title: Validation of molecularly imprinted polymers for side chain selective phosphopeptide enrichment.
abstract: Selective enrichment techniques are essential for mapping of protein posttranslational modifications (PTMs). Phosphorylation is one of the PTMs which continues to be associated with significant analytical challenges. Particularly problematic are tyrosine-phosphorylated peptides (pY-peptides) resulting from tryptic digestion which commonly escape current chemo- or immuno- affinity enrichments and hence remain undetected. We here report on significant improvements in this regard using pY selective molecularly imprinted polymers (pY-MIPs). The pY-MIP was compared with titanium dioxide (TiO2) affinity based enrichment and immunoprecipitation (IP) with respect to selective enrichment from a mixture of 13 standard peptides at different sample loads. At a low sample load (1pmol of each peptide), IP resulted in enrichment of only a triply phosphorylated peptide whereas TiO2enriched phosphopeptides irrespective of the amino acid side chain. However, with increased sample complexity, TiO2failed to enrich the doubly phosphorylated peptides. This contrasted with the pY-MIP showing enrichment of all four tyrosine phosphorylated peptides at 1pmol sample load of each peptide with a few other peptides binding unselectively. At an increased sample complexity consisting of the standard peptides spiked into mouse brain digest, the MIP showed clear enrichment of all four pY- peptides.
authors.affiliation: Medizinisches Proteom-Center, Ruhr-University Bochum, Universitätsstr. 150, 44801 Bochum, Germany.
authors.forename: Jing
authors.initials: J
authors.lastname: Chen
authors.affiliation: Department of Biomedical Sciences, Faculty of Health and Society, Malmö University, SE 205 06 Malmö, Sweden. Electronic address: sudhirkumar.shinde@mah.se.
authors.forename: Sudhirkumar
authors.initials: S
authors.lastname: Shinde
authors.affiliation: Medizinisches Proteom-Center, Ruhr-University Bochum, Universitätsstr. 150, 44801 Bochum, Germany.
authors.forename: Prabal
authors.initials: P
authors.lastname: Subedi
authors.affiliation: Department of Biomedical Sciences, Faculty of Health and Society, Malmö University, SE 205 06 Malmö, Sweden.
authors.forename: Celina
authors.initials: C
authors.lastname: Wierzbicka
authors.affiliation: Department of Biomedical Sciences, Faculty of Health and Society, Malmö University, SE 205 06 Malmö, Sweden.
authors.forename: Börje
authors.initials: B
authors.lastname: Sellergren
authors.affiliation: Medizinisches Proteom-Center, Ruhr-University Bochum, Universitätsstr. 150, 44801 Bochum, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Helling
authors.affiliation: Medizinisches Proteom-Center, Ruhr-University Bochum, Universitätsstr. 150, 44801 Bochum, Germany. Electronic address: katrin.marcus@rub.de.
authors.forename: Katrin
authors.initials: K
authors.lastname: Marcus
mesh_terms: D002623:Chemistry Techniques, Analytical / Q000379:methods* / Q000592:standards; D054802:Molecular Imprinting; D010748:Phosphopeptides / Q000737:chemistry / Q000302:isolation & purification*; D010766:Phosphorylation; D011108:Polymers / Q000737:chemistry*; D014025:Titanium / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry
chemical_list: D010748:Phosphopeptides; D011108:Polymers; C009495:titanium dioxide; D014443:Tyrosine; D014025:Titanium
keywords: Antibody; Molecularly imprinted polymer; Phosphopeptide enrichment; Phosphotyrosine; TiO(2)