bims-prodis 2019-03-24 papers

bims-prodis

Biomed News

on Proteomics in disease

Issue of 2019–03–24
24 papers selected by
Nancy Gough, Bioserendipity

[Changes in mass spectrometry of proteins in patients with 1-bromopropane poisoning after treatment].
Serum Proteomic Changes as Candidate Biomarkers of Intermediate Liver Fibrosis in Chronic Hepatitis B Infection.
Clinical protein science in translational medicine targeting malignant melanoma.
Integrated Lipidomics and Proteomics Point to Early Blood-Based Changes in Childhood Preceding Later Development of Psychotic Experiences: Evidence From the Avon Longitudinal Study of Parents and Children.
Transcriptomic and proteomic analysis of iris tissue and aqueous humor in juvenile idiopathic arthritis-associated uveitis.
A proteomic analysis of serum-derived exosomes in rheumatoid arthritis.
Multiplex targeted mass spectrometry assay for one-shot flavivirus diagnosis.
Proteomic investigations of human HERC2 mutants: Insights into the pathobiology of a neurodevelopmental disorder.
Isobaric tags for relative and absolute quantitation‑based proteomics reveals potential novel biomarkers for the early diagnosis of acute myocardial infarction within 3 h.
Proteomic Analysis Applied to Human Plasma During Intermittent Fasting.
Significant Down-regulation of Urea Cycle Generates Clinically Relevant Proteomic Signature in Hepatocellular Carcinoma Patients with Macrovascular Invasion.
Sperm functional aspects and enriched proteomic pathways of seminal plasma of adult men with obesity.
Autoantibody Biomarker Discovery in Primary Open Angle Glaucoma Using Serological Proteome Analysis (SERPA).
Isobaric tags for relative and absolute quantitation-based proteome analysis of Vietnamese colorectal carcinoma tissues.
Isotopologue multi-point calibration for proteomics biomarker quantification in clinical practice.
Serum Biomarkers for Racial Disparities in Breast Cancer Progression.
The autoantibody response to cyclic citrullinated collagen type II peptides in rheumatoid arthritis.
Translation and evaluation of a pre-clinical 5-protein response prediction signature in a breast cancer phase Ib clinical trial.
The Proteogenomic Landscape of Curable Prostate Cancer.
Interleukin-1 in cerebrospinal fluid for evaluating the neurological outcome in traumatic brain injury.
Proteomic profiling of whole-saliva reveals correlation between Burning Mouth Syndrome and the neurotrophin signaling pathway.
Identification of red blood cell membrane defects in a patient with hereditary spherocytosis using next‑generation sequencing technology and matrix‑assisted laser desorption/ionization time‑of‑flight mass spectrometry.
Molecular Profiling of Ulcerative Colitis Subjects from the TURANDOT Trial Reveals Novel Pharmacodynamic/Efficacy Biomarkers.
[Comparative Analysis of the Performance of Mascot and IdentiPy Algorithms on a Benchmark Dataset Obtained by Tandem Mass Spectrometry Analysis of Testicular Biopsies].

Zhonghua Lao Dong Wei Sheng Zhi Ye Bing Za Zhi. 2019 Jan 20. 37(1): 34-37

[Changes in mass spectrometry of proteins in patients with 1-bromopropane poisoning after treatment].

R M Miao, B M Ding, X T Yu, B L Zhu.

  Objective: To investigate the changes in mass spectrometry of proteins in patients with 1-bromopropane (1-BP) poisoning after treatment and their biological functions. Methods: From May 2016 to December 2017, 3 male patients aged 31-47 years with 1-BP poisoning in Bao'an District of Shenzhen, China were enrolled in this study. The whole blood sample (2 ml) was collected before and after treatment. Label-free mass spectrometry-based proteomics was used for protein identification and quantification. The differentially expressed proteins after treatment were analyzed. Bioinformatics tools were used to analyze the functions of the identified proteins and the biological processes they were involved in. Results: Proteomic analysis showed that there were 47 proteins that were differentially expressed more than 2-fold (P<0.05) after treatment in the patients with 1-BP poisoning; of them, 27 were up-regulated and 20 were down-regulated in the serum of treated patients. The identified proteins were mainly involved in proteolysis, protein modification, immune response, complement activation, lipoprotein metabolism, signal transduction, and coagulation. Conclusion: The differentially expressed proteins after treatment can help with the diagnosis, treatment, and prognosis monitoring of 1-BP poisoning and provide potential therapeutic and prognostic markers for 1-BP poisoning treatment.

Keywords:  1-Bromopropane; Occupational diseases; Poisoning; Proteome

DOI:  https://doi.org/10.3760/cma.j.issn.1001-9391.2019.01.007
OMICS. 2019 Mar;23(3): 167-179

Serum Proteomic Changes as Candidate Biomarkers of Intermediate Liver Fibrosis in Chronic Hepatitis B Infection.

Yi-Ning Dai, Yue-Xing Tu, Di Meng, Mei-Juan Chen, Jia-Jie Zhang, Yu-Han Gong, Yong-Xi Tong, Ming-Shan Wang, Hong-Ying Pan, Hai-Jun Huang.

  Chronic hepatitis B (CHB) is a major global health burden. Liver fibrosis, an insidious process, is the main histopathological change in CHB that might lead to the end-stage liver disease if left untreated. The intermediate liver fibrosis (S2) is the optimal time to start antiviral therapy. The aim of the present study was to examine the proteomic changes in patients with CHB at different fibrotic stages, with a view to identify future serum biomarkers for S2. Ninety CHB patients were grouped into mild (S0-1), intermediate (S2), and severe liver fibrosis (S3-4) (61 men and 29 women; age 25-63 years). Isobaric tagging for relative and absolute quantitation was applied to screen proteins differentially expressed among the patient groups. Another 46 patients with CHB (age 25-59 years; 31 men and 15 women), and 16 healthy controls (age 26-61 years; 11 men and 5 women) were enrolled in a validation group. Enzyme-linked immunosorbent assay was used to verify the diagnostic value of the candidate biomarkers. We found 139 proteins that were differentially expressed between various fibrotic stage-paired comparisons. Five protein candidates were selected as potential biomarkers of S2 for further verification. Notably, ficolin-2 (FCN2) and carboxypeptidase B2 (CPB2) showed differential expression between patients and healthy controls. In conclusion, serum proteomic changes reported here offer new molecular leads for future research on biomarker candidates to identify liver fibrotic stages in CHB. In particular, FCN2 and CPB2 warrant further research on their possible mechanistic involvement in CHB pathogenesis.

