Towards a 44-plex cPILOT

Abstract

Event Abstract Back to Event Towards a 44-plex cPILOT David A. Vassallo Rodríguez1 and Renā A. Robinson1* 1 Vanderbilt University, United States Our laboratory created a novel method, “combined precursor isotopic labeling and isobaric tagging (cPILOT)” to increase the multiplexing capability of quantitative proteomics. This scheme takes advantage of the pka difference between the N-terminal and Lysine primary amines to selectively incorporate chemical tags through manipulation of buffer pH. The use of heavy isotopic tags and isobaric reagents such as tandem mass tags (TMT) has made it possible to multiplex 24 samples in a single experiment to date. The current work aims to develop novel chemical strategies to expand the multiplexing capability of cPILOT. Specifically, we are evaluating if other amine chemistries, such as diethylation and propionylation, can be incorporated into the cPILOT workflow. For this work, we are using mouse brain tissue to generate complex tryptic peptide mixtures that we can independently diethylate or propionylate. Diethylation and propionylation increases peptide masses by 56.0626 and 56.0262 Da, respectively. Experimental variables such as buffer composition, buffer pH, reaction time and temperature are optimized in order to increase the specificity and labelling efficiency for N-terminal primary amines. For diethylation, peptides are resuspended in a coupling buffer, to which acetaldehyde is later added and this final solution is incubated at 37°C for 25 minutes. For propionylation, propionic anhydride is added to the peptides, pH adjusted, the solution is incubated at 37°C for 15 minutes, and these steps are performed twice more for high labelling efficiency. Samples are then TMT tagged and taken through a cPILOT workflow on a liquid chromatography coupled tandem mass spectrometry system. This poster will present our preliminary data on the incorporation of diethylation and propionylation in to the cPILOT workflow. Keywords: multiplexing, cPILOT, Enhanced multiplexing, Proteomics, reductive amination Conference: National Organization for the Professional Advancement of Black Chemists and Chemical Engineers (NOBCChE) 45th Annual Conference , Orlando, Florida, United States, 17 Sep - 20 Sep, 2018. Presentation Type: Poster Presentation Topic: Analytical Chemsitry Citation: Vassallo Rodríguez DA and Robinson RA (2019). Towards a 44-plex cPILOT. Front. Chem. Conference Abstract: National Organization for the Professional Advancement of Black Chemists and Chemical Engineers (NOBCChE) 45th Annual Conference . doi: 10.3389/conf.fchem.2018.01.00059 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 20 Oct 2018; Published Online: 17 Jan 2019. * Correspondence: Prof. Renā A Robinson, Vanderbilt University, Nashville, United States, rena.as.robinson@vanderbilt.edu Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers David A Vassallo Rodríguez Renā A Robinson Google David A Vassallo Rodríguez Renā A Robinson Google Scholar David A Vassallo Rodríguez Renā A Robinson PubMed David A Vassallo Rodríguez Renā A Robinson Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.