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Enhanced Sample Multiplexing of Tissues Using Combined Precursor Isotopic Labeling and Isobaric Tagging (cPILOT)

King, Christina D. and Dudenhoeffer, Joseph D. and Gu, Liqing and Evans, Adam R. and Robinson, Renã A. S. (2017) Enhanced Sample Multiplexing of Tissues Using Combined Precursor Isotopic Labeling and Isobaric Tagging (cPILOT). Journal of Visualized Experiments (123). ISSN 1940-087X

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There is an increasing demand to analyze many biological samples for disease understanding and biomarker discovery. Quantitative proteomics strategies that allow simultaneous measurement of multiple samples have become widespread and greatly reduce experimental costs and times. Our laboratory developed a technique called combined precursor isotopic labeling and isobaric tagging (cPILOT), which enhances sample multiplexing of traditional isotopic labeling or isobaric tagging approaches. Global cPILOT can be applied to samples originating from cells, tissues, bodily fluids, or whole organisms and gives information on relative protein abundances across different sample conditions. cPILOT works by 1) using low pH buffer conditions to selectively dimethylate peptide N-termini and 2) using high pH buffer conditions to label primary amines of lysine residues with commercially-available isobaric reagents (see Table of Materials/Reagents). The degree of sample multiplexing available is dependent on the number of precursor labels used and the isobaric tagging reagent. Here, we present a 12-plex analysis using light and heavy dimethylation combined with six-plex isobaric reagents to analyze 12 samples from mouse tissues in a single analysis. Enhanced multiplexing is helpful for reducing experimental time and cost and more importantly, allowing comparison across many sample conditions (biological replicates, disease stage, drug treatments, genotypes, or longitudinal time-points) with less experimental bias and error. In this work, the global cPILOT approach is used to analyze brain, heart, and liver tissues across biological replicates from an Alzheimer's disease mouse model and wild-type controls. Global cPILOT can be applied to study other biological processes and adapted to increase sample multiplexing to greater than 20 samples.


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Item Type: Article
Status: Published
CreatorsEmailPitt UsernameORCID
King, Christina D.
Dudenhoeffer, Joseph D.
Gu, Liqing
Evans, Adam R.
Robinson, Renã A. S.
Date: 1 May 2017
Date Type: Publication
Journal or Publication Title: Journal of Visualized Experiments
Number: 123
Publisher: Journal of Visualized Experiments (JOVE)
DOI or Unique Handle: 10.3791/55406
Schools and Programs: Dietrich School of Arts and Sciences > Chemistry
Refereed: No
ISSN: 1940-087X
Official URL:
Article Type: Research Article
Date Deposited: 13 May 2020 14:34
Last Modified: 13 May 2020 14:34


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