Keywords:  diagnostic medicine; hepatitis B; liver fibrosis biomarkers; microbial proteomics; precision medicine; proteomics

DOI:  https://doi.org/10.1089/omi.2018.0179
Cell Biol Toxicol. 2019 Mar 21.

Clinical protein science in translational medicine targeting malignant melanoma.

Jeovanis Gil, Lazaro Hiram Betancourt, Indira Pla, Aniel Sanchez, Roger Appelqvist, Tasso Miliotis, Magdalena Kuras, Henriette Oskolas, Yonghyo Kim, Zsolt Horvath, Jonatan Eriksson, Ethan Berge, Elisabeth Burestedt, Göran Jönsson, Bo Baldetorp, Christian Ingvar, Håkan Olsson, Lotta Lundgren, Peter Horvatovich, Jimmy Rodriguez Murillo, Yutaka Sugihara, Charlotte Welinder, Elisabet Wieslander, Boram Lee, Henrik Lindberg, Krzysztof Pawłowski, Ho Jeong Kwon, Viktoria Doma, Jozsef Timar, Sarolta Karpati, A Marcell Szasz, István Balázs Németh, Toshihide Nishimura, Garry Corthals, Melinda Rezeli, Beatrice Knudsen, Johan Malm, György Marko-Varga.

  Melanoma of the skin is the sixth most common type of cancer in Europe and accounts for 3.4% of all diagnosed cancers. More alarming is the degree of recurrence that occurs with approximately 20% of patients lethally relapsing following treatment. Malignant melanoma is a highly aggressive skin cancer and metastases rapidly extend to the regional lymph nodes (stage 3) and to distal organs (stage 4). Targeted oncotherapy is one of the standard treatment for progressive stage 4 melanoma, and BRAF inhibitors (e.g. vemurafenib, dabrafenib) combined with MEK inhibitor (e.g. trametinib) can effectively counter BRAFV600E-mutated melanomas. Compared to conventional chemotherapy, targeted BRAFV600E inhibition achieves a significantly higher response rate. After a period of cancer control, however, most responsive patients develop resistance to the therapy and lethal progression. The many underlying factors potentially causing resistance to BRAF inhibitors have been extensively studied. Nevertheless, the remaining unsolved clinical questions necessitate alternative research approaches to address the molecular mechanisms underlying metastatic and treatment-resistant melanoma. In broader terms, proteomics can address clinical questions far beyond the reach of genomics, by measuring, i.e. the relative abundance of protein products, post-translational modifications (PTMs), protein localisation, turnover, protein interactions and protein function. More specifically, proteomic analysis of body fluids and tissues in a given medical and clinical setting can aid in the identification of cancer biomarkers and novel therapeutic targets. Achieving this goal requires the development of a robust and reproducible clinical proteomic platform that encompasses automated biobanking of patient samples, tissue sectioning and histological examination, efficient protein extraction, enzymatic digestion, mass spectrometry-based quantitative protein analysis by label-free or labelling technologies and/or enrichment of peptides with specific PTMs. By combining data from, e.g. phosphoproteomics and acetylomics, the protein expression profiles of different melanoma stages can provide a solid framework for understanding the biology and progression of the disease. When complemented by proteogenomics, customised protein sequence databases generated from patient-specific genomic and transcriptomic data aid in interpreting clinical proteomic biomarker data to provide a deeper and more comprehensive molecular characterisation of cellular functions underlying disease progression. In parallel to a streamlined, patient-centric, clinical proteomic pipeline, mass spectrometry-based imaging can aid in interrogating the spatial distribution of drugs and drug metabolites within tissues at single-cell resolution. These developments are an important advancement in studying drug action and efficacy in vivo and will aid in the development of more effective and safer strategies for the treatment of melanoma. A collaborative effort of gargantuan proportions between academia and healthcare professionals has led to the initiation, establishment and development of a cutting-edge cancer research centre with a specialisation in melanoma and lung cancer. The primary research focus of the European Cancer Moonshot Lund Center is to understand the impact that drugs have on cancer at an individualised and personalised level. Simultaneously, the centre increases awareness of the relentless battle against cancer and attracts global interest in the exceptional research performed at the centre.

Keywords:  Cancer moonshot; Clinical proteomics; Malignant melanoma; Post-translational modifications; Translational medicine

DOI:  https://doi.org/10.1007/s10565-019-09468-6
Biol Psychiatry. 2019 Jan 30. pii: S0006-3223(19)30064-2. [Epub ahead of print]

Integrated Lipidomics and Proteomics Point to Early Blood-Based Changes in Childhood Preceding Later Development of Psychotic Experiences: Evidence From the Avon Longitudinal Study of Parents and Children.

Francisco Madrid-Gambin, Melanie Föcking, Sophie Sabherwal, Meike Heurich, Jane A English, Aoife O'Gorman, Tommi Suvitaival, Linda Ahonen, Mary Cannon, Glyn Lewis, Ismo Mattila, Caitriona Scaife, Sean Madden, Tuulia Hyötyläinen, Matej Orešič, Stanley Zammit, Gerard Cagney, David R Cotter, Lorraine Brennan.

   BACKGROUND: The identification of early biomarkers of psychotic experiences (PEs) is of interest because early diagnosis and treatment of those at risk of future disorder is associated with improved outcomes. The current study investigated early lipidomic and coagulation pathway protein signatures of later PEs in subjects from the Avon Longitudinal Study of Parents and Children cohort.
METHODS: Plasma of 115 children (12 years of age) who were first identified as experiencing PEs at 18 years of age (48 cases and 67 controls) were assessed through integrated and targeted lipidomics and semitargeted proteomics approaches. We assessed the lipids, lysophosphatidylcholines (n = 11) and phosphatidylcholines (n = 61), and the protein members of the coagulation pathway (n = 22) and integrated these data with complement pathway protein data already available on these subjects.
RESULTS: Twelve phosphatidylcholines, four lysophosphatidylcholines, and the coagulation protein plasminogen were altered between the control and PEs groups after correction for multiple comparisons. Lipidomic and proteomic datasets were integrated into a multivariate network displaying a strong relationship between most lipids that were significantly associated with PEs and plasminogen. Finally, an unsupervised clustering approach identified four different clusters, with one of the clusters presenting the highest case-control ratio (p < .01) and associated with a higher concentration of smaller low-density lipoprotein cholesterol particles.
CONCLUSIONS: Our findings indicate that the lipidome and proteome of subjects who report PEs at 18 years of age are already altered at 12 years of age, indicating that metabolic dysregulation may contribute to an early vulnerability to PEs and suggesting crosstalk between these lysophosphatidylcholines, phosphatidylcholines, and coagulation and complement proteins.

