEC.
    CONCLUSIONS: Differences in the glycomodification of albumin from HF patients were found and specifically characterized in comparison with albumin from healthy subjects. Functionally, in vivo glycated albumin in patients with HF induced an increase in adhesion molecules expression on HUVEC, which supported an increase in peripheral blood mononuclear cells adhesion to endothelial cells.
    Keywords:  Human endothelial cells; In vivo glycated albumin; Mononuclear cells adhesion; Peptide mapping of glycomodified proteins; Vascular endothelial dysfunction
    DOI:  https://doi.org/10.1016/j.biopha.2019.108763
  23. Sci Rep. 2019 Mar 14. 9(1): 4587
    High-throughput epitope profiling of antibodies in the plasma of Alzheimer's disease patients using random peptide microarrays.
    Kyu-Young Sim, Sang-Heon Park, Kyu Yeong Choi, Jung Eun Park, Jung Sup Lee, Byeong C Kim, Jeonghwan Gwak, Woo Keun Song, Kun Ho Lee, Sung-Gyoo Park.
      The symptoms of Alzheimer's disease (AD), a major cause of dementia in older adults, are linked directly with neuronal cell death, which is thought to be due to aberrant neuronal inflammation. Autoantibodies formed during neuronal inflammation show excellent stability in blood; therefore, they may be convenient blood-based diagnostic markers of AD. Here, we performed microarray analysis of 29,240 unbiased random peptides to be used for comprehensive screening of AD-specific IgG and IgM antibodies in the blood. The results showed that (1) sequence-specific and isotype-specific antibodies are regulated differentially in AD, and combinations of these antibodies showing high area under the receiver operating characteristic curve values (0.862-0.961) can be used to classify AD, (2) AD-specific IgG antibodies arise from IgM antibody-secreting cells that existed before disease onset and (3) target protein profiling of the antibodies identified some AD-related proteins, some of which are involved in AD-related signalling pathways. Therefore, we propose that these epitopes may facilitate the development of biomarkers for AD diagnosis and form the basis for a mechanistic study related to AD progression.
    DOI:  https://doi.org/10.1038/s41598-019-40976-x
  24. Oncol Lett. 2019 Mar;17(3): 3517-3522
    Decorin as a prognostic biomarker in patients with malignant peripheral nerve sheath tumors.
    Xiaotian Jia, Chao Chen, Lin Chen, Cong Yu, Tadashi Kondo.
      Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive soft-tissue sarcomas. The prognosis of MPNSTs has been reported to differ among previous studies. However, there have been a number of reported prognostic biomarkers associated with MPNSTs. In the present study, a proteomics study was performed to discover the differential protein expression in patients with MPSNTs with different prognoses. The clinical data of 30 primary extremities of patients with MPNSTs, who underwent surgery at the Department of Hand Surgery, Huashan Hospital, Fudan University between January 2002 and December 2011, were acquired. A total of 16 patients succumbed to their diseases within 5 years, whereas 14 patients were disease-free for >5 years. Samples from the 9 patients who succumbed within 2 years were assigned to Group D, while samples from the 8 patients who were continuously disease-free for >5 years following diagnosis were assigned to Group L for the proteomics study. Label-free quantitative proteomics and mass spectrometry were performed to filtrate differential protein in patients with MPSNTs with different prognoses. Decorin was filtrated as a differential protein of note. The expression level of decorin was significantly lower in Group D compared with that in Group L (D/L=0.0948; P=0.0004). The result was verified by immunohistochemical staining in the 30 primary extremities of patients with MPNSTs. The 5-year survival rate of patients with positive expression of decorin was 78.57%, while the 5-year survival rate of patients negative for decorin expression was 18.75% (P=0.0014). Overall, a high level of decorin indicted a better prognosis in patients with MPNSTs. With further investigation, decorin may be a reliable prognostic biomarker for MPNSTs.
    Keywords:  decorin; formalin-fixed paraffin-embedded; label-free quantitative proteomics; malignant peripheral nerve sheath tumor; prognostic biomarker
    DOI:  https://doi.org/10.3892/ol.2019.9959
  25. Clin Chim Acta. 2019 Mar 08. pii: S0009-8981(19)30348-1. [Epub ahead of print]
    Development of parallel reaction monitoring assays for cerebrospinal fluid proteins associated with Alzheimer's disease.
