Bischoff, R.

Franciosi, L.

Fusetti, F.

Govorukhina, N.

Hacken, N. ten

Poolman, B.

Postma, D.

English

RATIONALEChronic Obstructive Pulmonary Disease (COPD) is the third most frequent disease worldwide with increasing mortality. Cigarette smoking is the principle risk factor and 15-20% of smokers develop COPD.Epithelial Lining Fluid (ELF) covers the internal part of the airways and can be collected during bronchoscopy. ELF appears to be well-suited for proteomic analysis, since it contains a higher concentration of proteins (150-300 μg /mL) than other lung fluids and can be obtained from different locations of the lungs. No comprehensive proteomic analysis of human ELF has been performed to date, which makes ELF a highly interesting fluid for biomarker discovery in COPD.AIMTo discover proteins that change in abundance in ELF from COPD patients versus healthy controls using a quantitative proteomics approach.METHODSThe ELF proteome from COPD patients and healthy controls was studied by 1D polyacrylamide gel electrophoresis in the presence of SDS followed by in-gel tryptic digestion to establish the methodology and assess the feasibility of such an approach. Approximately 40 gel slices were obtained from each lane of the gel (corresponding to one patient). Digested samples were analyzed by nanoChip-LC-MS/MS using an ion trap.We performed a quantitative pilot study of ELF from 4 COPD patients and 4 healthy controls (table 1) to test for statistically significant differences in protein levels. ELF samples were digested by trypsin, labeled with stable isotope-containing reagents (iTRAQ®, 8-plex) and processed by strong cation-exchange chromatography followed by nanoLC-MS/MS. In order to validate the results, a second quantitative analysis of an independent sample set (4 COPD vs 4 healthy) using the same methodological approach was done.RESULTSThe 1D electrophoretic approach resulted in more than 300 identified proteins. Most of the identified proteins were present in both COPD and healthy samples, although some proteins were only identified either in healthy control or in COPD samples.The quantitative studies showed that a number of proteins was significantly different between ELF of COPD patients and controls, including 4 up-regulated proteins in common in both studies.CONCLUSIONSThis is the first study in ELF of COPD patients and healthy controls in which such a large number of proteins has been identified.The obtained results show the feasibility of this proteomic approach and the possibility to discover proteins that are differentially expressed in ELF of COPD patients and controls. We are currently validating these proteins further by western blot and immunohistochemistry

Proceedings - University of Groningen

   University of GroningenBiomarker Discovery In Chronic Obstructive Pulmonary Disease (COPD) Using EpithelialLining FluidFranciosi, L.; Govorukhina, N.; Fusetti, F.; Poolman, B.; Hacken, N. ten; Postma, D.; Bischoff,R.Published in:American Journal of Respiratory and Critical Care MedicineIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:2011Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Franciosi, L., Govorukhina, N., Fusetti, F., Poolman, B., Hacken, N. T., Postma, D., & Bischoff, R. (2011).Biomarker Discovery In Chronic Obstructive Pulmonary Disease (COPD) Using Epithelial Lining Fluid: AProteomic Approach. American Journal of Respiratory and Critical Care Medicine, 183(1), A1492-A1493.CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum. / Thematic Poster Session / Sunday, May 15/8:15 AM-4:30 PM / Area D, Hall B (Upper Level), ColoradoA40 OUTCOMES IN COPDConvention CenterBiomarker Discovery In Chronic Obstructive Pulmonary Disease (COPD) Using Epithelial Lining Fluid: AProteomic Approach, N. govorukhina , F. fusetti , B. poolman , N. hacken ten , D. Postma , R. bischoffL. Franciosi1 1 1 1 2 3 1University of Groningen, groningen, Netherlands, University Medical Center Groningen, groningen, Netherlands, University Medical1 2 3Center Groningen, Groningen, NetherlandsCorresponding author's email: L.Franciosi@rug.nlRATIONALEChronic Obstructive Pulmonary Disease (COPD) is the third most frequent disease worldwide with increasing mortality. Cigarette smokingis the principle risk factor and 15-20% of smokers develop COPD.Epithelial Lining Fluid (ELF) covers the internal part of the airways and can be collected during bronchoscopy. ELF appears to bewell-suited for proteomic analysis, since it contains a higher concentration of proteins (150-300 μg /mL) than other lung fluids and can beobtained from different locations of the lungs. No comprehensive proteomic analysis of human ELF has been performed to date, whichmakes ELF a highly interesting fluid for biomarker discovery in COPD.AIMTo discover proteins that change in abundance in ELF from COPD patients versus healthy controls using a quantitative proteomicsapproach.METHODSThe ELF proteome from COPD patients and healthy controls was studied by 1D polyacrylamide gel electrophoresis in the presence of SDSfollowed by in-gel tryptic digestion to establish the methodology and assess the feasibility of such an approach. Approximately 40 gelslices were obtained from each lane of the gel (corresponding to one patient). Digested samples were analyzed by nanoChip-LC-MS/MSusing an ion trap.We performed a quantitative pilot study of ELF from 4 COPD patients and 4 healthy controls (table 1) to test for statistically significantdifferences in protein levels. ELF samples were digested by trypsin, labeled with stable isotope-containing reagents (iTRAQ®, 8-plex) andprocessed by strong cation-exchange chromatography followed by nanoLC-MS/MS. In order to validate the results, a second quantitativeanalysis of an independent sample set (4 COPD vs 4 healthy) using the same methodological approach was done.RESULTSThe 1D electrophoretic approach resulted in more than 300 identified proteins. Most of the identified proteins were present in both COPDand healthy samples, although some proteins were only identified either in healthy control or in COPD samples.The quantitative studies showed that a number of proteins was significantly different between ELF of COPD patients and controls,including 4 up-regulated proteins in common in both studies.CONCLUSIONSThis is the first study in ELF of COPD patients and healthy controls in which such a large number of proteins has been identified.The obtained results show the feasibility of this proteomic approach and the possibility to discover proteins that are differentiallyexpressed in ELF of COPD patients and controls. We are currently validating these proteins further by western blot andimmunohistochemistry.Patient characteristics, lung function, smoking historyPilot study Pilot study Validation study Validation studyCOPD (n=4)Healthy(n=4)COPD (n=4)Healthy(n=4)Female, number 0 2 2 2Age, years 63.75 62.75 60 55.5Co-morbidity, CCI 2.25 0.25 1.5 0.25Current smoker, number 1 0 1 2Ex smoker, number 3 0 3 1Pack years, number 37.5 0 37.5 9.52Inhaled corticosteroids, number 2 0 1 0FEV /FVC, %1 58.5 76 33.25 84.25FEV  %predicted1, 71.5 116.3 40.25 111.25This abstract is funded by: NoneAm J Respir Crit Care Med 183;2011:A1492Internet address: www.atsjournals.org Online Abstracts Issue

Biomarker Discovery In Chronic Obstructive Pulmonary Disease (COPD) Using Epithelial Lining Fluid:A Proteomic Approach

https://pure.rug.nl/ws/files/2549918/2011AmJRespirCritCareMedFranciosi.pdf

Biomarker Discovery In Chronic Obstructive Pulmonary Disease (COPD) Using Epithelial Lining Fluid:A Proteomic Approach

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