Keywords:  ALSPAC; Early life; Integration; Lipidomics; Proteomics; Psychotic episode

DOI:  https://doi.org/10.1016/j.biopsych.2019.01.018
J Autoimmun. 2019 Mar 15. pii: S0896-8411(19)30038-1. [Epub ahead of print]

Transcriptomic and proteomic analysis of iris tissue and aqueous humor in juvenile idiopathic arthritis-associated uveitis.

Lena Wildschütz, Doreen Ackermann, Anika Witten, Maren Kasper, Martin Busch, Shirin Glander, Harutyun Melkonyan, Karoline Walscheid, Christoph Tappeiner, Solon Thanos, Andrei Barysenka, Jörg Koch, Carsten Heinz, Björn Laffer, Dirk Bauer, Monika Stoll, Simone König, Arnd Heiligenhaus.

  Gene and protein expression profiles of iris biopsies, aqueous humor (AqH), and sera in patients with juvenile idiopathic arthritis-associated uveitis (JIAU) in comparison to control patients with primary open-angle glaucoma (POAG) and HLA-B27-positive acute anterior uveitis (AAU) were investigated. Via RNA Sequencing (RNA-Seq) and mass spectrometry-based protein expression analyses 136 genes and 56 proteins could be identified as being significantly differentially expressed (DE) between the JIAU and POAG group. Gene expression of different immunoglobulin (Ig) components as well as of the B cell-associated factors ID3, ID1, and EBF1 was significantly upregulated in the JIAU group as compared to POAG patients. qRT-PCR analysis showed a significantly higher gene expression of the B cell-related genes CD19, CD20, CD27, CD138, and MZB1 in the JIAU group. At the protein level, a significantly higher expression of Ig components in JIAU than in POAG was confirmed. The B cell-associated protein MZB1 showed a higher expression in JIAU patients than in POAG which was confirmed by western blot analysis. Using bead-based immunoassay analysis we were able to detect a significantly higher concentration of the B cell-activating and survival factors BAFF, APRIL, and IL-6 in the AqH of JIAU and AAU patients than in POAG patients. The intraocularly upregulated B cell-specific genes and proteins in iris tissue suggest that B cells participate in the immunopathology of JIAU. The intracameral environment in JIAU may facilitate local effector and survival functions of B cells, leading to disease course typical for anterior uveitis.

Keywords:  B cells; Cytokines; HLA-B27; Juvenile idiopathic arthritis; Plasma cell niche; Uveitis

DOI:  https://doi.org/10.1016/j.jaut.2019.03.004
BMC Rheumatol. 2018 ;2 35

A proteomic analysis of serum-derived exosomes in rheumatoid arthritis.

Hirotaka Tsuno, Mitsumi Arito, Naoya Suematsu, Toshiyuki Sato, Atsushi Hashimoto, Toshihiro Matsui, Kazuki Omoteyama, Masaaki Sato, Kazuki Okamoto, Shigeto Tohma, Manae S Kurokawa, Tomohiro Kato.

   Background: To understand the roles of serum exosomes in rheumatoid arthritis (RA), we comprehensively investigated the protein profiles of serum exosomes in patients with RA.
Methods: Exosomes were isolated from serum samples obtained from 33 patients (12 with active RA [aRA], 11 with inactive RA [iRA], 10 with osteoarthritis [OA]) and 10 healthy donors (HLs). Proteins extracted from the exosomes were separated by two-dimensional differential gel electrophoresis (2D-DIGE) and identified by mass spectrometry.
Results: In total, 204 protein spots were detected by 2D-DIGE. In the aRA, iRA, and OA groups, 24, 5, and 7 spots showed approximately ≥ ±1.3-fold intensity differences compared with the HL group, respectively. We were able to identify proteins in six protein spots. Among them, the protein spot identified as Toll-like receptor 3 (TLR3) showed approximately 6-fold higher intensity in the aRA group than in the other groups.
Conclusions: Patients with active RA possessed considerably different protein profiles of serum exosomes from patients with iRA, patients with OA, and healthy donors. The unique protein profile of serum exosomes, such as the possession of abundant TLR3 fragments, may reflect the pathophysiology of active RA.

Keywords:  Exosome; Osteoarthritis; Proteomics; Rheumatoid arthritis; Toll like receptor 3

DOI:  https://doi.org/10.1186/s41927-018-0041-8
Proc Natl Acad Sci U S A. 2019 Mar 18. pii: 201817867. [Epub ahead of print]

Multiplex targeted mass spectrometry assay for one-shot flavivirus diagnosis.

Sheena Wee, Asfa Alli-Shaik, Relus Kek, Hannah L F Swa, Wei-Ping Tien, Vanessa W Lim, Yee-Sin Leo, Lee-Ching Ng, Hapuarachchige C Hapuarachchi, Jayantha Gunaratne.

  Targeted proteomic mass spectrometry is emerging as a salient clinical diagnostic tool to track protein biomarkers. However, its strong analytical properties have not been exploited in the diagnosis and typing of flaviviruses. Here, we report the development of a sensitive and specific single-shot robust assay for flavivirus typing and diagnosis using targeted mass spectrometry technology. Our flavivirus parallel reaction monitoring assay (fvPRM) has the ability to track secreted flaviviral nonstructural protein 1 (NS1) over a broad diagnostic and typing window with high sensitivity, specificity, extendibility, and multiplexing capability. These features, pivotal and pertinent to efficient response toward flavivirus outbreaks, including newly emerging flavivirus strains, circumvent the limitations of current diagnostic assays. fvPRM thus carries high potential in positioning itself as a forerunner in delivering early and accurate diagnosis for disease management.

Keywords:  PRM; dengue; diagnostics; flavivirus; targeted proteomics

DOI:  https://doi.org/10.1073/pnas.1817867116
Biochem Biophys Res Commun. 2019 Mar 19. pii: S0006-291X(19)30364-X. [Epub ahead of print]

Proteomic investigations of human HERC2 mutants: Insights into the pathobiology of a neurodevelopmental disorder.

