Andersen, V.

Bennike, Tue Bjerg

Birkelund, Svend

Bonderup, O. K.

Bøgsted, M.

Carlsen, Thomas Gelsing

Christiansen, Gunna

Ellingsen, T.

Glerup, H.

Stensballe, Allan

VBN (Videnbasen)  Aalborg Universitets forskningsportal

    Aalborg UniversitetNeutrophil extracellular traps in ulcerative colitisBennike, Tue Bjerg; Carlsen, Thomas Gelsing; Ellingsen, T.; Bonderup, O. K.; Glerup, H.;Bøgsted, M.; Christiansen, G.; Birkelund, Svend; Stensballe, Allan; Andersen, V.Published in:Proceedings, Targeted Proteomics Workshop &amp; International Symposium, TPWIS 2015, 10-14 December2015, Mumbai, IndiaPublication date:2015Document VersionPublisher's PDF, also known as Version of recordLink to publication from Aalborg UniversityCitation for published version (APA):Bennike, T. B., Carlsen, T. G., Ellingsen, T., Bonderup, O. K., Glerup, H., Bøgsted, M., ... Andersen, V. (2015).Neutrophil extracellular traps in ulcerative colitis: A proteome analysis of intestinal biopsies. In Proceedings,Targeted Proteomics Workshop & International Symposium, TPWIS 2015, 10-14 December 2015, Mumbai,India (pp. 97-98, No. D-1-07). Indian Institute of Technology.General rightsCopyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright ownersand it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights.            ? Users may download and print one copy of any publication from the public portal for the purpose of private study or research.            ? You may not further distribute the material or use it for any profit-making activity or commercial gain            ? You may freely distribute the URL identifying the publication in the public portal ?Take down policyIf you believe that this document breaches copyright please contact us at vbn@aub.aau.dk providing details, and we will remove access tothe work immediately and investigate your claim.Downloaded from vbn.aau.dk on: April 30, 201797TPWIS-2015rameters and their effect on DIA. Protein digest ob-tained from human cell line was analyzed in tripli-cates with different sample amounts from 250ng–2µg. DIA acquisition was obtained using MS scans analyz-ing 400-1000m/z with a resolution of 60k (400m/z). 30 MS/MS scans were acquired with 20m/z isolation window, loop count of 10 and orbitrap resolution of 30k (400m/z). For two fold change, we observed rela-tively better quantitative accuracy for lower protein amount compared to higher. For higher fold changes, we observed good quantitative accuracy irrespective of protein amount. Although DIA offers several ad-vantages over DDA, informatics tools to deconvolute the data for identification and quantitation need sig-nificant improvement.D-1-06:Targted Interaction Analysis of FtsZ with Small Molecules and the Effect of Six Point Mutations of ClpX on BsFtsZ PolymerizationReddy PJ, Khanzode M, Shah VG, Panda D, Srivas-tava SDepartment of Biosciences and Bioengineering, Indian In-stitute of Technology Bombay, Powai, Mumbai-400076, IndiaDrug resistance is the major concern for healthcare professionals and looking deep into the existing and new chemical libraries to explore the new drugs nov-el mechanism of action. Bacterial cell division process is highly conserved mechanism in prokaryotes and FtsZ is the key player involved in cytokinesis process along with many accessory proteins. Many natural compounds have showed potential antimicrobial activity by targeting the FtsZ polymerization. The protein expression profile of FtsZ has not changed much under curcumin, totarol and plumbagin treat-ment whereas mRNA expression analysis of FtsZ showed significant repression. In the present study, we have performed real-time interaction analysis of both BsFtsZ and EcFtsZ with curcumin, totarol and plumbagin for binding and kinetic analysis. Further, molecular docking has provided the putative bind-ing sites for the small molecules in FtsZ. Besides, we also tested the effect of wild type and six point mu-tants ClpX on BsFtsZ polymerization. Interestingly, mutated ClpX has induced BsFtsZ polymerization in concentration dependent manner however wild type hasn’t changed much. The real-time SPR analysis also showed that both wild type and mutated ClpX binds to BsFtsZ strongly in a concentration dependent man-ner. In summary, our study showed that the men-tioned small molecules did not affect the FtsZ protein expression levels rather bind with FtsZ at molecular level and regulate the FtsZ polymerization dynamics. This study also provided the KD for both BsFtsZ and EcFtsZ with the small molecules using SPR analysis. In addition, our results demonstrated that both wild type and mutant (six mutations) ClpX leads to inhi-bition of FtsZ polymerization with equal affinity. In depth analysis is necessary for conclusive prospective on role of mutated ClpX on bacterial cell division.D-1-07:Neutrophil Extracellular Traps in Ulcerative Coli-tis–A Proteome Analysis of Intestinal BiopsiesBennike TB1, Carlsen TG1, Ellingsen T2, Bonderup OK3, Glerup H3, Boegsted M1, Christiansen G4, Birkelund S1, Stensballe A1, Andersen V5,61 Department of Health Science and Technology, Aalborg University, Denmark: 2 Department of Rheumatology, Odense University Hos-pital, Denmark 3 Department of Clinical Medicine, Silkeborg Hospital, Denmark 4 Department of Clinical Medicine, Aalborg University Hospital, Denmark 5 Institute of Regional Health Research, University of Southern Denmark 6 Organ Center, Hospital of Southern Jutland, Aabenraa, DenmarkInflammatory bowel diseases, including Ulcerative colitis affects 2.5–3 million people in Europe. The life-long conditions significantly reduce the quality of life of the patients, and compose a significant economic burden for society. The etiology of these diseases remains incompletely explained. We, therefore per-formed the most thorough proteome-based study of ulcerative colitis colon tissue, based on number of an-alyzed proteins, and have for the first time observed neutrophil extracellular traps in