    Annika Andersson, Julia Remnestål, Bengt Nellgård, Helian Vunk, David Kotol, Fredrik Edfors, Mathias Uhlén, Jochen M Schwenk, Leopold L Ilag, Henrik Zetterberg, Kaj Blennow, Anna Månberg, Peter Nilsson, Claudia Fredolini.
      Detailed knowledge of protein changes in cerebrospinal fluid (CSF) across healthy and diseased individuals would provide a better understanding of the onset and progression of neurodegenerative disorders. In this study, we selected 20 brain-enriched proteins previously identified in CSF by antibody suspension bead arrays (SBA) to be potentially biomarkers for Alzheimer's disease (AD) and verified these using an orthogonal approach. We examined the same set of 94 CSF samples from patients affected by AD (including preclinical and prodromal), mild cognitive impairment (MCI), non-AD dementia and healthy individuals, which had previously been analyzed by SBA. Twenty-eight parallel reaction monitoring (PRM) assays were developed and 13 of them could be validated for protein quantification. Antibody profiles were verified by PRM. For seven proteins, the antibody profiles were highly correlated with the PRM results (r > 0.7) and GAP43, VCAM1 and PSAP were identified as potential markers of preclinical AD. In conclusion, we demonstrate the usefulness of targeted mass spectrometry as a tool for the orthogonal verification of antibody profiling data, suggesting that these complementary methods can be successfully applied for comprehensive exploration of CSF protein levels in neurodegenerative disorders.
    Keywords:  AD; Alzheimer's disease; Biomarkers; Cerebrospinal fluid; Parallel reaction monitoring (PRM); Suspension bead array (SBA)
    DOI:  https://doi.org/10.1016/j.cca.2019.03.243
  26. Exp Mol Med. 2019 Mar 15. 51(3): 30
    Mass spectrometry-based proteome profiling of extracellular vesicles and their roles in cancer biology.
    Raju Bandu, Jae Won Oh, Kwang Pyo Kim.
      Over the past three decades, extracellular vesicles (EVs) have arisen as important mediators of intercellular communication that are involved in the transmission of biological signals between cells to regulate various biological processes. EVs are largely responsible for intercellular communication through the delivery of bioactive molecules, such as proteins, messenger RNAs (mRNAs), microRNAs (miRNAs), DNAs, lipids, and metabolites. EVs released from cancer cells play a significant role in signal transduction between cancer cells and the surrounding cells, which contributes to the formation of tumors and metastasis in the tumor microenvironment. In addition, EVs released from cancer cells migrate to blood vessels and flow into various biological fluids, including blood and urine. EVs and EV-loaded functional cargoes, including proteins and miRNAs, found in these biological fluids are important biomarkers for cancer diagnosis. Therefore, EV proteomics greatly contributes to the understanding of carcinogenesis and tumor progression and is critical for the development of biomarkers for the early diagnosis of cancer. To explore the potential use of EVs as a gateway to understanding cancer biology and to develop cancer biomarkers, we discuss the mass spectrometric identification and characterization of EV proteins from different cancers. Information provided in this review may help in understanding recent progress regarding EV biology and the potential roles of EVs as new noninvasive biomarkers and therapeutic targets.
    DOI:  https://doi.org/10.1038/s12276-019-0218-2
  27. Pathol Res Pract. 2019 Feb 28. pii: S0344-0338(18)30723-4. [Epub ahead of print]
    Identification of a novel biomarker-CCL5 using antibody microarray for colorectal cancer.
    Mingxia Chen, Xiaoqing Yang, Min Yang, Wei Zhang, Lei Li, Qing Sun.
      PURPOSE: To screen novel candidate biomarkers in primary colorectal cancer (CRC), and indentify their clinical valuation in progress of colorectal cancer.METHODS: By using antibody microarray, 274 target proteins in tissue samples from primary colorectal cancer patients were detected. Among differently expressed proteins in CRC tissues, As promising candidate biomarker, RANTES/CCL5 was validated by enzyme-linked immunosorbentassay and immunohistochemistry (IHC), and the clinical significance of CCL5 was analyzed.
    RESULTS: Totally, 25 differentially expressed proteins were indentified between colorectal cancers and matched normal mucosa. CCL5 expression was significantly associated with adverse pathological progress, apt to lymph node metastasis and higher T stage.
    CONCLUSIONS: CCL5 may contribute to promoting tumor growth, and CCL5 is a promising target that may help in understanding the pathogenesis of CRC.