Joseph R Abraham, John Barnard, Heng Wang, Garey H Noritz, Mehdi Yeganeh, Daniela Buhas, Marvin R Natowicz.

  HERC2 is a giant protein with E3 ubiquitin ligase activity and other known and suspected functions. Mutations of HERC2 are implicated in the pathogenesis of various cancers and result in severe neurological conditions in Herc2-mutant mice. Recently, a pleotropic autosomal recessive HERC2-associated syndrome of intellectual disability, autism and variable neurological deficits was described; its pathogenetic basis is largely unknown. Using peripheral blood-derived lymphoblasts from 3 persons with homozygous HERC2 variants and 14 age- and gender-matched controls, we performed label-free unbiased HPLC-tandem mass spectrometry-based proteomic analyses to provide insights into HERC2-mediated pathobiology. We found that out of 3427 detected proteins, there were 812 differentially expressed proteins between HERC2-cases vs. controls. 184 canonical pathways were enriched after FDR adjustment, including mitochondrial function, energy metabolism, EIF2 signaling, immune functions, ubiquitination and DNA repair. Ingenuity Pathway Analysis® identified 209 upstream regulators that could drive the differential expression, prominent amongst which were neurodegeneration-associated proteins. Differentially expressed protein interaction networks highlighted themes of immune function/dysfunction, regulation of cell cycle/cell death, and energy metabolism. Overall, the analysis of the HERC2-associated proteome revealed striking differential protein expression between cases and controls. The large number of differentially expressed proteins likely reflects HERC2's multiple domains and numerous interacting proteins. Our canonical pathway and protein interaction network findings suggest derangements of multiple pathways in HERC2-associated disease.

Keywords:  Autism; Bioinformatics; HERC2; Neurodevelopment; Proteomics

DOI:  https://doi.org/10.1016/j.bbrc.2019.02.149
Int J Mol Med. 2019 Mar 19.

Isobaric tags for relative and absolute quantitation‑based proteomics reveals potential novel biomarkers for the early diagnosis of acute myocardial infarction within 3 h.

Changqing Du, Yingzheng Weng, Jiangjie Lou, Guangzhong Zeng, Xiaowei Liu, Hongfeng Jin, Senna Lin, Lijiang Tang.

Acute myocardial infarction (AMI) is one of the most common and life‑threatening cardiovascular diseases. However, the ability to diagnose AMI within 3 h is currently lacking. The present study aimed to identify the differentially expressed proteins of AMI within 3 h and to investigate novel biomarkers using isobaric tags for relative and absolute quantitation (ITRAQ) technology. A total of 30 beagle dogs were used for establishing the MI models successfully by injecting thrombin powder and a polyethylene microsphere suspension. Serum samples were collected prior to (0 h) and following MI (1, 2 and 3 h). ITRAQ‑coupled liquid chromatography‑mass spectrometry (LC‑MS) technology was used to identify the differentially expressed proteins. The bioinformatics analysis selected several key proteins in the initiation of MI. Further analysis was performed using STRING software. Finally, western blot analysis was used to evaluate the results obtained from ITRAQ. In total, 28 proteins were upregulated and 23 were downregulated in the 1 h/0 h group, 28 proteins were upregulated and 26 were downregulated in the 2 h/0 h group, and 24 proteins were upregulated and 19 were downregulated in the 3 h/0 h group. The Gene Ontology (GO) annotation and functional enrichment analysis identified 19 key proteins. Protein‑protein interactions (PPIs) were investigated using the STRING database. GO enrichment analysis revealed that a number of key proteins, including ATP synthase F1 subunit β (ATP5B), cytochrome c oxidase subunit 2 and cytochrome c, were components of the electron transport chain and were involved in energy metabolism. The western blot analysis demonstrated that the expression of ATP5B decreased significantly at all three time points (P<0.01), which was consistent with the ITRAQ results, whereas the expression of fibrinogen γ chain increased at 2 and 3 h (P<0.01) and the expression of integrator complex subunit 4 increased at all three time points (P<0.01), which differed from the ITRAQ results. According to the proteomics of the beagle dog MI model, ATP5B may serve as the potential biomarkers of AMI. Mitochondrial dysfunction and disruption of the electron transport chain may be critical indicators of early MI within 3 h. These finding may provide a novel direction for the diagnosis of AMI.

DOI: https://doi.org/10.3892/ijmm.2019.4137
J Proteome Res. 2019 Mar 20.

Proteomic Analysis Applied to Human Plasma During Intermittent Fasting.

Dylan J Harney, Amy T Hutchison, Luke Hatchwell, Sean J Humphrey, David E James, Samantha Hocking, Leonie K Heilbronn, Mark Larance.

Intermittent fasting (IF) increases lifespan and decreases metabolic disease phenotypes and cancer risk in model organisms, but the health benefits of IF in humans are less clear. Human plasma derived from clinical trials is one of the most difficult sample sets to analyze using mass spectrometry-based proteomics due to the extensive sample preparation required and the need to process many samples to achieve statistical significance. Here, we describe an optimized and accessible device (Spin96) to accommodate up to 96 StageTips, a widely used sample preparation medium enabling efficient and consistent processing of samples prior to LC-MS/MS. We have applied this device to the analysis of human plasma from a clinical trial of IF. In this longitudinal study employing 8-weeks IF, we identified significant abundance differences induced by the IF intervention, including increased apolipoprotein A4 (APOA4) and decreased apolipoprotein C2 (APOC2) and C3 (APOC3). These changes correlated with a significant decrease in plasma triglycerides after the IF intervention. Given that these proteins have a role in regulating apolipoprotein particle metabolism, we propose that IF had a positive effect on lipid metabolism through modulation of HDL particle size and function. In addition, we applied a novel human protein variant database to detect common protein variants across the participants. We show that consistent detection of clinically-relevant peptides derived from both alleles of many proteins is possible, including some that are associated with human metabolic phenotypes. Together, these findings illustrate the power of accessible workflows for proteomics analysis of clinical samples to yield significant biological insight.

DOI: https://doi.org/10.1021/acs.jproteome.9b00090
J Proteome Res. 2019 Mar 22.

