Phosphotyrosine biased enrichment of tryptic peptides from cancer cells by combining pY-MIP and TiO2 affinity resins

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Phosphotyrosine biased enrichment of tryptic peptides from cancer cells by combining pY-MIP and TiO2 affinity resins

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Publication Article, peer reviewed scientific
Title Phosphotyrosine biased enrichment of tryptic peptides from cancer cells by combining pY-MIP and TiO2 affinity resins
Author Bllaci, Loreta ; Torsetnes, Silje ; Shinde, Sudhirkumar ; Sellergren, Börje ; Rogowska-Wrzesinska, Adelina ; Jensen, Ole N.
Research Centre Biofilms - Research Center for Biointerfaces
Date 2017
English abstract
Protein phosphorylation at distinct tyrosine residues (pY) is essential for fast, specific, and accurate signal transduction in cells. Enrichment of pY-containing peptides derived from phosphoproteins is commonly facilitated by use of immobilized anti-pY antibodies prior to phosphoproteomics analysis by mass spectrometry. We here report on an alternative approach for pY-peptide enrichment using inexpensive pY-imprinted polymer (pY-MIP). We assessed by mass spectrometry the performance of pY-MIP for enrichment and sequencing of phosphopeptides obtained by tryptic digestion of protein extracts from HeLa cells. The combination of pY-MIP- and TiO2-based phosphopeptide enrichment provided more than 90% selectivity for phosphopeptides. Mass spectrometry signal intensities were enhanced for most pY-phosphopeptides (approximately 70%) when using the pY-MIP-TiO2 combination as compared to TiO2 alone. pY constituted up to 8% of the pY-MIP-TiO2-enriched phosphopeptide fractions. The pY-MIP-TiO2 and the TiO2 protocols yielded comparable numbers of distinct phosphopeptides, 1693 and 1842, respectively, from microgram levels of peptide samples. Detailed analysis of physicochemical properties of pY-MIP-TiO2-enriched phosphopeptides demonstrated that this protocol retrieved phosphopeptides that tend to be smaller (<24 residues), less acidic, and almost exclusively monophosphorylated, as compared to TiO2 alone. These unique properties render the pY-MIP-based phosphopeptide enrichment technique an attractive alternative for applications in phosphoproteomics research.
DOI https://doi.org/10.1021/acs.analchem.7b02091 (link to publisher's fulltext.)
Publisher American Chemical Society
Host/Issue Analytical Chemistry;21
Volume 89
ISSN 0003-2700
Language eng (iso)
Subject Sciences
Research Subject Categories::NATURAL SCIENCES
Handle http://hdl.handle.net/2043/24207 Permalink to this page
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