    Keywords:  Antibody microarray; Colorectal cancer; ELISA; Immunochemistry; RANTES/CCL5
    DOI:  https://doi.org/10.1016/j.prp.2019.02.011
  28. Methods Mol Biol. 2019 ;1959 1-22
    Pre- and Post-analytical Factors in Biomarker Discovery.
    Frank Klont, Peter Horvatovich, Natalia Govorukhina, Rainer Bischoff.
      The translation of promising biomarkers, which were identified in biomarker discovery experiments, to clinical assays is one of the key challenges in present-day proteomics research. Many so-called "biomarker candidates" fail to progress beyond the discovery phase, and much emphasis is placed on pre- and post-analytical variability in an attempt to provide explanations for this bottleneck in the biomarker development pipeline. With respect to such variability, there is a large number of pre- and post-analytical factors which may impact the outcomes of proteomics experiments and thus necessitate tight control. This chapter highlights some of these factors and provides guidance for addressing them on the basis of examples from previously published proteomics studies.
    Keywords:  Biomarker; Liquid chromatography; Mass spectrometry; Post-analytical variability; Pre-analytical variability; Proteomics; Regulated bioanalysis; Sample preparation; Stability
    DOI:  https://doi.org/10.1007/978-1-4939-9164-8_1
  29. Elife. 2019 Mar 12. pii: e41431. [Epub ahead of print]8
    Functional proteomic atlas of HIV infection in primary human CD4+ T cells.
    Adi Naamati, James C Williamson, Edward Jd Greenwood, Sara Marelli, Paul J Lehner, Nicholas J Matheson.
      Viruses manipulate host cells to enhance their replication, and the identification of cellular factors targeted by viruses has led to key insights into both viral pathogenesis and cell biology. In this study, we develop an HIV reporter virus (HIV-AFMACS) displaying a streptavidin-binding affinity tag at the surface of infected cells, allowing facile one-step selection with streptavidin-conjugated magnetic beads. We use this system to obtain pure populations of HIV-infected primary human CD4+ T cells for detailed proteomic analysis, and quantitate approximately 9000 proteins across multiple donors on a dynamic background of T cell activation. Amongst 650 HIV-dependent changes (q < 0.05), we describe novel Vif-dependent targets FMR1 and DPH7, and 192 proteins not identified and/or regulated in T cell lines, such as ARID5A and PTPN22. We therefore provide a high-coverage functional proteomic atlas of HIV infection, and a mechanistic account of host factors subverted by the virus in its natural target cell.
    Keywords:  HIV; human; immunology; infectious disease; inflammation; magnetic cell selection; microbiology; primary human CD4+ T cell; proteomics; virus
    DOI:  https://doi.org/10.7554/eLife.41431
  30. Methods Mol Biol. 2019 ;1955 309-314
    Cytometric Bead Array (CBA) for Measuring Cytokine Levels in Chagas Disease Patients.
    Nayara I Medeiros, Juliana A S Gomes.
      Cytometric bead array (CBA) is a flow cytometry application that allows users to quantify multiple proteins simultaneously. Compared to other quantifier assays, as enzyme-linked immunosorbent assay (ELISA) and Western blot, CBA significantly reduces sample requirements and time to results. This technology allows for the design and creation of assays to measure a variety of analytes including inflammatory mediators, chemokines, immunoglobulin isotypes, intracellular signaling molecules, apoptotic mediators, adhesion molecules, and antibodies. Here we describe CBA steps to measure soluble cytokine levels in body fluids of infected patients by the protozoan Trypanosoma cruzi, morbidity known as Chagas disease.
    Keywords:  CBA; Cytokine measurement; Cytometric bead array; Flow cytometry
    DOI:  https://doi.org/10.1007/978-1-4939-9148-8_23
  31. Sci Rep. 2019 Mar 12. 9(1): 4202
    A comparison between the abdominal and femoral adipose tissue proteome of overweight and obese women.
    M A A Vogel, P Wang, F G Bouwman, N Hoebers, E E Blaak, J Renes, E C Mariman, G H Goossens.