Significant Down-regulation of Urea Cycle Generates Clinically Relevant Proteomic Signature in Hepatocellular Carcinoma Patients with Macrovascular Invasion.

Yin Cao, WenWen Ding, Jingzi Zhang, Qi Gao, HaoXiang Yang, Wangsen Cao, Zhongxia Wang, Lei Fang, Ronghui Du.

Vascular invasion is considered as the critical risk factors of hepatocellular carcinoma (HCC). To reveal the molecular mechanisms underlying macrovascular invasion (MaVI) in HCC, we performed an iTRAQ based proteomic study to identify notably dysregulated proteins from 8 HCC patients with differential vascular invasion and further confirmed them in the other 53 HCC patients. 47 proteins were found significantly down-regulated in HCC with MaVi. More importantly, 30 of them were not changed in HCC without MaVI. Gene ontology analysis of these 47 proteins shows the top 3 enriched biological processes are urea cycle, gluconeogenesis and arginine biosynthetic process. We validated 9 remarkably dysregulated candidates in HCC patients with MaVI by Western blot, including 8 down-regulated proteins (CPS1, ASS1, ASL, ARG1, BHMT, DMGDH, Annexin A6 and CES1) and 1 up-regulated protein (CKAP4). Furthermore, dysregulation of CPS1, ASL and ARG1, key enzymes involved in urea cycle, together with Annexin A6 and CES1, major proteins in regulating cholesterol homeostasis and fatty acid ester metabolism were verified using immunohistochemical staining. The significant down-regulation of urea cycle generates clinically relevant proteomic signature in HCC patients with macrovascular invasion, which may provide possible insights into the molecular mechanisms of metastasis and new therapeutic targets of HCC.

DOI: https://doi.org/10.1021/acs.jproteome.8b00921
Andrology. 2019 Mar 19.

Sperm functional aspects and enriched proteomic pathways of seminal plasma of adult men with obesity.

P C Ferigolo, M B Ribeiro de Andrade, M Camargo, V M Carvalho, K H M Cardozo, R P Bertolla, R Fraietta.

   BACKGROUND: Previous studies have demonstrated an association between obesity and the decreased male fertility.
OBJECTIVE: to observe the mechanisms by which obesity affects semen quality.
MATERIALS AND METHODS: A prospective study was performed including 47 male volunteers, of which 27 were obese group (body mass index >30 kg/m2 ) and 20 were eutrophic (body mass index between 18.5 and 25 kg/m2 ) controls. Sperm functional analysis was performed. The remaining seminal plasma was pooled-four pools per group- and submitted to proteomic analysis by liquid chromatography coupled to tandem mass spectrometry. Groups were compared by an unpaired Student's t-test. Differentially expressed proteins were submitted to functional enrichment analysis using the online platform PantherDB.
RESULTS: Obese men presented decreased non-progressive motility, morphology, acrosome integrity, mitochondrial activity, and increased sperm DNA fragmentation. In proteomics analysis, 69 proteins were differentially expressed between the two groups. Among them, one protein was absent, 19 were down-regulated, 49 were up-regulated, and one was exclusive in the study group. The main functions enriched were as follows: negative regulation of the intrinsic pathway of apoptosis, activation of immune and inflammatory, antioxidant activity, among others.
CONCLUSION: molecular pathways suggest there is a causative link, and that the effector mechanisms alter sperm metabolic status and defective testicular selection 5 mechanisms.

Keywords:  DNA fragmentation; obesity; proteomics; seminal plasma; spermatozoa

DOI:  https://doi.org/10.1111/andr.12606
Front Immunol. 2019 ;10 381

Autoantibody Biomarker Discovery in Primary Open Angle Glaucoma Using Serological Proteome Analysis (SERPA).

Vanessa M Beutgen, Natarajan Perumal, Norbert Pfeiffer, Franz H Grus.

  Glaucoma is an optic neurological disorder and the leading cause of irreversible blindness worldwide, with primary open angle glaucoma (POAG) as its most prevalent form. An early diagnosis of the disease is crucial to prevent loss of vision. Mechanisms behind glaucoma pathogenesis are not completely understood, but disease related alterations in the serological autoantibody profile indicate an immunologic component. These changes in immunoreactivity may serve as potential biomarkers for glaucoma diagnostics. We aimed to identify novel disease related autoantibodies targeting antigens in the trabecular meshwork as biomarkers to support early detection of POAG. We used serological proteome analysis (SERPA) for initial autoantibody profiling in a discovery sample set. The identified autoantibodies were validated by protein microarray analysis in a larger cohort with 60 POAG patients and 45 control subjects. In this study, we discovered CALD1, PGAM1, and VDAC2 as new biomarker candidates. With the use of artificial neural networks, the panel of these candidates and the already known markers HSPD1 and VIM was able to classify subjects into POAG patients and non-glaucomatous controls with a sensitivity of 81% and a specificity of 93%. These results suggest the benefit of these potential autoantibody biomarkers for utilization in glaucoma diagnostics.

Keywords:  autoantibodies; biomarker; glaucoma; immunoproteomics; microarray; trabecular meshwork

DOI:  https://doi.org/10.3389/fimmu.2019.00381
J Cancer Res Ther. 2019 Jan-Mar;15(1):15(1): 96-103

Isobaric tags for relative and absolute quantitation-based proteome analysis of Vietnamese colorectal carcinoma tissues.

Thi Thu Hien Pham, Kim Phuong Uyen Le, Phuong Uyen Vo, Kieu Minh Le, Teck Kwang Lim, Qinsong Lin, Thi Thu Hoai Nguyen.

   Context: Colorectal cancer (CRC) is one of the most common malignancies and one of the leading causes of cancer death worldwide. Establishing early detection methods or markers of CRC is central to improve the survival rate of CRC patients. Nowadays, new molecular tools have been developed to acquire further knowledge on tumor progression.
Aims: Comparative proteomics analysis of Vietnamese colorectal carcinoma in different stages was performed to gain an insight into the molecular events taking place in CRC and metastasis.
Subjects and Methods: In this study, the comparative protein expression analysis of ten paired CRC and its corresponding noncancerous tissue samples was performed using the combination of isobaric tags for relative and absolute quantitation labeling and mass spectrometry (MS). The data obtained were further analyzed with Ingenuity Pathways Analysis (IPA) system.
Results: Based on the MS/MS spectra analyzed by ProteinPilot software, 684 proteins were identified, out of which 215 were observed to be differentially expressed in at least 1 sample pair. Individual protein expression and variation have been identified for each patient. IPA system demonstrated cytoskeletal signaling as the top-ranked functional pathway network associated with the oncogenic function.
Conclusions: Our study supplemented the understanding about proteome of Vietnamese CRC patients and identified statistically protein expression differences among samples assisting in finding effective biomarkers for CRC diagnostics.

