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Updated: Apr 18, 2026
Enhanced Sample Multiplexing of Tissues Using Combined Precursor Isotopic Labeling and Isobaric Tagging cPILOT
Published on: May 1, 2017
Global cPILOT analysis of the APP/PS-1 mouse liver proteome
Adam R Evans1, Liqing Gu1, Rodolfo Guerrero1
1Department of Chemistry, University of Pittsburgh, Pittsburgh, PA, USA.
Purpose:
A quantitative proteomics strategy called combined precursor isotopic labeling and isobaric tagging (cPILOT) was designed to discover alterations in the amyloid precursor protein/presenilin-1 (APP/PS-1) mouse liver proteome. The multiplexing strategy allows simultaneous quantitation of 12 samples in a single experiment.
Experimental Design:
For cPILOT samples, six APP/PS-1 and six heterozygous mouse livers were modified using precursor dimethylation (pH 2.5) followed by isobaric tagging (pH 8.0). Samples were pooled, fractioned with strong cation exchange, and analyzed using RPLC-MS(3) for protein identification and relative quantitation. In order to increase proteome coverage, a two-tiered data collection strategy was employed. Six duplex precursor dimethylation experiments were also performed to verify cPILOT protein quantitation.
Results:
The combination of cPILOT with precursor dimethylation data resulted in 2437 total liver proteins identified and 77 differentially expressed proteins in APP/PS-1 liver. Differentially expressed proteins are involved in metabolic processes such as B-oxidation, pyruvate metabolism, and glucose regulation.
Conclusions And Clinical Relevance:
cPILOT expands protein quantitation using isobaric tags and can be applied to any clinical laboratory interested in enhanced multiplexing strategies. Differentially expressed proteins in APP/PS-1 mouse liver suggest the potential use of ketone bodies to alleviate metabolic dysregulation in Alzheimer's disease brain. Our work also suggests alterations in the alanine cycle potentially leading to hyperammonia production, may contribute to Alzheimer's disease pathogenesis.
