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Simultaneous Detection of Human C‑Terminal p53 Isoforms by Single Template Molecularly Imprinted Polymers (MIPs) Coupled with Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS)-Based Targeted Proteomics
journal contribution
posted on 2018-02-15, 20:20 authored by Wenting Jiang, Liang Liu, Yun ChenAbnormal expression of C-terminal
p53 isoforms α, β,
and γ can cause the development of cancers including breast
cancer. To date, much evidence has demonstrated that these isoforms
can differentially regulate target genes and modulate their expression.
Thus, quantification of individual isoforms may help to link clinical
outcome to p53 status and to improve cancer patient treatment. However,
there are few studies on accurate determination of p53 isoforms, probably
due to sequence homology of these isoforms and also their low abundance.
In this study, a targeted proteomics assay combining molecularly imprinted
polymers (MIPs) and liquid chromatography-tandem mass spectrometry
(LC-MS/MS) was developed for simultaneous quantification of C-terminal
p53 isoforms. Isoform-specific surrogate peptides (i.e., KPLDGEYFTLQIR
(peptide-α) for isoform α, KPLDGEYFTLQDQTSFQK
(peptide-β) for isoform β, and KPLDGEYFTLQMLLDLR
(peptide-γ) for isoform γ) were first selected and used
in both MIPs enrichment and mass spectrometric detection. The common
sequence KPLDGEYFTLQ of these three surrogate peptides
was used as single template in MIPs. In addition to optimization of
imprinting conditions and characterization of the prepared MIPs, binding
affinity and cross-reactivity of the MIPs for each surrogate peptide
were also evaluated. As a result, a LOQ of 5 nM was achieved, which
was >15-fold more sensitive than that without MIPs. Finally, the
assay
was validated and applied to simultaneous quantitative analysis of
C-terminal p53 isoforms α, β, and γ in several human
breast cell lines (i.e., MCF-10A normal cells, MCF-7 and MDA-MB-231
cancer cells, and drug-resistant MCF-7/ADR cancer cells). This study
is among the first to employ single template MIPs and cross-reactivity
phenomenon to select isoform-specific surrogate peptides and enable
simultaneous quantification of protein isoforms in LC-MS/MS-based
targeted proteomics.
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Keywords
template MIPsKPLDimprinting conditionsC-terminal p 53 isoformspeptideMDA-MB -231 cancer cellsproteomics assayp 53 isoformsbreast cell linesSimultaneous Detectionchromatography-tandem mass spectrometrytarget genes-10ALiquid Chromatography-Tandem Mass Spectrometry5 nMsequence homologyp 53 statusSingle Template Molecularly Imprinted Polymerscancer patient treatmentbreast cancercross-reactivity phenomenonbinding affinityprotein isoformsLOQMCF -7LC-MSMIPs enrichment
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