Keywords:  Colorectal cancer; isobaric tags for relative and absolute quantitation; proteomics

DOI:  https://doi.org/10.4103/0973-1482.202889
Anal Chem. 2019 Mar 18.

Isotopologue multi-point calibration for proteomics biomarker quantification in clinical practice.

Cristina Chiva, Olga Pastor, Lucía Trilla-Fuertes, Angelo Gámez-Pozo, Juan Ángel Fresno Vara, Eduard Sabidó.

Targeted proteomics has become the method of choice for biomarker validation in human biopsies due to its high sensitivity, reproducibility, accuracy and precision. However, for targeted proteomics technologies to be transferred to clinical routine there is the need to reduce its complexity, make its procedures simpler, increase its throughput and improve its analytical performance. Here we present the Isotopologue Multiple-point Calibration (ImCal) quantification strategy, which uses a mix of isotopologue peptides to generate internal multiple-point calibration curves for each individual sample and to accurately quantify biomarker peptides in clinical applications without the need of expert supervision. ImCal relies on the use of five different isotopically-labelled peptides of different nominal mass mixed at different concentrations to be used as internal calibration curve for each endogenous peptide. The use of internal multi-point calibration curves is well-suited for the generation of ready-to-use biomarker kits for clinical applications as it is compatible with both high- and low-resolution mass spectrometers and different levels of endogenous peptide, it eliminates the need for blank matrices required in external curves, it allows the evaluation of matrix effects and the valid quantification range in each individual sample, and does not require expert adjustment. We used the ImCal method to quantify HER2 in 35 breast cancer formalin-fixed paraffin-embedded patient samples revealing a high degree of heterogeneity among patients, which contrast with the homogeneous immunohistochemistry patient classification. Our work illustrates how an improvement of mass spectrometry methods for biomarker quantification can provide fine-grain patient stratification, and thus better disease diagnostic and prognosis.

DOI: https://doi.org/10.1021/acs.analchem.8b05802
Mil Med. 2019 Mar 01. 184(Supplement_1): 652-657

Serum Biomarkers for Racial Disparities in Breast Cancer Progression.

Meera Srivastava, Ofer Eidelman, James Craig, Joshua Starr, Leonid Kvecher, Jianfang Liu, Matthew Hueman, Harvey B Pollard, Hai Hu, Craig D Shriver.

  African American (AA) women are often diagnosed with more aggressive breast cancers and have worse survival outcomes than their Caucasian American (CA) counterparts. However, a comprehensive understanding of this disparity remains unclear. In this study, we attempted to identify the race-specific non-invasive protein biomarkers that may particularly benefit interventions aimed at reducing the risk of recurrence and metastasis in breast cancers (BrCa). Our technical strategy has been to discover candidate protein biomarkers in patient sera using a high throughput antibody microarray platform. A total of 240 subjects were selected, composed of controls and all immunohistochemistry-based subtypes of breast cancer cases, subdivided by pre- and post-menopausal status and by race. A global Wilcoxon analysis comparing no-cancer controls and cancer patients identified Pyk2, SAPK/JNK, and phosphatase and tensin homolog as present in higher concentrations in cancer patient serum. A paired t-test revealed that c-kit and Rb are significantly over-represented in AA cancer serum when compared to CA cancer serum. Interestingly, VEGFR2, a protein linked to BrCa metastasis and poor prognosis, was significantly over-represented in AA cancer serum compared to AA controls; however, this was not found in CA cancer serum compared to CA controls, suggesting a possible explanation for the higher incidence of aggressive BrCa in AA versus CA patients. Through examining race-specific differences in the protein landscape of BrCa patient serum, the identified proteins could lay the groundwork for the development of an all-inclusive "liquid mammogram test."

Keywords:  Human; biomarkers; breast cancer; race

DOI:  https://doi.org/10.1093/milmed/usy417
Rheumatology (Oxford). 2019 Mar 19. pii: kez073. [Epub ahead of print]

The autoantibody response to cyclic citrullinated collagen type II peptides in rheumatoid arthritis.

Bibo Liang, Changrong Ge, Erik Lönnblom, Xiaoyin Lin, Hui Feng, Lianbo Xiao, Jing'an Bai, Burcu Ayoglu, Peter Nilsson, Kutty Selva Nandakumar, Ming Zhao, Rikard Holmdahl.

   OBJECTIVES: The detection of anti-citrullinated peptide antibodies (ACPAs) is a serological hallmark of RA. Autoantibodies reactive with collagen type II (CII) are present in RA sera and synovial fluid and are potentially pathogenic. Here, we investigate the prevalence and specificity of the autoantibody responses to defined citrullinated cyclic peptides derived from CII in a China RA cohort.
METHODS: Using bead-based multiplex assay, we examined the presence of autoantibodies binding to 54 cyclic 17-mer citrullinated CII peptides, encompassing all citrullinate epitopes in CII, and the corresponding unmodified peptides in 415 RA patients, in addition to 304 patients with OA. Furthermore, the autoantibody responses to a selected set of 10 cyclic citrullinated peptides were also examined in 203 healthy individuals.
RESULTS: Autoantibody responses to cyclic citrullinated CII peptides were higher in RA patients as compared with OA patients or healthy individuals, whereas little or negligible antibody responses to cyclic unmodified CII peptides were observed. Interestingly, several novel citrullinated CII epitopes were identified. Antibodies to these novel citrullinated CII epitopes showed not only substantial overlapping reactivities but also had unique specificities.
CONCLUSION: We found a high prevalence of autoantibodies against cyclic citrullinated CII in the sera of patients in a China RA cohort. The present study revealed heterogeneous binding patterns against novel citrullinated CII epitopes, which may help to stratify RA patients into different subgroups.

Keywords:  anti-citrullinated peptide antibody; collagen type II; osteoarthritis; rheumatoid arthritis

DOI:  https://doi.org/10.1093/rheumatology/kez073
PLoS One. 2019 ;14(3): e0213892

Translation and evaluation of a pre-clinical 5-protein response prediction signature in a breast cancer phase Ib clinical trial.