ulcerative colitis, de-tected by proteomics and verified by microscopy.We performed a comparative proteome analysis of non-inflamed mucosal colon biopsies from 10 patients with ulcerative colitis and 10 controls.The proteome of the biopsies were characterized by high through-put label-free quantitative proteomics, and the biopsy histology was analyzed by microscopy.We quantified 5,711 different proteins in the colon biopsies. Forty-six proteins had a statistically sig-nificant difference in abundance between the ulcer-ative colitis colon tissue and controls. Eleven of the proteins with increased abundances in the ulcerative colitis biopsies were associated with neutrophils and neutrophil extracellular traps. The abundance of cal-98TPWIS-2015protectin and lactotransferrin in the tissue correlated with the degree of tissue inflammation as determined by histology. The findings were validated by micros-copy where an increased abundance of neutrophils within the mucosal tissue was found. Additionally, we confirmed the presence of neutrophil extracellu-lar traps by extracellular DNA in the ulcerative colitis colon tissue.We found an increased abundance of several proteins hitherto not associated with innate immunity and neutrophils in non-inflamedmucosal colon biopsies from ulcerative colitis patients. The increased abun-dance of these antimicrobial compounds points to the stimulation of the innate immune system in the eti-ology of ulcerative colitis. Our findings demonstrate that even though remission has been achieved on the surface of the ulcerative colitis colon tissue, a chronic condition is still present within the morphologically normal tissue.D-1-08:Mass Spectrometric N-Terminal Sequencing of Pep-tides Using a Bacterial AminopeptidaseKishor N, Guptasarma PDepartment of Biological Sciences, Indian Institute of Sci-ence Education and Research (IISER) MohaliAminopeptidases are exo-peptidases that catalyze the hydrolysis of peptide bonds joining the N-termi-nal amino acid of any peptide to the next amino acid in the sequence. Non- processive aminopeptidases are specific for certain residues and cannot progres-sively hydrolyze successively placed amino acid resi-dues, whereas processive aminopeptidases tend to be non-specific and can ‘progressively’ cleave up to 30-35 amino acid residues in any peptide. Theoretically, therefore, beginning with a homogenous population of molecules of a defined mass constituting a single peptide of defined amino acid sequence, any proces-sive aminopeptidase can be used to create a popula-tion of peptides of different lengths differing by one amino acid mass, so that the entire population can be studied in a single mass spectrum, to determine the peptide’s amino acid sequence. However, in practice, it is difficult to control the reaction from progress-ing rapidly and also some peptides have blocked N-termini which prevent facile proteolysis.  Sometimes, structure present in the peptide can also interfere. In this study, we describe the use of a non-specific, pro-cessive, deblocking Bacillus subtillis-derived amino-peptidase (BsuAP) in a reaction conducted at 70 de-grees Centigrade for N-terminus sequencing.Glu fibrino peptide (GFP B) from Waters, USA, was used as substrate. GFP B is used as a standard (lock mass) in mass spectrometry and has a mass of 1570.67 Da. BsuAP was a recombinant protein expressed in, and purified from, E. coli in our own lab [3]. The reac-tion mixture of enzyme and substrate was prepared by mixing 1 nanomole of the aminopeptidase to 1.6 nanomoles of the substrate (GFP B) in water. The re-action mixture and control reaction (lacking amino-peptidase) were incubated at 700c for 1 hour. After incubation, the reaction-mixture and control reaction were spotted on the MALDI plate. GFP B was used as a lock mass control. For spotting, 1µl of sample was spotted with 1µl CHCA matrix.  Samples were ana-lyzed on the Q-TOF Synapt G2S HDMS system (from Waters, USA), with laser desorption achieved by the instrument’s 355nm laser. The spectra analyzed and presented are lock mass-corrected spectra. The GFP B peptide is 14 residues-long. A sufficiently slow reaction would produce a mass spectrum con-taining the mass corresponding to the complete pep-tide, and all smaller masses progressively truncating the peptide from its N-terminus, assuming that the reaction does not proceed to completion on all sub-strate molecules. We found all expected masses down to 480.25 Da in the reaction spectrum. This data sug-gests that under the conditions used, BsuAP is non-specific and quite processive, progressively digesting 11 amino acid residues in the peptide substrate, be-ginning with the N-terminal glycine.  Masses are seen to have different relative intensities which could be indicative of differences in specificity. It may be not-ed that the differences in masses of successive peaks (moving leftwards from the control GFP B mass) cor-respond to masses of the amino acids removed, after addition of the mass of one hydrogen atom. D-1-09:MRM Based Identification and Relative Quanti-tation of Vimentin in Different Grades of Menin-giomasGhantasala Saicharan1, Mukherjee Shuvolina1, Singh Vedita Anand1, Gahoi Nikita1, Datar Ajit2, Bhandarkar Deepti2, Rane Shailendra2, Moyadi Aliasgar3, Srivastava Sanjeeva11 Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Powai, Mumbai-400076, India2 Shimadzu Analytical (India) Pvt. Ltd., 1 A/B, Rushabh Chambers, Makwana Road, Marol, Andheri East, Mum-bai-400059, India3 Department of Neurosurgery, Advanced Center for Treatment Research and Education in Cancer, Tata Memo-rial Center, Kharghar, Navi Mumbai-410210, India

Neutrophil extracellular traps in ulcerative colitis:A proteome analysis of intestinal biopsies

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Neutrophil extracellular traps in ulcerative colitis:A proteome analysis of intestinal biopsies

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