      Body fat distribution is an important determinant of cardiometabolic health. Lower-body adipose tissue (AT) has protective characteristics as compared to upper-body fat, but the underlying depot-differences remain to be elucidated. Here, we compared the proteome and morphology of abdominal and femoral AT. Paired biopsies from abdominal and femoral subcutaneous AT were taken from eight overweight/obese (BMI ≥ 28 kg/m2) women with impaired glucose metabolism after an overnight fast. Proteins were isolated and quantified using liquid chromatography-mass spectrometry, and protein expression in abdominal and femoral subcutaneous AT was compared. Moreover, correlations between fat cell size and the proteome of both AT depots were determined. In total, 651 proteins were identified, of which 22 proteins tended to be differentially expressed between abdominal and femoral AT after removal of blood protein signals (p < 0.05). Proteins involved in cell structure organization and energy metabolism were differently expressed between AT depots. Fat cell size, which was higher in femoral AT, was significantly correlated with ADH1B, POSTN and LCP1. These findings suggest that there are only slight differences in protein expression between abdominal and femoral subcutaneous AT. It remains to be determined whether these differences, as well as differences in protein activity, contribute to functional and/or morphological differences between these fat depots.
    DOI:  https://doi.org/10.1038/s41598-019-40992-x
  32. Biosens Bioelectron. 2019 Feb 23. pii: S0956-5663(19)30137-X. [Epub ahead of print]132 47-54
    Graphically encoded suspension array for multiplex immunoassay and quantification of autoimmune biomarkers in patient sera.
    Kexiao Zheng, Chao Chen, Xiuli Chen, Menghong Xu, Linqi Chen, Yining Hu, Yanan Bai, Binghuan Liu, Cheng Yan, Hong Wang, Jiong Li.
      In precision medicine, clinical decisions and pharmaceutical evaluations tends to be made upon parallel analysis of multiple protein biomarkers. Currently, the growing needs of high-throughput multiplex immunoassay is partially satisfied by spectrally encoded bead flow suspension arrays and other platforms, yet there is still room for progress in terms of encoding capacity, decoding accuracy, ease-of-manufacture/operation, and cost-effectiveness, for which graphical suspension arrays could make substantial contributions. Here we described a suspension array system made up of graphically encoded silica particles, an automated microplate imager and an in-house data processing program. The micro-fabricated, highly uniform planar particles provide a code space of 128-plex with further extendibility. The derived multiplex immunoassay reaches sub-picogram per milliliter sensitivity level (lowest LoD = 80 fg/ml) with wide dynamic range, as well as high precision and accuracy. The potential of clinical diagnostics was demonstrated by parallel measurement of three serum biomarkers for type 1 diabetes patients. Importantly, use of standard microplates as assay vessel extends its power to high-throughput applications, such as disease screening or drug discovery.
    Keywords:  Autoimmune diabetes; Cytokines; Graphical encoding; Multiplex immunoassay; Suspension array
    DOI:  https://doi.org/10.1016/j.bios.2019.02.030
  33. Methods Mol Biol. 2019 ;1959 113-122
    Lys-C/Trypsin Tandem-Digestion Protocol for Gel-Free Proteomic Analysis of Colon Biopsies.
    Armin Schniers, Yvonne Pasing, Terkel Hansen.
      The protocol presented was specifically optimized for in-depth analysis of the human colon mucosa proteome. After cell lysis in a sodium deoxycholate/urea buffer, a tandem digestion with Lys-C and trypsin was performed. Prior to LC-MS/MS analysis, peptides were TMT-labeled and fractionated by high pH reversed-phase spin columns. This protocol is a powerful, reproducible, sample-saving, and cost-effective option when an in-depth quantitative proteome analysis is desired.
    Keywords:  Colon mucosa proteome; High pH reversed-phase fractionation; Lys-C; Sodium deoxycholate; TMT labeling
    DOI:  https://doi.org/10.1007/978-1-4939-9164-8_7
  34. Cancer Treat Res Commun. 2019 Feb 26. pii: S2468-2942(18)30186-2. [Epub ahead of print]19 100126
    Phase II study of stereotactic radiosurgery for the treatment of patients with oligoprogression on erlotinib.
    Jared Weiss, Brian Kavanagh, Allison Deal, Liza Villaruz, James Stevenson, Ross Camidge, Hossein Borghaei, Jack West, Padmini Kirpalani, David Morris, Carrie Lee, Chad V Pecot, Timothy Zagar, Thomas Stinchcombe, Nathan Pennell.