Axel Ducret, Ian James, Sabine Wilson, Martina Feilke, Andreas Tebbe, Nikolaj Dybowski, Sarah Elschenbroich, Martin Klammer, Adele Blackler, Wei-Li Liao, Yuan Tian, Thomas Friess, Birgit Bossenmaier, Gabriele Dietmann, Christoph Schaab, Todd Hembrough, Maurizio Ceppi.

Human protein biomarker discovery relies heavily on pre-clinical models, in particular established cell lines and patient-derived xenografts, but confirmation studies in primary tissue are essential to demonstrate clinical relevance. We describe in this study the process that was followed to clinically translate a 5-protein response signature predictive for the activity of an anti-HER3 monoclonal antibody (lumretuzumab) originally measured in fresh frozen xenograft tissue. We detail the development, qualification, and validation of the multiplexed targeted mass spectrometry assay used to assess the signature performance in formalin-fixed, paraffin-embedded human clinical samples collected in a phase Ib trial designed to evaluate lumretuzumab in patients with metastatic breast cancer. We believe that the strategy delineated here provides a path forward to avoid the time- and cost-consuming step of having to develop immunological reagents against unproven targets. We expect that mass spectrometry-based platforms may become part of a rational process to rapidly test and qualify large number of candidate biomarkers to identify the few that stand a chance for further development and validation.

DOI: https://doi.org/10.1371/journal.pone.0213892
Cancer Cell. 2019 Mar 18. pii: S1535-6108(19)30100-X. [Epub ahead of print]35(3): 414-427.e6

The Proteogenomic Landscape of Curable Prostate Cancer.

Ankit Sinha, Vincent Huang, Julie Livingstone, Jenny Wang, Natalie S Fox, Natalie Kurganovs, Vladimir Ignatchenko, Katharina Fritsch, Nilgun Donmez, Lawrence E Heisler, Yu-Jia Shiah, Cindy Q Yao, Javier A Alfaro, Stas Volik, Anna Lapuk, Michael Fraser, Ken Kron, Alex Murison, Mathieu Lupien, Cenk Sahinalp, Colin C Collins, Bernard Tetu, Mehdi Masoomian, David M Berman, Theodorus van der Kwast, Robert G Bristow, Thomas Kislinger, Paul C Boutros.

  DNA sequencing has identified recurrent mutations that drive the aggressiveness of prostate cancers. Surprisingly, the influence of genomic, epigenomic, and transcriptomic dysregulation on the tumor proteome remains poorly understood. We profiled the genomes, epigenomes, transcriptomes, and proteomes of 76 localized, intermediate-risk prostate cancers. We discovered that the genomic subtypes of prostate cancer converge on five proteomic subtypes, with distinct clinical trajectories. ETS fusions, the most common alteration in prostate tumors, affect different genes and pathways in the proteome and transcriptome. Globally, mRNA abundance changes explain only ∼10% of protein abundance variability. As a result, prognostic biomarkers combining genomic or epigenomic features with proteomic ones significantly outperform biomarkers comprised of a single data type.

Keywords:  biomarker; epigenome; genome; multi-omic features; prostate cancer; proteome; transcriptome

DOI:  https://doi.org/10.1016/j.ccell.2019.02.005
Biosci Rep. 2019 Mar 21. pii: BSR20181966. [Epub ahead of print]

Interleukin-1 in cerebrospinal fluid for evaluating the neurological outcome in traumatic brain injury.

Yingming Yue, Chongzhi Shang, Huajiang Dong, Kun Meng.

  Objective Severe traumatic brain injury (TBI) is associated with unfavorable outcomes secondary to injury from activation of the inflammatory cascade, the release of excitotoxic neurotransmitters, and changes in the reactivity of cerebral vessels, causing ischemia. Inflammation induced by TBI is complex, individual-specific, and associated with morbidity and mortality. The aim of this study was to discover the differentially expressed cerebrospinal fluid (CSF) proteins and identify which can improve the clinical outcomes in TBI patients. Methods In this study, we report 145 patients with TBI and we found the change of patients' leukocyte in serum and interleukin-1( IL-1) in CSF, which strongly correlated with the neurological outcome. In terms of results of leukocyte in blood and IL-1 in CSF, we retained the patient's cerebrospinal fluid specimens and conducted a proteomic analysis.Results A total of 119 differentially expressed proteins were detected between samples of TBI and the normal, which were commonly expressed in all samples, indicating the differentially expressed proteins. When the patients' glasgow outcome score (GOS) improved, IL-1 was downregulated, and when the patients' GCS score deteriorated, IL-1 was upregulated accompanied with the progression TBI. Conclusions The differentially expressed proteins in CSF may be the novel therapeutic targets for TBI treatment. The leukocyte in blood samples and the IL-1 in CSF may be two important indicators for predicting the prognosis of TBI patients.

Keywords:  cerebrospinal fluid; differential expressed proteins; interleukin-1; traumatic brain injury

DOI:  https://doi.org/10.1042/BSR20181966
Sci Rep. 2019 Mar 18. 9(1): 4794

Proteomic profiling of whole-saliva reveals correlation between Burning Mouth Syndrome and the neurotrophin signaling pathway.

Guy Krief, Yaron Haviv, Omer Deutsch, Naama Keshet, Galit Almoznino, Batia Zacks, Aaron Palmon, Doron J Aframian.

Burning mouth syndrome (BMS) is characterized by a spontaneous and chronic sensation of burning in the oral mucosa, with no apparent signs. The underlying pathophysiological and neuropathic mechanisms remain unclear. Here, we attempt to elucidate some of these mechanisms using proteomic profiling and bioinformatic analyses of whole-saliva (WS) from BMS patients compared to WS from healthy individuals. Qualitative and quantitative proteomic profiling was performed using two dimensional gel electrophoresis (2-DE) and quantitative mass spectrometry (q-MS). In order to improve protein visibility, 21 high abundance proteins were depleted before proteomic profiling. Quantitative proteomic analysis revealed 100 BMS specific proteins and an additional 158 proteins up-regulated by more than threefold in those with BMS. Bioinformatic analyses of the altered protein expression profile of BMS group indicated high correlations to three cellular mechanisms including the neurotrophin signaling pathway. Based on this finding, we suggest that neurotrophin signaling pathway is involved in the pathophysiology of BMS by amplifying P75NTR activity, which in turn increases neural apoptosis thereby reducing sub-papillary nerve fiber density in the oral mucosa.

DOI: https://doi.org/10.1038/s41598-019-41297-9
Mol Med Rep. 2019 Mar 14.

Identification of red blood cell membrane defects in a patient with hereditary spherocytosis using next‑generation sequencing technology and matrix‑assisted laser desorption/ionization time‑of‑flight mass spectrometry.

Leyla Türker Şener, Melih Aktan, Gürcan Albeniz, Aziz Şener, Duran Üstek, Işıl Albeniz.

Hereditary spherocytosis (HS) is characterized by the morphological transformation of erythrocytes into a spherical shape due to a hereditary defect in cell membrane proteins (ghosts) associated with disruption of erythrocyte skeletal structures. Contrary to the literature, pores were detected in the erythrocytes of a patient with HS. The aim of the present study was to determine the affected proteins and genes that were responsible for the pores. Ghost isolation was performed to determine the proteins responsible for the pores observed on the erythrocytes of the patient. Erythrocyte membrane proteins were visualized using SDS‑PAGE. Exome and matrix‑assisted laser desorption/ionization time‑of‑flight mass spectrometry (MALDI TOF MS) analyses were used to identify the genes and proteins responsible for the observed defect. Quantitative protein assessments were performed using MALDI TOF MS. A difference was detected in the components of the erythrocyte membrane proteins. Band 3 and protein 4.2, which serve a particular role in membrane structure, decreased 4.573 and 4.106 fold, respectively. Through proteomic analyses, a non‑synonymous exonic mutation region was identified in the Golgi membrane protein 1 (GOLM1) gene (Chr9 rs142242230). Sorting Intolerant From Tolerant and Polymorphism Phenotyping Scores, Likelihood Ratio Tests and MutationTaster revealed that the mutation was deleterious. The pores observed in the morphology of the erythrocytes may have developed due to the decrease in these proteins, which reside in the erythrocyte membrane structure. Furthermore, genetic profiling of the patient with HS and her family was conducted in the present study. Next‑generation sequencing was used, and the genetic source of HS was identified as a GOLM1 gene mutation. The assessment of specific molecular defects is often not performed as the majority of mutations are unique to a family. However, molecular analyses should be performed in severe cases where prenatal diagnosis is required, or for unique HS phenotypes to aid scientific investigation.

DOI: https://doi.org/10.3892/mmr.2019.10036
J Crohns Colitis. 2019 Mar 19. pii: jjy217. [Epub ahead of print]

Molecular Profiling of Ulcerative Colitis Subjects from the TURANDOT Trial Reveals Novel Pharmacodynamic/Efficacy Biomarkers.

Huanyu Zhou, Li Xi, Daniel Ziemek, Shawn O'Neil, Julie Lee, Zachary Stewart, Yutian Zhan, Shanrong Zhao, Ying Zhang, Karen Page, Austin Huang, Mateusz Maciejewski, Baohong Zhang, Kenneth J Gorelick, Lori Fitz, Vivek Pradhan, Fabio Cataldi, Michael Vincent, David Von Schack, Kenneth Hung, Mina Hassan-Zahraee.

   BACKGROUND AND AIMS: To define pharmacodynamic and efficacy biomarkers in ulcerative colitis [UC] patients treated with PF-00547659, an anti-human mucosal addressin cell adhesion molecule-1 [MAdCAM-1] monoclonal antibody, in the TURANDOT study.
METHODS: Transcriptome, proteome and immunohistochemistry data were generated in peripheral blood and intestinal biopsies from 357 subjects in the TURANDOT study.
RESULTS: In peripheral blood, C-C motif chemokine receptor 9 [CCR9] gene expression demonstrated a dose-dependent increase relative to placebo, but in inflamed intestinal biopsies CCR9 gene expression decreased with increasing PF-00547659 dose. Statistical models incorporating the full RNA transcriptome in inflamed intestinal biopsies showed significant ability to assess response and remission status. Oncostatin M [OSM] gene expression in inflamed intestinal biopsies demonstrated significant associations with, and good accuracy for, efficacy, and this observation was confirmed in independent published studies in which UC patients were treated with infliximab or vedolizumab. Compared with the placebo group, intestinal T-regulatory cells demonstrated a significant increase in the intermediate 22.5-mg dose cohort, but not in the 225-mg cohort.
CONCLUSIONS: CCR9 and OSM are implicated as novel pharmacodynamic and efficacy biomarkers. These findings occur amid coordinated transcriptional changes that enable the definition of surrogate efficacy biomarkers based on inflamed biopsy or blood transcriptomics data.ClinicalTrials.gov identifierNCT01620255.

Keywords:  MAdCAM-1; PF-00547659; Ulcerative colitis; biomarkers; inflammatory bowel disease

DOI:  https://doi.org/10.1093/ecco-jcc/jjy217
Mol Biol (Mosk). 2019 Jan-Feb;53(1):53(1): 166-176

[Comparative Analysis of the Performance of Mascot and IdentiPy Algorithms on a Benchmark Dataset Obtained by Tandem Mass Spectrometry Analysis of Testicular Biopsies].

A V Lisitsa, N A Petushkova, L I Levitsky, V G Zgoda, O V Larina, Yu S Kisrieva, V E Frankevich, S I Gamidov.

  Proteome profiling of human testicular biopsies was performed using tandem mass spectrometry with electrospray ionization. Protein identification results were compared for the Mascot commercial search engine, the SearchGUI noncommercial package, and their analog IdentiProt based on the open-source IdentiPy algorithm (http://hg.theorchromo.ru/identipy). A feature of IdentiPy is an automatic optimization of MS/MS search parameters. A set of protein identifications obtained with IdentiPy was consequently greater by one third than the sets with the other search engines. For the first time, an IdentiPy/IdentiProt search was conducted within the Progenesis LC-MS framework, which allows spectrum alignment, and the proteome profile obtained with alignment was compared with that obtained using the ProteoWizard converter. A total of 16 human chromosome 18 proteins were identified, including the myelin basic protein, which is not characteristic of testicular tissue.

Keywords:  IdentiProt; Mascot; SearchGUI; biopsy; chromosome 18; proteomics; tandem mass spectroscopy; testicular tissue

DOI:  https://doi.org/10.1134/S0026898419010099