      INTRODUCTION: Retrospective studies have evaluated the approach of stereotactic radiotherapy (SRT) to address oligoprogression in patients with EGFR mutant NSCLC on TKI therapy, it has never been prospectively studied.MATERIALS AND METHODS: We treated 25 patients with EGFR mutant NSCLC on erlotinib who had 3 or fewer sites of extra-cranial progression with SRT to progressing sites, followed by re-initiation of erlotinib.
    RESULTS: Median PFS from the initiation of SRT was 6 months (95% CI 2.5 to 11.6) and median OS was 29 months (95% CI 21.7 to 36.3). Neither baseline nor changes in the Veristrat proteomic predicted PFS.
    CONCLUSIONS: SRT and TKI continuation may be considered for select patients with EGFR mutant NSCLC and oligo-progression on EGFR TKI therapy.
    Keywords:  EGFR; Erlotinib; Oligoprogression; Stereotactic radiosurgery
    DOI:  https://doi.org/10.1016/j.ctarc.2019.100126
  35. Onco Targets Ther. 2019 ;12 1521-1538
    Development of treatment options for Chinese patients with advanced squamous cell lung cancer: focus on afatinib.
    Shun Lu.
      Lung cancer is the leading cause of cancer death in China, and approximately one third of these cancers are squamous cell carcinoma (SqCC) of the lung. Ethnic diversity and country-specific environmental factors can account for interindividual variations in response to and tolerability of anticancer therapies. Although several targeted therapies have recently been approved for patients with relapsed/refractory SqCC of the lung, only afatinib, an irreversible ErbB family blocker, has data of Chinese patients. In the Phase III LUX-Lung 8 trial, afatinib demonstrated a significant clinical benefit vs the reversible first-generation EGFR tyrosine kinase inhibitor erlotinib in both the overall population and the Chinese subset, with a manageable safety profile. Emerging biomarker data from LUX-Lung 8 suggest that patients with ErbB mutations, especially ErbB2, and those classified as "good" in the VeriStrat® proteomic test, may benefit from afatinib treatment in particular, regardless of ethnicity, and may get a long-term response. In conclusion, afatinib is a valid second-line option for Chinese patients with SqCC of the lung, and specific biomarkers may help guide in treatment decision-making. Ongoing studies will provide further guidance on afatinib's place in the treatment algorithm, alongside the other novel targeted therapies.
    Keywords:  Chinese; EGFR; ErbB; NSCLC; afatinib; biomarker; squamous cell carcinoma
    DOI:  https://doi.org/10.2147/OTT.S188296
  36. Nature. 2019 Mar 13.
    Glucocorticoids promote breast cancer metastasis.
    Milan M S Obradović, Baptiste Hamelin, Nenad Manevski, Joana Pinto Couto, Atul Sethi, Marie-May Coissieux, Simone Münst, Ryoko Okamoto, Hubertus Kohler, Alexander Schmidt, Mohamed Bentires-Alj.
      Diversity within or between tumours and metastases (known as intra-patient tumour heterogeneity) that develops during disease progression is a serious hurdle for therapy1-3. Metastasis is the fatal hallmark of cancer and the mechanisms of colonization, the most complex step in the metastatic cascade4, remain poorly defined. A clearer understanding of the cellular and molecular processes that underlie both intra-patient tumour heterogeneity and metastasis is crucial for the success of personalized cancer therapy. Here, using transcriptional profiling of tumours and matched metastases in patient-derived xenograft models in mice, we show cancer-site-specific phenotypes and increased glucocorticoid receptor activity in distant metastases. The glucocorticoid receptor mediates the effects of stress hormones, and of synthetic derivatives of these hormones that are used widely in the clinic as anti-inflammatory and immunosuppressive agents. We show that the increase in stress hormones during breast cancer progression results in the activation of the glucocorticoid receptor at distant metastatic sites, increased colonization and reduced survival. Our transcriptomics, proteomics and phospho-proteomics studies implicate the glucocorticoid receptor in the activation of multiple processes in metastasis and in the increased expression of kinase ROR1, both of which correlate with reduced survival. The ablation of ROR1 reduced metastatic outgrowth and prolonged survival in preclinical models. Our results indicate that the activation of the glucocorticoid receptor increases heterogeneity and metastasis, which suggests that caution is needed when using glucocorticoids to treat patients with breast cancer who have developed cancer-related complications.
    DOI:  https://doi.org/10.1038/s41586-019-1019-4
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